Proteopedia - User contributions [en]

Highlighted Proteins of Lyme Disease

2012-09-25T19:09:00Z

Marvin O'Neal:

<font size='4'>Highlighted Proteins of Lyme Disease</font size>
<p>
[http://en.wikipedia.org/wiki/Lyme_disease Lyme disease] is caused by three species of bacteria belonging to the <i>Borrelia</i> genus, with <i>Borrelia burgdorferi</i> being the most common in the US. The bacteria are transmitted via hard-bodied ticks of the [http://en.wikipedia.org/wiki/Ixodidae <i>Ixodidae</i>] family. <i>Borrelia</i> spirochetes are motile, helical bacteria that have many lipoproteins exposed on the surfaces of their membranes. Two predominant groups of surface lipoproteins are classified as the outer surface proteins (Osps) and the variable major protein-like sequence expressed (VlsE). Both groups of proteins play important roles in pathogenesis, the the life cycle of <i>Borrelia</i>, and eliciting an immune response from the host (Figure 1).<ref name="connolly">PMID: 15864264</ref>
</p>
<p>
In a introductory biology course at Stony Brook University, undergraduates are modeling and exploring <i>Borrelia burgdorferi</i> surface proteins, as well as host produced antibodies to these proteins. This Proteopedia page is the product of their efforts, with a focus on highlighted proteins from five categories: [[#Osp C and Lyme Disease|Osp C]], [[#Antibodies to Osp A and Osp B and Lyme Disease|antibodies to Osp A and Osp B]], [[#Osp A and Lyme Disease|Osp A]], [[#Osp B and Lyme Disease|Osp B]], and [[#VlsE and Lyme Disease|VlsE]].
</p>
<p>
The goal of this Proteopedia page is to describe Lyme disease from a structural biology perspective. What do <i>Borrelia burgdorferi</i> outer surface proteins look like? How does the structure/function of these proteins relate to the infection cycle of <i>Borrelia burgdorferi</i>? What are the structural targets of the human immune system and how have these targets evolved? What are the ideal structural targets for a vaccine to protect against Lyme disease?
</P>
[[Image:BorreliaGeneExpressionCycle.png|500px|right|thumb|Borrelia Gene Expression Over Life Cycle.]]

== Osp C and Lyme Disease ==

<font size='4'>Imporance of OspC in Lyme disease</font size>
[[Image:Spirochetes in infected unfed and feeding nymph.png|300px|right|thumb|Migration of infected nymph from midgut to salivary glands]]

OspC, one of the major outer surface proteins of ''B. burgdoferi'', plays a pivotal role in transmission of ''B. burgdoferi'' from the tick vector to mammalian host. The protein gets upregulated when the tick feeds, allowing for the ''B. burgdoferi'' to adhere to the tick's saliva and move to the tick's mouth and into the host.<ref>PMID:14981110</ref> The upregulation of OspC is accompanied by a downregulation of OspA and OspB, which is thought to be induced by changes in environmental temperature and pH. <ref>PMID:14981110</ref>

OspC is a highly variable protein and strains of ''B. burgdoferi'' are classified according to the sequence of the OspC locus into 19 outer surface major groups (oMGs), denoted by type A to S, only four of which are invasive (disease causing).<ref name=kumaran>PMID:11230121</ref> Polymorphism of OspC and abundance of invasive strains in a population of ''B. burgdoferi''are driven by ecological factors, such as host mammalian community composition, and is a determinant of human Lyme disease risk<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>.

Researchers are attempting to take advantage of the presence of OspC on Borrelia's surface, while the bacteria is in the host, to develop an OspC-based vaccine. However, development of OspC-based vaccination has presented difficulties due to the highly variable nature of OspC. <ref name='protein'>PMID:17921702</ref>

<h3>Exploring the Structure of OspC</h3>

{{STRUCTURE_1ggq| PDB=1ggq | SCENE=Studio:G4SecL04/Dimer_with_mg/1 }}

The model presented to the right is the B31 strain (residues 38-201), which is also known as oMG A. This is one of four invasive oMGs that are responsible for systematic Lyme disease. In crystal structure, OspC exists as a dimer with the coordination of divalent ion, which is modeled as a magnesium ion. Each subunit is predominantly helical, consisting of five parallel
<scene name='Studio:G4SecL04/Helix_blue_in_ribbon/1'> α-helices </scene>
, two short antiparallel
<scene name='Studio:G4SecL04/Sheet_yellow_in_ribbon/1'> β-sheets</scene>
and six
<scene name='Studio:G4SecL04/Random_coils/2'>random coils</scene>
. The '''N and C termini''' at the membrane proximal end of two long alpha helices,
<scene name='Studio:G4SecL04/Alpha_1_with_pointer/1'>α1</scene> (residues 38-76) and
<scene name='Studio:G4SecL04/Alpha_5_with_pointer/1'>α5</scene> (residues 170-201) are in close proximity to each other. At the membrane distal end, there are three remaining alpha helices,
<scene name='Studio:G4SecL04/Alpha_2_with_pointer/1'>α2</scene> (residues 95-112),
<scene name='Studio:G4SecL04/Alpha_3_with_pointer/1'>α3</scene> (residues 121-145), including a short
<scene name='Studio:G4SecL04/Alpha_4_with_pointer/1'>α4</scene> (residues 152-159). At the end of membrane surface, the connection between helices α1 and α2 forms two short anti-parallel β-strands,
<scene name='Studio:G4SecL04/Beta_1_with_pointer/2'>β1</scene> (residues 79-80),and
<scene name='Studio:G4SecL04/Beta_2_with_pointer/1'>β2</scene> (residues 88-89) are formed.

While most of the OspC locus is highly variable, the sequence alignment of all oMGs reveals that towards the membrane proximal end, the surface-exposed residues on α1 and α5 are highly <scene name='Studio:G4SecL04/Conserved_region/1'>conserved</scene>, resulting in a positively charged surface. Other than those on helices, α1 and α5, the surface-exposed residues on the remaining regions of OspC molecule are variable.<ref name= variable>PMID: 11139584 </ref>

{{Template:ColorKey_ConSurf_NoYellow}}

<h3>OspC Structure and Antigenicity</h3>

At the membrane distal region, the six loop regions, including two β-strands illustrates the
<scene name='Studio:G4SecL04/Most_antigenic_site/1'>most antigenic sites</scene>
of OspC due to the presence of variable surface-exposed residues among OspC isolates. <ref>Earnhart C, LeBlanc D, Alix K, Desrosiers D, Radolf J, and Marconi R. 2010. Identification of residues within ligand-binding domain 1 (LBD1) of the ''Borrelia burgdorferi'' OspC protein required for function in the mammalian environment. Molecular Microbiology 76(2): 393-408. [http://dx.crossref.org/10.1111%2Fj.1365-2958.2010.07103.x DOI: 10.1111/j.1365-2958.2010.07103.x]</ref> However, among these variable regions, the outer surface-exposed residues connecting the helices α1 and α2, forming the loops,
<scene name='Studio:G4SecL04/L1/4'>L1</scene> (residues 74-78),
<scene name='Studio:G4SecL04/L2/3'>L2</scene> (residues 81-87),
<scene name='Studio:G4SecL04/L3/3'>L3</scene> (residues 90-93)and two short beta strands, β1 and β2, and also
<scene name='Studio:G4SecL04/L5/3'>L5</scene> (residues 146-150)
are more highly variable than those present in the loops,
<scene name='Studio:G4SecL04/L4/2'>L4</scene> (residues 115-119)and
<scene name='Studio:G4SecL04/L6/3'>L6</scene> (residues 161-169). Consequently, the surface potential of red region that projects away from the membrane is negatively charged and mainly involved in the protein-protein or protein-ligand interactions.<ref name= variable>PMID: 11139584 </ref> Only four types of oMGs (A, B, I and K), whose surface potential in red region is highly negative relative to non-invasive one plays a major role in pathogenesis of human Lyme disease. <ref name=kumaran>PMID:11230121</ref>The residue, <scene name='Studio:G4SecL04/His_82/1'>His82</scene>, located on the red region at the membrane distal end is unique that the replacement of other residues except His82, Lys82, Gln82, which are present only in four invasive oMGs enhances the possibility of turning invasive strains to non-invasive one. Thus, the stronger the electrostatic potential on red region, the higher the chance for OspC to bind with positively charged host ligands. Therefore, the alternation of an amino acid residue at the 82nd position on red region not only demonstrates OspC polymorphism, but also points out the probability for turning invasive strains to non-invasive strains.<ref name=kumaran>PMID:11230121</ref>

<h3>Lyme Disease and Ecology</h3>

[[Image:Life cycle of tick.png|300px|right|thumb|Life cycle of tick.[[http://www.cdc.gov/ticks/life_cycle_and_hosts.html]]]]

The number of reported cases of Lyme disease is increasing annually in highly focused geographic locations of the United States (CITE). The occurrence of Lyme disease is dependent upon the abundance of ticks infected with ''B. burgdorferi'' in natural ecosystems. Ticks are born without ''B. burgdorferi'' and acquire the bacteria while feeding on the blood of natural reservoir hosts such as mice, squirrels, shrews and other small vertebrates (Figure XX). After growth and development, the infected nymphal ticks can transmit ''B. burgdorferi'' to incidental vertebrates, including humans. The ecological interaction between the competence of reservoir hosts and the ticks is an underlying measure of human Lyme disease risk.<ref>PMID: 12525705</ref>

<h3>Ecological factors responsible for human Lyme disease risk</h3>

* '''Vertebrate Community Composition'''<ref>PMID:15514047</ref>: Two types of environment that the vertebrate hosts reside, which is also called vertebrate host density are interspecific community, which involves organisms of different species and intraspecific community, which is composed of organisms of same species. The hosts living in the community within different species or same species strongly affects the proportion of infected nymphal tick that can cause human Lyme disease.
* '''Distribution frequency of particular oMGs'''<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>: After taking blood meal from their hosts, the proportion of host-seeking nymphs infected with each oMG differs among oMGs. As only four types of oMGs (A, B, I and K) are responsible for systemic human Lyme disease, the host-seeking nymphs that have high distribution frequency of four invasive oMGs is one of the standard measures of human Lyme disease risk.
* '''Transmission Probability'''<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>: The transmission probability of each oMGs from individual species differs. The higher the transmission probability of a particular oMG from vertebrate host, the higher the chance of carrying that particular oMG by the ticks after receiving blood meal from their hosts is. Thus, it is one of the parameters that contributes the prevalence of human Lyme disease.

<h3>Using Ecological Models to Predict Lyme Disease Risk</h3>

[[Image:Lyme_Disease_Risk_Map.gif|300px|right|thumb|Map illustrating prevalence of Lyme disease in the Untied States by CDC.[[http://www.cdc.gov/mmwr/preview/mmwrhtml/rr4807a2.htm]]]]

Conceptual and mathematical models have been developed by researchers to characterize the ecological interaction between vertebrate host community and distribution frequency of invasive oMGs and predict the cases of human Lyme disease. In one model, the principal natural reservoir host used in the model for the epidemic of Lyme disease in northeastern and central United States is the presence of the white-footed mice (''Peromyscus leucopus'') population, which has both high frequency distribution in all four human infectious oMGs and high transmission probabilities of oMGs A, B, I and K.<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref> Ticks are least likely to parasitize inefficient reservoir hosts, thereby increasing high infection prevalence in the tick population, which enhances the risk of exposure of Lyme disease in humans. Many studies have found support for this "dilution-effect model" which proposes that maintaining high diversity of vertebrate host community may dilute the power of one host, such as the white-footed mouse by increasing the degree of specialization of ticks on inefficient hosts. This model strongly demonstrates the relationship between species diversity in the community of hosts and the risk of human exposure to Lyme disease. These ecological driving forces described in the model are useful tools in predicting the prevalence and risk of human Lyme disease.

<h3>OspC-based vaccine against Lyme disease</h3>

An OspC-based vaccine against Lyme disease is currently being developed. Because of the variability of OspC, the recombinant OspC vaccine, targeting the antigenic site of one specific OspC type is ineffective for ''B. burgdorferi'' with different OspC types. Therefore, the development of vaccine that recognizes the antigenic determinant on the variable regions of multiple OspC types is required in order to effectively activate human immune response. Based on the mapping of epitope-containing regions from oMGs: A, B, K and D, the experiment-based tetravalent chimeric vaccine is being developed to tigger anti-ABKD response. <ref>Christopher G. Earnhart, Eric L. Buckles, Richard T. Marconi. "Development of an OspC-based tetravalent, recombinant, chimeric vaccinogen that elicits bactericidal antibody against diverse Lyme disease spirochete strains, Vaccine." 25(3) 466-480 (2007). [http://dx.doi.org/10.1016/j.bbr.2011.03.031 DOI: 10.1016/j.vaccine.2006.07.052]</ref> Taking advantage of tetravalent ABKD construct, octavalent chimeric vaccine also known as OspC-A8.1, recognizing additional epitopes of oMGs: C, E, N and K, has been tested in mice. <ref name='protein'>PMID:17921702</ref>

== Antibodies to Osp A and Osp B and Lyme Disease ==

[[Image:Fab reigon.png|right|thumb|Digestion of an antibody by Papain separates the fab reigons from the antibody]]
A factor contributing to the severity of Lyme disease is its resistance to certain forms of complement-dependent immune response by the evasion of the [http://en.wikipedia.org/wiki/Alternative_complement_pathway alternative complement pathway] and the blocking of complement [http://en.wikipedia.org/wiki/Complement_component_3 C3].<ref>PMID:18080415</ref> This resistance increases the importance of the complement independent immune response when combating ''B. burgdorferi''. Certain fragment antigen binding regions ([http://en.wikipedia.org/wiki/Fragment_antigen-binding fab]) of IgG and IgM monoclonal antibodies (mAbs) are bactericidal even in the absence of complement. Binding of these fabs to their corresponding outer surface protein (OspA and OspB) of ''B. burgdoferi'' leads to the lysis of the bacteria.<ref name="connolly">PMID: 15864264</ref>

<h3>Fragment Antigen Binding (fab)</h3>
Fab consists of a [http://en.wikipedia.org/wiki/Immunoglobulin_heavy_chain heavy chain] and [http://en.wikipedia.org/wiki/Immunoglobulin_light_chain light chain] and each chain is composed of a variable and a constant region. The [http://en.wikipedia.org/wiki/Paratope paratope] is located in the N terminal of the variable region of the heavy and light chains of the fab. H6831 and CB2 are IgG mAbs that targets the C-terminal of OspB and LA-2 is an IgM mAb that targets the C-terminal of OspA.<ref>PMID:107164</ref>

<h3>OspB and H6831</h3>

<h4>Interaction between OspB and H6831</h4>
<table width='450' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1RJL' size='400' frame='true' align='center' scene='Studio:G1SecL01/1/10' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''OspB-H6831 Complex''' (<scene name='Studio:G1SecL01/1/10'>Initial Scene</scene>)<br>
<scene name='Studio:G1SecL01/1/11'>OspB</scene>''':''' <scene name='Studio:G1SecL01/1/31'>Loop 1</scene> <scene name='Studio:G1SecL01/1/32'>(w/ His 52)</scene> ·· <scene name='Studio:G1SecL01/1/33'>Loop 2</scene> ·· <scene name='Studio:G1SecL01/1/34'>Lysine 253</scene> <scene name='Studio:G1SecL01/1/35'>(CPK coloring)</scene> ·· <scene name='Studio:G1SecL01/1/36'>Loop 3</scene> <br>
<scene name='Studio:G1SecL01/1/12'>H6831</scene>''': '''
<scene name='Studio:G1SecL01/1/14'>Heavy Chain</scene> ·· <scene name='Studio:G1SecL01/1/13'>Light Chain</scene> ·· <scene name='Studio:G1SecL01/1/37'>Tyr-Trp-Glu-His</scene> <br> <scene name='Studio:G1SecL01/1/30'>OspB-H6831 Complex</scene>''': ''' <scene name='Studio:G1SecL01/1/38'>Residues 218-220</scene><br>
<scene name='Studio:G1SecL01/3/2'>OspB Unbound</scene>''':''' <scene name='Studio:G1SecL01/3/3'> Central β Sheet Strands 1-4 </scene>

</center></td></tr></table>
<scene name='Studio:G1SecL01/1/10'>Restore Original Scene</scene>

The <scene name='Studio:G1SecL01/1/30'>OspB-H6831 complex</scene> consist of two components, the outer surface protein <scene name='Studio:G1SecL01/1/11'>OspB</scene> and the <scene name='Studio:G1SecL01/1/12'>fab</scene>, which is subdivided into the <scene name='Studio:G1SecL01/1/14'>heavy chain</scene> and the <scene name='Studio:G1SecL01/1/13'>light chain</scene>. Most hydrogen bonds and electrostatic interactions that are responsible for the binding of H6831 to OspB are between the <scene name='Studio:G1SecL01/1/15'>three adjacent surface-exposed loops</scene> at the C-terminal of OspB and some <scene name='Studio:G1SecL01/1/37'>residues on the fab heavy chain</scene> that include tyrosine, tryptophan, glutamate, and histidine.<ref name=becker>PMID:15713683</ref>

The majority of hydrogen bonds and electrostatic interactions are between <scene name='Studio:G1SecL01/1/33'>Loop2</scene> (residues 250-254) and the fab heavy chain. <scene name='Studio:G1SecL01/1/34'>Lys 253</scene> in loop 2 of OspB has a necessary and major role due to its central position in the exposed loops. A mutation at its position abrogates the binding interaction and causes the resistance of the bacteria to the bactericidal effect of the fab. Lys 253 interacts with the two aromatic residues on the fab heavy chain, tyrosine and tryptophan. It also makes hydrogen bonds with the glutamate 50 in the heavy chain of the fab and forms an ionic bond. Carbonyl in <scene name='Studio:G1SecL01/1/31'>loop 1</scene> of the OspB interacts with <scene name='Studio:G1SecL01/1/32'>histidine 52</scene> in the fab heavy chain. <scene name='Studio:G1SecL01/1/36'>Loop 3</scene> of OspB interacts with fab light chain.<ref name=becker />

<h4>Structural changes to OspB in the complexed form</h4>
The binding of H6831 to OspB leads to some conformational changes in OspB compared to its <scene name='Studio:G1SecL01/3/2'> unbound form </scene>. [http://en.wikipedia.org/wiki/Crystallography Crystallography] has shown that the most significant difference is the loss of the <scene name='Studio:G1SecL01/3/3'>central β sheet strands 1-4 </scene>.<ref name=becker /> The loss of these β sheets may be due to conformational change as a result of the binding or a disorder that could have occurred during a crystallization of the complex. Both small positional shifts near the fab binding site and a few larger structural changes away from the binding site were observed. The largest shifts (7– 8 Å) correspond to the repositioning of a loop opposite the fab-binding site <scene name='Studio:G1SecL01/1/38'>at residues 218-220</scene>. In the free OspB structure, all regions that exhibit shifts are adjacent to the central sheet; in the OspB-H6831 complex they all shift toward, and slightly overlap the position of the missing sheet.

<h4>Bactericidal action</h4>
The fab binding destabilizes the [http://en.wikipedia.org/wiki/Bacterial_outer_membrane outer membrane] (OM) of B. burdorferi, with subsequent formation of [http://en.wikipedia.org/wiki/Spheroplast spheroplasts]. It has been observed that the bactericidal action, but not the binding, requires the presence of divalent cations (Mg<sup>2+</sup> and Ca<sup>2+</sup>), and
fab is unable to clear bacteria in the absence of these cations.<ref name=ding /> It is speculated that OspB-Cb2 (a fab similar to H6831) complexes could lead to the lysis of the cell by creating physical openings in the OM, allowing for rapid infusion of electrolytes and increasing the [http://en.wikipedia.org/wiki/Osmolarity osmolarity] of the [http://en.wikipedia.org/wiki/Periplasm periplasm].<ref name="escudero">PMID:9125579</ref>

<h3>OspA and LA-2</h3>

<table width='450' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='400' frame='true' align='center' scene='Studio:G1SecL01/2/1' />
</td></tr><tr><td bgcolor='#eeeeee'><center>'''OspA-LA2 Complex''' (<scene name='Studio:G1SecL01/2/1'>Initial Scene</scene>)<br>
<scene name='Studio:G1SecL01/2/1'>OspA-LA2 Complex</scene>''':''' <scene name='Studio:G1SecL01/2/2'>Three Loops</scene> '''··''' <scene name='Studio:G1SecL01/2/3'>Ser 206</scene>
</center></td></tr></table>

<h4>OspA</h4>
<scene name='Studio:G1SecL01/2/1'>Restore Original Scene</scene>

OspA is usually undetectable in the early stages of Lyme disease, and is down regulated when OspC is expressed.<ref name=kumaran>PMID:11230121</ref> OspA is 53% similar to OspB. Despite their similarity, OspB is susceptible to cleavage by exogenous [http://en.wikipedia.org/wiki/Protease proteases] both ''in vivo'' and ''in vitro'', whereas OspA is relatively resistant in both cases.<ref name=becker />

OspA is used in adhering to the tick's gut by binding with the tick receptor for OspA (TROSPA), a receptor necessary for the colonization of <i>Borrelia</i> inside the tick. High levels of both OspA and TROSPA are found prior to feeding, but are downregulated once the feeding process begins in order to initiate transmission into the host.<ref name="pal">PMID: 15537536</ref>
</p>

<h4>Interaction between OspA and LA-2</h4>
LA-2 is an IgM murine monoclonal antibody that interacts with <scene name='Studio:G1SecL01/2/2'> three exposed loops </scene> on the C-terminal of OspA. These interactions include eight direct [[hydrogen bonds]], four solvent-bridged hydrogen bonds, three ion pairs, and numerous van der Waals interactions.<ref name=ding />

<h4>Structural changes to OspA in the complexed form</h4>
Conformational changes upon the binding of OspA and LA-2 show that LA-2 recognition of OspA involves an induced fit mechanism where the conformations of loops 1-3 shift to optimize complementarity to the antigen-combining site.<ref name=ding /> The overall structure of the C-terminal of OspA is unchanged upon the binding of LA-2 with comparison to the free OspA. The maximum atomic shift is 4.7Å at the site of <scene name='Studio:G1SecL01/2/3'>Ser 206</scene>.<ref name=ding>PMID: 11183781</ref>

<h3>Medical Application</h3>

''B. burgdoferi'' is able to escape human immune response because the outer surface proteins, to which the immune system responds, are variable. The effectiveness of different Osp molecules as [http://en.wikipedia.org/wiki/Vaccine vaccines] can vary depending on their variability. OspC and OspB are highly polymorphic, with variability of OspC observed between strains collected from within a single geographical area.<ref name=kumaran>PMID:11230121</ref> Observed variations of OspB in ''B. burgdoferi'' (including its absence from the bacteria) are not accounted for by major [http://en.wikipedia.org/wiki/Dna DNA] arrangements or failure in [http://en.wikipedia.org/wiki/Transcription_(genetics) transcription] of the OspB gene. This indicates that the OspB gene may code for a variety of proteins, making OspB a poor candidate for use in vaccines.<ref>PMID:2668185</ref> OspA is the most conserved; of the three exposed loops, only loop 1 is variable while loops 2 and 3 are conserved. This makes OspA a more consistent antigen (compared to OspB and OspC) for the immune system to target and usable as a vaccine to Lyme disease.<ref name=ding />

== Osp A and Lyme Disease ==

<Structure load="1fj1" size="350" frame="true" align="right" name="OspA-secondary" caption="Outer surface protein A (OspA) ([[1fj1|1FJ1]], chain F) showing secondary structures." scene="Studio:G2SecL03/Ospa_default/5"/>
<p>
<b>Outer Surface Protein A (OspA)</b> is a major lipoprotein found on the surface of [http://en.wikipedia.org/wiki/Spirochaete spirochetes] from the genus [http://en.wikipedia.org/wiki/Borrelia <i>Borrelia</i>] and is comprised of 21 anti-parallel β-sheets and a single α-helix. OspA's expression is regulated at different points in time, from being downregulated during the tick's feeding process on its host to being upregulated in the host's cerebrospinal fluid (CSF) to induce inflammatory response, resulting in acute Lyme [http://en.wikipedia.org/wiki/Neuroborreliosis neuroborreliosis]. OspA has also been used as a vector in working towards the development of a vaccine for [http://en.wikipedia.org/wiki/Lyme_disease Lyme disease].
</p>
<p>
While feeding, OspA is downregulated in order to evade an immune response from the incoming host blood into the gut, releasing the <i>Borrelia</i> from the gut wall and migrating into the tick's salivary glands, thereby allowing it to enter the host through the bite. This is evidenced by the fact that patients with Lyme disease have been found to not possess OspA antibodies in the early stages of the disease.<ref name="connolly">PMID: 15864264</ref><ref name="rupprecht">PMID: 18097481</ref> OspA is the protein most related to acute Lyme neuroborreliosis (LNB), the neurological manifestations of Lyme disease.
</p>

<h3>OspA's Role in Invasion</h3>
<p>
Once inside the host, the <i>Borrelia</i> has a great number of mechanisms available to actively suppress the host's immune system response and neutralize its effector mechanisms, such as the expression of another outer surface protein, OspC, which prevents susceptibility to the host's [http://en.wikipedia.org/wiki/Innate_immune_system innate immunity] and [http://en.wikipedia.org/wiki/Complement_system complement systems]. Additionally, <i>Borrelia</i> is capable of suppressing many of its surface proteins to reduce its detectability, but can also utilize protective means by temporarily expressing them when needed.
</p>

<h4>Acute Lyme Neuroborreliosis (LNB)</h4>
<p>
Acute Lyme Neuroborreliosis (LNB) is part of the second stage of Lyme disease in which the spirochete invades the peripheral and central nervous systems (CNS). Symptoms of LNB include: meningoradiculitis with inflammation of the nerve roots and [http://en.wikipedia.org/wiki/Radicular_pain radiculitis] (Bannwarth’s syndrome), lymphocytic meningitis, and cranial and [http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001619/ peripheral neuritis]. In Europe, the strain predominantly found in the CSF of patients with Bannwarth's syndrome is <i>B. garinii</i>. However, in the United States, Bannwarth's syndrome is rare and the most common manifestations of Lyme neuroborreliosis is [http://en.wikipedia.org/wiki/Meningitis meningitis], caused by <i>B. burgdorferi</i>. The presence of OspA in the cerebrospinal fluid (CSF) is responsible for this complex inflammatory response in the brain that leads to the neuroborreliosis.<ref name="rupprecht">PMID: 18097481</ref>
</p>

<h4>Evasion and the Extracellular Matrix</h4>
<p>
The <i>Borrelia</i> are able to hide in the [http://en.wikipedia.org/wiki/Extracellular_matrix extracellular matrix], allowing it to survive by avoiding [http://en.wikipedia.org/wiki/Leukocytes leukocytes] circulating in the bloodstream. OspA can rapidly bind to plasminogen, which becomes [http://en.wikipedia.org/wiki/Plasmin plasmin] once activated, and degrades the extracellular matrix. By binding to plasminogen, <i>Borrelia</i> could be exploiting its function and utilizing it to invade the extracellular matrix. However, due to the fact that OspA is downregulated during feeding, and stays unexpressed, a different mechanism may be used instead. Additionally, <i>Borrelia</i> induces the local upregulation of matrix metalloproteinase-9, causing the digestion of the surrounding extracellular matrix. <i>Borrelia</i> can also bind to several proteins in the extracellular matrix, such as [http://en.wikipedia.org/wiki/Fibronectin fibronectin], [http://en.wikipedia.org/wiki/Integrins integrins] or [http://en.wikipedia.org/wiki/Decorin decorin], which can aid in the spread and survival of the spirochetes in these tissues.<ref name="rupprecht">PMID: 18097481</ref>
</p>

<h4>Migration Across the Blood-Brain Barrier</h4>
<p>
It is not fully understood how <i>Borrelia</i> get past the [http://en.wikipedia.org/wiki/Blood-brain_barrier blood-brain barrier], though some researchers suggest a paracellular route, which involves a process using transient tether-type associations, short-term dragging interactions, and stationary adhesion. There is evidence that <i>Borrelia</i> utilizes OspA in the transient tethering stage. The blood-brain barrier is composed of brain microvascular endothelial cells, astrocytes, a basement membrane, pericytes, and neurons. OspA is a major adherent molecule to brain microvascular cells by binding to the [[1aly|CD40]] receptors outside, which results in events that are typically seen when leukocytes cross the blood brain barrier.

Activation of CD40 receptors leads to the production of proinflammatory cytokines and enhanced expression of ICAM-1, E-selectin and VCAM-1, resulting in increased cell binding, and the formation of fenestrations due to increased vascular endothelial growth factor, and vascular permeability factor. OspA might be mimicking leukocytes in order to cross the blood-brain barrier. However not all strains of <i>Borrelia</i> can utilize OspA to do this, OspA only contributes about 70% to adherence, and other <i>Borrelia</i> proteins are also needed in this process. It has also been seen that OspA mediates the adhesion of <i>Borrelia</i> to murine neural and glial cell lines. <ref name="pulzova">PMID: 22355605</ref>
</p>

<h4>Role in Inflammation</h4>

[[Image:G2L03-OspA-mechanism.jpg|thumb|300px|Mechanism of the host inflammatory response to OspA]]

There are six steps involved in the host's inflammatory response to OspA: <ref name="rupprecht">PMID: 18097481</ref><br>
<ol>
<li>When the <i>Borrelia</i> enter the host’s CNS they encounter several different types of immune cells such as [http://en.wikipedia.org/wiki/Monocyte monocytes], [http://en.wikipedia.org/wiki/Macrophages macrophages], and [http://en.wikipedia.org/wiki/Dendritic_cells dendritic cells]. While in the CSF, outer surface protein A (OspA) is upregulated and it’s increased expression promotes recognition by a specific receptor on a monocyte.</li>

<li>The OspA-bound monocyte then releases proinflammatory [http://en.wikipedia.org/wiki/Cytokine cytokines] (i.e. [http://en.wikipedia.org/wiki/Interferon interferon]), as well as [http://en.wikipedia.org/wiki/Chemokine chemokines], such as [http://en.wikipedia.org/wiki/CXCL13 CXCL13]. In patients with LNB, there is an observed increase in the levels of these cytokines and chemokines in their CSF. The production of chemokines leads to the recruitment of other immune cells to the site of infection.</li>

<li>[http://en.wikipedia.org/wiki/B_lymphocyte B-lymphocytes] respond to the new concentration gradient of CXCL13 between the blood and CSF and migrate into the CSF.</li>

<li>B-lymphocytes undergo [http://en.wikipedia.org/wiki/Receptor-mediated_endocytosis receptor-mediated endocytosis], consuming the OspA antigens present in the CSF, thereby triggering its activation. The B-lymphocytes then are able to differentiate and mature into [http://en.wikipedia.org/wiki/Plasma_cells plasma cells].</li>

<li>The plasma cells create large quantities of anti-OspA antibodies specific to this strain of <i>Borrelia</i> and release them into the CSF.</li>

<li>The anti-OspA antibodies will then bind to the OspA on the spirochete’s membrane, thus killing the <i>Borrelia</i>.</li>
</ol>

<p>
This process is two-sided in the sense that the OspA aids in the pathogenesis of new symptoms (neuroborreliosis) through the chemokine’s actions, as well as initiating the signaling cascade to destroy itself.
</p>

<h3>OspA Vaccination</h3>

<table width='400' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='350' frame='true' align='center' name='complex' caption='Outer Surface Protein A (Osp A) in complex with the LA-2 Fab antibody ([[1fj1|1FJ1]]).' scene='Studio:G2SecL03/Ospafab-orig/3'/>
</td></tr><tr><td bgcolor='#eeeeee'><center><scene name='Studio:G2SecL03/Ospafab-orig/3' target="complex">Reset model</scene>,<scene name='Studio:G2SecL03/Ospafab-fab/3' target="complex">LA-2</scene> Fab antibody (Bluish regions indicate heavy chains (chains B & D of 1FJ1) and greenish regions indicate light chains (chains A & C of 1FJ1))<scene name='Studio:G2SecL03/Ospafab-ospa/3' target="complex">OspA</scene> proteins in complex with the LA-2 Fab antibody (chains E & F of 1FJ1)<scene name='Studio:G2SecL03/Ospafab-interaction/3' target="complex">Closeup</scene> of the OspA antigen : LA-2 Fab antibody interactions
</center></td></tr></table>

<p>
Risk of developing Lyme disease can be mitigated by staying clear of areas with populations of ticks, wearing proper attire to minimize easily bitten areas of the body, and using insect repellents containing [http://en.wikipedia.org/wiki/DEET DEET] (N,N-diethy-m-toluamide). However, another effective means for prevention could be possible by using an outer surface protein from <i>Borrelia</i> in the creation of a vaccine.<ref name="nigrovic">PMID: 16893489</ref>
</p>
<p>
The membrane composition of <i>Borrelia</i> is abundant in both OspA and OspB, and the two proteins share a 53% similarity in their primary sequences. Both OspA and OspB are expressed in the tick's gut and downregulated during feeding and aid in its survivability; however, OspA is overall less varied and reactive than OspB, which has greater variability.<ref name="becker">PMID: 15713683</ref> The relatively conserved sequence of OspA thus lends itself better to study and application toward the development of a vaccine for a broader range of <i>Borrelia</i> strains in the treatment of Lyme disease than that of OspB. The first vaccine used a purified recombinant form of OspA and functioned in blocking transmission of the spirochetes expressing OspA from tick to host during feeding, killing them while still attached to the tick's gut.<ref name="connolly">PMID: 15864264</ref><ref name="battisti">PMID: 18779341</ref> The vaccine, Lymerix, had shown 76% and 92% effectiveness in separate clinical trials in which patients were treated for two years following a three-dose schedule. However, the vaccination was suspended from use in 2002 when opponents claimed the [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG antibodies] for OspA were associated with the onset of severe chronic arthritis, as well as other side effects affecting immunity.<ref name="connolly">PMID: 15864264</ref><ref name="plotkin">PMID: 21217175</ref> This fact, in conjunction with the desire for a more widespread vaccine treating multiple strains of <i>Borrelia</i>, has spurred research towards a new vaccine.
</p>
<p>
To address the concerns of vaccine with broader protection, creation of a chimera, mixing the OspA of different strains of <i>Borrelia</i> would be ideal. Study of the epitope of <scene name='Studio:G2SecL03/Ospafab-ospa/3' target="complex">OspA</scene> and its <scene name='Studio:G2SecL03/Ospafab-interaction/3' target="complex">interactions</scene> with the murine monoclonal antibody <scene name='Studio:G2SecL03/Ospafab-fab/3' target="complex">LA-2</scene> have proved useful in determining effectiveness of a given vaccine trial as high levels of antibodies in test sera compete against LA-2 for binding with OspA. LA-2 makes direct contact with three exposed loops of the C-terminus of OspA. The recognition of OspA by LA-2 requires an induced fit mechanism where these three loops undergo conformational changes to optimize their interaction in the complex. <ref name="ding">PMID: 11183781</ref>
</p>

<h3>Structure of OspA</h3>

<table width='400' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='350' frame='true' align='center' name='OspA-manip' caption='Outer surface protein A (OspA) ([[1fj1|1FJ1]], chain F).' scene='Studio:G2SecL03/Ospa_3loopscartoon/3'/>
</td>
</tr>
<tr>
<td bgcolor='#eeeeee'>
<center>
<scene name='Studio:G2SecL03/Ospa_3loopscartoon/3' target="OspA-manip">Reset model</scene>,<br><scene name='Studio:G2SecL03/Ospa-3loops/4' target="OspA-manip">Three loops</scene> in C-terminus (close up)<scene name='Studio:G2SecL03/Ospa-loop1/1' target="OspA-manip">including Loop 1</scene> (residues 203-220)<scene name='Studio:G2SecL03/Ospa-loop2/1' target="OspA-manip">, Loop 2</scene> (residues 224-233)<scene name='Studio:G2SecL03/Ospa-loop3/1' target="OspA-manip">and Loop 3</scene> (residues 246-257)<scene name='Studio:G2SecL03/Ospa-3residues-nor/3' target="OspA-manip">Three residues</scene> in C-terminus (Ala208, Ala215 and Asn251 in <i>B. burgdorferi</i>; also hides R-groups). <scene name='Studio:G2SecL03/Ospa-3residues-r/2' target="OspA-manip">Display R-groups</scene> of Ala208, Ala215 and Asn251<scene name='Studio:G2SecL03/Ospa-ala208/1' target="OspA-manip">Alanine (Ala) 208</scene><scene name='Studio:G2SecL03/Ospa-ala215/1' target="OspA-manip">Alanine (Ala) 215</scene><scene name='Studio:G2SecL03/Ospa-asn251/1' target="OspA-manip">Asparagine (Asn) 251</scene><scene name='Studio:G2SecL03/Ospa-3loops3res/1' target="OspA-manip">Three loops and all three residues (with R-groups included)</scene>
</center>
</td>
</tr>
</table>

<p>
OspA is made up of 273 residues over 21 anti-parallel β-sheets and a single α-helix. It's folded conformation is divided into three main sections: a N-terminus "sandwich," a central region comprising of several β-sheets and a C-terminus "barrel" domain.<ref name="ding">PMID: 11183781</ref> The folded regions at its ends are connected by a single β-sheet layer in the middle, giving the protein the unique shape of a dumbell.<ref name="makabe">PMID: 16823038</ref>

There are <scene name='Studio:G2SecL03/Ospa-3loops/4' target="OspA-manip">three loops</scene> at the C-terminus of OspA that are important in binding with the LA-2 Fab antibody, whose interactions provide great insight into vaccine research and effectiveness. These three loops are linearly arranged and form protruding ridge at the C-terminus of OspA. Within these loops, there are <scene name='Studio:G2SecL03/Ospa-3residues-nor/3' target="OspA-manip">three residues</scene> <scene name='Studio:G2SecL03/Ospa-3residues-r/2' target="OspA-manip">(show residue R-groups)</scene> where there are distinct variations between the different strains of <i>Borrelia</i> and serve as potential targets for the creation of a broader vaccine.<ref name="ding">PMID: 11183781</ref> <scene name='Studio:G2SecL03/Ospa-3loops3res/1' target="OspA-manip">(display both the three loops and three residues together)</scene>
</p>
<p>
<scene name='Studio:G2SecL03/Ospa-loop1/1' target="OspA-manip">Loop 1</scene>, (residues 203-220), is important in showing variation amongst the different strains of <i>Borrelia</i> as well as being optimally conformed for binding without steric hindrance. <scene name='Studio:G2SecL03/Ospa-loop2/1' target="OspA-manip">Loop 2</scene> (residues 224-233) and <scene name='Studio:G2SecL03/Ospa-loop3/1' target="OspA-manip">Loop 3</scene> (residues 246-257) are more strongly conserved than Loop 1 but also help to show some variation amongst strains. The LA-2 Fab antibody readily recognizes OspA from <i>B. burgdorferi</i>, but does not recognize that from <i>B. afzelii</i> or <i>B. garinii</i>.

Between <i>B. burgdorferi</i> and <i>B. afzelii</i> genetic sequences are generally invariant, but two residues change between the species: <scene name='Studio:G2SecL03/Ospa-ala208/1' target="OspA-manip">Alanine (Ala) 208</scene> in <i>B. burgdorferi</i> is a Glutamine (Gln) in <i>B. afzelii</i>, and <scene name='Studio:G2SecL03/Ospa-asn251/1' target="OspA-manip">Asparagine (Asn) 251</scene> in <i>B. burgdorferi</i> is an Alanine (Ala) in <i>B. afzelii</i>. <i>B. garinii</i> has more variation and in addition to the previous two differences, having at least one more difference, where <scene name='Studio:G2SecL03/Ospa-ala215/1' target="OspA-manip">Alanine (Ala) 215</scene> in <i>B. burgdorferi</i> is a Lysine (Lys), and sometimes also has a deletion at <i>B. burgdorferi</i>’s Alanine 208. LA-2 and OspA of <i>B. burgdorferi</i> form a tight interface when binding, and the longer Glutamine (Gln) sidechain found in <i>B. afzelii</i> and <i>B. garinii</i> is more difficult to accommodate, causing less binding. A chimera that was weakly recognized by LA-2 was made with parts of loop 1 from <i>B. burgdorferi</i>, and loops 2 and 3 from <i>B. garinii</i>.<ref name="ding">PMID: 11183781</ref> Recently, a different kind of chimera has been made which combined the proximal region of <i>B. burgdorferi</i> and distal region of <i>B. afzelii</i>, and was able to successfully protect mice from both species.<ref name="livey">PMID: 21217174</ref>
</p>

== Osp B and Lyme Disease ==

<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="becker">PMID:15713683</ref>.

<h3>OspB Interaction with Fab of H6831</h3>
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="becker"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/7'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="becker"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="becker"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

<h4>Binding</h4>
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />
H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="becker"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="becker"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="becker"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref name="escudero">PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="becker"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain.

<h3>Potential Mechanism of Lysis</h3>

<h4>Potential Catalytic Triad</h4>

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]
The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/2'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease.

<h4>Potential Oxidative Mechanism</h4>
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement.

== VlsE and Lyme Disease ==

<table width='400' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><applet load='1l8w.pdb' size='400' frame='true' align='right' scene='Studio:G5SecL01/Main_image_vlse/1' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''Variable Major Protein (VMP)-like sequence Expressed''' ([[1l8w]]), resolution 2.3Å
[[Image:VLSE PRIMARY STRUCTURE4343.png|400px]]<br>
<nowiki>*</nowiki><small>'''This representation of VlsE illustrates the only crystal structure available on the [http://www.rcsb.org/pdb/explore/explore.do?structureId=1l8w PDB site]. There are 10<sup>30</sup> possible combinations of the VR.'''</small>
<br></center></td></tr></table>

<h3>Structural Overview</h3>

Variable Major Protein (VMP)-like sequence Expressed ('''VlsE''') is a surface lipoprotein of ''Borrelia burgdorferi''. It undergoes [http://en.wikipedia.org/wiki/Antigenic_variation antigenic variation] seemingly important in evasion of the host’s immune system. In addition, the protein is used for Lyme disease diagnosis. It is composed of four similar subunits each possessing two invariable domains and one variable domain.<ref name="A">PMID:10569796</ref> The variable domain contains six variable regions (VR<sub>1</sub>-VR<sub>6</sub>), and six invariable regions (IR<sub>1</sub>-IR<sub>6</sub>). Research suggests that the protein may exist as a dimer where each monomeric C & N termini neighbor each other forming the membrane proximal portion of the protein, and the variable regions form the membrane distal portion.<ref name="B">PMID:11923306</ref> <ref name="C">PMID:11716485</ref> The invariable regions are largely embedded in the protein and remain relatively unchanged within the host and across strains. The variable regions encompass 37% of the VlsE’s exposed surface area despite comprising only 25% of the protein.<ref name="A" /> <ref name="B" /> However, 50% of the VR surface area is exposed while IR<sub>6</sub>, a strong [http://en.wikipedia.org/wiki/Antigen antigen], exposes just 13.7% of its surface area. This leaves only <scene name='Studio:G5SecL01/Ir_6_4_residues/1'>four amino residues</scene> of the antigenic IR<sub>6</sub> unprotected: lysine-276, glutamine-279, lysine-291, and lysine-294. Thus, it is almost entirely embedded in the protein and <scene name='Studio:G5SecL01/Ir6_embedded/1'>sheilded by the variable regions </scene>.<ref name="B" />

<h3>Antigenicity</h3>

The variable regions undergo a recombination event stimulated by the host’s cytokines and absence of those cytokines results in a decreased bacterial burden.<ref name="D">PMID:11544329</ref> This leads to variation with an estimated 10<sup>30</sup> possible combinations, far exceeding the number of antibodies found in the human immune system. While the VR does exhibit antigenicity, this recombination makes it unlikely that a sufficient amount of a single VR variation will be present in large enough supply to lead to an immunodominant variable region.<ref name="E">PMID:10553085</ref> IR<sub>6</sub>, however, exhibits immunodominance while IR<sub>1</sub>-IR<sub>5</sub> are primarily nonantigenic in humans. Thus, shielding of the immunodominant IR<sub>6</sub> by VR regions not subject to antibody response allows for IR<sub>6</sub> to elicit an immune response while remaining inaccessible to antibody binding. Research suggests that the 26 amino residues of <scene name='Studio:G5SecL01/Ir6_with_epitope/1'>IR6</scene> may function as a single epitope with a central alpha helical core.<ref name="B" /> <ref name="D" /> <ref name="F">PMID:10722641</ref>

<h3>Function in Immune System Evasion</h3>

VlsE is essential to the persistence and virulence of Lyme disease and is upregulated under humoral immune pressure.<ref name="G">PMID:17714442</ref> <ref name="H">PMID:15385475</ref> While the exact mechanism for immune evasion remains unknown, several theories have been put forth. One popular theory maintains that VlsE masks other surface antigens by coating the surface of the bacteria, thereby sterically blocking the antigens from antibody binding. This is similar to other pathogens with variable regions, such as [http://en.wikipedia.org/wiki/Trypanosoma_brucei Trypanosoma brucei], the protozoa responsible for African sleeping sickness and [http://en.wikipedia.org/wiki/Neisseria_gonorrhoeae Neisseria gonorrhea], the bacterial cause of gonorrhea. However, recent studies have cast doubt on this theory. An alternate theory provides that VlsE directly stimulates B cell antibody production independent of T-cells. The robust response elicited is thought to override antibody production against other antigens.<ref name="G" />
<br>

<table width='400' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><applet load='1l8w.pdb' size='400' frame='true' align='right' scene='Studio:G5SecL01/Chart_main/4' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''VlsE''' ([[1l8w]]), resolution 2.3Å (<scene name='Studio:G5SecL01/Chart_main/4'>Original</scene>). <br>
Highlight: <scene name='Studio:G5SecL01/Chart_main/3'>IR6</scene>, <scene name='Studio:G5SecL01/Chart_vr_seagreen/1'>VR</scene>, <scene name='Studio:G5SecL01/Chart_ir1_yellow/1'>IR1</scene>, <scene name='Studio:G5SecL01/Chart_ir2_yellow/1'>IR2</scene>, <scene name='Studio:G5SecL01/Chart_ir3_yellow/1'>IR3</scene>, <scene name='Studio:G5SecL01/Chart_ir4_yellow/1'>IR4</scene>, <scene name='Studio:G5SecL01/Chart_ir5_yellow/1'>IR5</scene>. <br>
<br></center></td></tr></table>

<h3>C<sub>6</sub> Diagnostic Testing</h3>

Throughout the course of the disease, IR<sub>6</sub> produces a strong antibody response that can be identified from early to late phases. Applications in diagnostic testing have been identified as a result of this strong immune response and IR<sub>6</sub>’s relative invariability across strains.<ref name="I">PMID:10565920</ref> <ref name="F" /> A C<sub>6</sub> [http://en.wikipedia.org/wiki/ELISA ELISA] test has been developed which uses a 26 amino acid synthetic peptide, C<sub>6</sub>, containing the IR<sub>6</sub> sequence. Results show 99% specificity and 100% precision with high sensitivity. In fact, OspA vaccination does not influence C6 specificity; therefore, C<sub>6</sub> ELISA tests are valuable diagnostic tools.<ref name="I" /> The CDC currently recommends a [http://www.cdc.gov/lyme/healthcare/clinician_twotier.html two-step test] incorporating first a polyvalent, whole-cell [http://en.wikipedia.org/wiki/Sonicate sonicate] (WCS) [http://en.wikipedia.org/wiki/Immunofluorescence_assay immunofluorescent assay]. If results are positive, this is followed by IgG and IgM WCS [http://en.wikipedia.org/wiki/Western_blot Western blots] to eliminate false positives.<ref name="J">PMID:21865190</ref> Therefore, this one-step ELISA test presents an accurate and economical alternative to the current two-step model.<ref name="I" />

<br><br><br><br><br><br><br><br><br><br><br><br><br>
==References==

<references />

==Teaching at Stony Brook University==

This Proteopedia page is the product of a new introductory biology laboratory started in the spring of 2012 at Stony Brook University. Undergraduates model and print tactile 3-dimensional proteins involved in Lyme disease in order to understand and interpret contemporary structural biology research. The best student-authored summaries from spring and summer 2012 were selected for this Proteopedia page, thereby connecting students to scientists and facilitating further research experiences.<br>

Author contributions:<br>
Osp C: Irene Chen, Khine Tun<br>
Antibodies to Osp A and B: Safa Abdelhakim, Alexandros Konstantinidis, Philip J. Pipitone, Christopher Smilios<br>
Osp A: Jenny Kim Kim, Cara Lin, Andrea Mullen, Kimberly Slade<br>
Osp B: Olivia Cheng, Stephanie Maung, Ying Zhao<br>
VlsE: Frank J. Albergo, Rachel Cirineo, Tanya Turkewitz<br>
Editors, teachers: Jeff Ecklund, Joan M. Miyazaki, Christopher Morales, Carol Nicosia, Deborah A. Spikes, Raymond Suhandynata, La Zhong<br>
Technical support: Nancy A. Black, Jameson T. Crowley<br>
Collaborating research scientists, editors: Jorge L. Benach, Timothy J. LaRocca<br>
Course co-developer, writer, editor: Niamh B. O'Hara<br>
Course director, course co-developer, writer, editor: Marvin H. O'Neal III<br>
Supported by: Howard Hughes Medical Institute 52006940

==Proteopedia Page Authors==
[http://proteopedia.org/wiki/index.php/User:Safa_Abdelhakim Safa Abdelhakim],
[http://proteopedia.org/wiki/index.php/User:Frank_J._Albergo Frank J. Albergo],
[http://proteopedia.org/wiki/index.php/User:Irene_Chen Irene Chen],
[http://proteopedia.org/wiki/index.php/User:Olivia_Cheng Olivia Cheng],
[http://proteopedia.org/wiki/index.php/User:Rachel_Cirineo Rachel Cirineo],
[http://proteopedia.org/wiki/index.php/User:Jenny_Kim_Kim Jenny Kim Kim],
[http://proteopedia.org/wiki/index.php/User:Alexandros_Konstantinidis Alexandros Konstantinidis],
[http://proteopedia.org/wiki/index.php/User:Cara_Lin Cara Lin],
[http://proteopedia.org/wiki/index.php/User:Stephanie_Maung Stephanie Maung],
[http://proteopedia.org/wiki/index.php/User:Christopher_Morales Christopher Morales],
[http://proteopedia.org/wiki/index.php/User:Andrea_Mullen Andrea Mullen],
[http://proteopedia.org/wiki/index.php/User:Niamh_O'Hara Niamh B. O'Hara],
[http://proteopedia.org/wiki/index.php/User:Marvin_O'Neal Marvin H. O'Neal III],
[http://proteopedia.org/wiki/index.php/User:Philip_J._Pipitone Philip J. Pipitone],
[http://proteopedia.org/wiki/index.php/User:Kimberly_Slade Kimberly Slade],
[http://proteopedia.org/wiki/index.php/User:Christopher_Smilios Christopher Smilios],
[http://proteopedia.org/wiki/index.php/User:Raymond_Suhandynata Raymond Suhandynata],
[http://proteopedia.org/wiki/index.php/User:Khine_Tun Khine Tun],
[http://proteopedia.org/wiki/index.php/User:Tanya_Turkewitz Tanya Turkewitz],
[http://proteopedia.org/wiki/index.php/User:Ying_Zhao Ying Zhao],
[http://proteopedia.org/wiki/index.php/User:La_Zhong La Zhong].

Highlighted Proteins of Lyme Disease

2012-09-25T19:07:42Z

Marvin O'Neal:

<font size='4'>Highlighted Proteins of Lyme Disease</font size>
<p>
[http://en.wikipedia.org/wiki/Lyme_disease Lyme disease] is caused by three species of bacteria belonging to the <i>Borrelia</i> genus, with <i>Borrelia burgdorferi</i> being the most common in the US. The bacteria are transmitted via hard-bodied ticks of the [http://en.wikipedia.org/wiki/Ixodidae <i>Ixodidae</i>] family. <i>Borrelia</i> spirochetes are motile, helical bacteria that have many lipoproteins exposed on the surfaces of their membranes. Two predominant groups of surface lipoproteins are classified as the outer surface proteins (Osps) and the variable major protein-like sequence expressed (VlsE). Both groups of proteins play important roles in pathogenesis, the the life cycle of <i>Borrelia</i>, and eliciting an immune response from the host (Figure 1).<ref name="connolly">PMID: 15864264</ref>
</p>
<p>
In a introductory biology course at Stony Brook University, undergraduates are modeling and exploring <i>Borrelia burgdorferi</i> surface proteins, as well as host produced antibodies to these proteins. This Proteopedia page is the product of their efforts, with a focus on highlighted proteins from five categories: [[#Osp C and Lyme Disease|Osp C]], [[#Antibodies to Osp A and Osp B and Lyme Disease|antibodies to Osp A and Osp B]], [[#Osp A and Lyme Disease|Osp A]], [[#Osp B and Lyme Disease|Osp B]], and [[#VlsE and Lyme Disease|VlsE]].
</p>
<p>
The goal of this Proteopedia page is to describe Lyme disease from a structural biology perspective. What do <i>Borrelia burgdorferi</i> outer surface proteins look like? How does the structure/function of these proteins relate to the infection cycle of <i>Borrelia burgdorferi</i>? What are the structural targets of the human immune system and how have these targets evolved? What are the ideal structural targets for a vaccine to protect against Lyme disease?
</P>
[[Image:BorreliaGeneExpressionCycle.png|500px|right|thumb|Borrelia Gene Expression Over Life Cycle.]]

== Osp C and Lyme Disease ==

<font size='4'>Imporance of OspC in Lyme disease</font size>
[[Image:Spirochetes in infected unfed and feeding nymph.png|300px|right|thumb|Migration of infected nymph from midgut to salivary glands]]

OspC, one of the major outer surface proteins of ''B. burgdoferi'', plays a pivotal role in transmission of ''B. burgdoferi'' from the tick vector to mammalian host. The protein gets upregulated when the tick feeds, allowing for the ''B. burgdoferi'' to adhere to the tick's saliva and move to the tick's mouth and into the host.<ref>PMID:14981110</ref> The upregulation of OspC is accompanied by a downregulation of OspA and OspB, which is thought to be induced by changes in environmental temperature and pH. <ref>PMID:14981110</ref>

OspC is a highly variable protein and strains of ''B. burgdoferi'' are classified according to the sequence of the OspC locus into 19 outer surface major groups (oMGs), denoted by type A to S, only four of which are invasive (disease causing).<ref name=kumaran>PMID:11230121</ref> Polymorphism of OspC and abundance of invasive strains in a population of ''B. burgdoferi''are driven by ecological factors, such as host mammalian community composition, and is a determinant of human Lyme disease risk<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>.

Researchers are attempting to take advantage of the presence of OspC on Borrelia's surface, while the bacteria is in the host, to develop an OspC-based vaccine. However, development of OspC-based vaccination has presented difficulties due to the highly variable nature of OspC. <ref name='protein'>PMID:17921702</ref>

<h3>Exploring the Structure of OspC</h3>{{STRUCTURE_1ggq| PDB=1ggq | SCENE=Studio:G4SecL04/Dimer_with_mg/1 }}The model presented to the right is the B31 strain (residues 38-201), which is also known as oMG A. This is one of four invasive oMGs that are responsible for systematic Lyme disease. In crystal structure, OspC exists as a dimer with the coordination of divalent ion, which is modeled as a magnesium ion. Each subunit is predominantly helical, consisting of five parallel
<scene name='Studio:G4SecL04/Helix_blue_in_ribbon/1'> α-helices </scene>
, two short antiparallel
<scene name='Studio:G4SecL04/Sheet_yellow_in_ribbon/1'> β-sheets</scene>
and six
<scene name='Studio:G4SecL04/Random_coils/2'>random coils</scene>
. The '''N and C termini''' at the membrane proximal end of two long alpha helices,
<scene name='Studio:G4SecL04/Alpha_1_with_pointer/1'>α1</scene> (residues 38-76) and
<scene name='Studio:G4SecL04/Alpha_5_with_pointer/1'>α5</scene> (residues 170-201) are in close proximity to each other. At the membrane distal end, there are three remaining alpha helices,
<scene name='Studio:G4SecL04/Alpha_2_with_pointer/1'>α2</scene> (residues 95-112),
<scene name='Studio:G4SecL04/Alpha_3_with_pointer/1'>α3</scene> (residues 121-145), including a short
<scene name='Studio:G4SecL04/Alpha_4_with_pointer/1'>α4</scene> (residues 152-159). At the end of membrane surface, the connection between helices α1 and α2 forms two short anti-parallel β-strands,
<scene name='Studio:G4SecL04/Beta_1_with_pointer/2'>β1</scene> (residues 79-80),and
<scene name='Studio:G4SecL04/Beta_2_with_pointer/1'>β2</scene> (residues 88-89) are formed.

While most of the OspC locus is highly variable, the sequence alignment of all oMGs reveals that towards the membrane proximal end, the surface-exposed residues on α1 and α5 are highly <scene name='Studio:G4SecL04/Conserved_region/1'>conserved</scene>, resulting in a positively charged surface. Other than those on helices, α1 and α5, the surface-exposed residues on the remaining regions of OspC molecule are variable.<ref name= variable>PMID: 11139584 </ref>

{{Template:ColorKey_ConSurf_NoYellow}}

<h3>OspC Structure and Antigenicity</h3>

At the membrane distal region, the six loop regions, including two β-strands illustrates the
<scene name='Studio:G4SecL04/Most_antigenic_site/1'>most antigenic sites</scene>
of OspC due to the presence of variable surface-exposed residues among OspC isolates. <ref>Earnhart C, LeBlanc D, Alix K, Desrosiers D, Radolf J, and Marconi R. 2010. Identification of residues within ligand-binding domain 1 (LBD1) of the ''Borrelia burgdorferi'' OspC protein required for function in the mammalian environment. Molecular Microbiology 76(2): 393-408. [http://dx.crossref.org/10.1111%2Fj.1365-2958.2010.07103.x DOI: 10.1111/j.1365-2958.2010.07103.x]</ref> However, among these variable regions, the outer surface-exposed residues connecting the helices α1 and α2, forming the loops,
<scene name='Studio:G4SecL04/L1/4'>L1</scene> (residues 74-78),
<scene name='Studio:G4SecL04/L2/3'>L2</scene> (residues 81-87),
<scene name='Studio:G4SecL04/L3/3'>L3</scene> (residues 90-93)and two short beta strands, β1 and β2, and also
<scene name='Studio:G4SecL04/L5/3'>L5</scene> (residues 146-150)
are more highly variable than those present in the loops,
<scene name='Studio:G4SecL04/L4/2'>L4</scene> (residues 115-119)and
<scene name='Studio:G4SecL04/L6/3'>L6</scene> (residues 161-169). Consequently, the surface potential of red region that projects away from the membrane is negatively charged and mainly involved in the protein-protein or protein-ligand interactions.<ref name= variable>PMID: 11139584 </ref> Only four types of oMGs (A, B, I and K), whose surface potential in red region is highly negative relative to non-invasive one plays a major role in pathogenesis of human Lyme disease. <ref name=kumaran>PMID:11230121</ref>The residue, <scene name='Studio:G4SecL04/His_82/1'>His82</scene>, located on the red region at the membrane distal end is unique that the replacement of other residues except His82, Lys82, Gln82, which are present only in four invasive oMGs enhances the possibility of turning invasive strains to non-invasive one. Thus, the stronger the electrostatic potential on red region, the higher the chance for OspC to bind with positively charged host ligands. Therefore, the alternation of an amino acid residue at the 82nd position on red region not only demonstrates OspC polymorphism, but also points out the probability for turning invasive strains to non-invasive strains.<ref name=kumaran>PMID:11230121</ref>

<h3>Lyme Disease and Ecology</h3>

[[Image:Life cycle of tick.png|300px|right|thumb|Life cycle of tick.[[http://www.cdc.gov/ticks/life_cycle_and_hosts.html]]]]

The number of reported cases of Lyme disease is increasing annually in highly focused geographic locations of the United States (CITE). The occurrence of Lyme disease is dependent upon the abundance of ticks infected with ''B. burgdorferi'' in natural ecosystems. Ticks are born without ''B. burgdorferi'' and acquire the bacteria while feeding on the blood of natural reservoir hosts such as mice, squirrels, shrews and other small vertebrates (Figure XX). After growth and development, the infected nymphal ticks can transmit ''B. burgdorferi'' to incidental vertebrates, including humans. The ecological interaction between the competence of reservoir hosts and the ticks is an underlying measure of human Lyme disease risk.<ref>PMID: 12525705</ref>

<h3>Ecological factors responsible for human Lyme disease risk</h3>

* '''Vertebrate Community Composition'''<ref>PMID:15514047</ref>: Two types of environment that the vertebrate hosts reside, which is also called vertebrate host density are interspecific community, which involves organisms of different species and intraspecific community, which is composed of organisms of same species. The hosts living in the community within different species or same species strongly affects the proportion of infected nymphal tick that can cause human Lyme disease.
* '''Distribution frequency of particular oMGs'''<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>: After taking blood meal from their hosts, the proportion of host-seeking nymphs infected with each oMG differs among oMGs. As only four types of oMGs (A, B, I and K) are responsible for systemic human Lyme disease, the host-seeking nymphs that have high distribution frequency of four invasive oMGs is one of the standard measures of human Lyme disease risk.
* '''Transmission Probability'''<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>: The transmission probability of each oMGs from individual species differs. The higher the transmission probability of a particular oMG from vertebrate host, the higher the chance of carrying that particular oMG by the ticks after receiving blood meal from their hosts is. Thus, it is one of the parameters that contributes the prevalence of human Lyme disease.

<h3>Using Ecological Models to Predict Lyme Disease Risk</h3>

[[Image:Lyme_Disease_Risk_Map.gif|300px|right|thumb|Map illustrating prevalence of Lyme disease in the Untied States by CDC.[[http://www.cdc.gov/mmwr/preview/mmwrhtml/rr4807a2.htm]]]]

Conceptual and mathematical models have been developed by researchers to characterize the ecological interaction between vertebrate host community and distribution frequency of invasive oMGs and predict the cases of human Lyme disease. In one model, the principal natural reservoir host used in the model for the epidemic of Lyme disease in northeastern and central United States is the presence of the white-footed mice (''Peromyscus leucopus'') population, which has both high frequency distribution in all four human infectious oMGs and high transmission probabilities of oMGs A, B, I and K.<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref> Ticks are least likely to parasitize inefficient reservoir hosts, thereby increasing high infection prevalence in the tick population, which enhances the risk of exposure of Lyme disease in humans. Many studies have found support for this "dilution-effect model" which proposes that maintaining high diversity of vertebrate host community may dilute the power of one host, such as the white-footed mouse by increasing the degree of specialization of ticks on inefficient hosts. This model strongly demonstrates the relationship between species diversity in the community of hosts and the risk of human exposure to Lyme disease. These ecological driving forces described in the model are useful tools in predicting the prevalence and risk of human Lyme disease.

<h3>OspC-based vaccine against Lyme disease</h3>

An OspC-based vaccine against Lyme disease is currently being developed. Because of the variability of OspC, the recombinant OspC vaccine, targeting the antigenic site of one specific OspC type is ineffective for ''B. burgdorferi'' with different OspC types. Therefore, the development of vaccine that recognizes the antigenic determinant on the variable regions of multiple OspC types is required in order to effectively activate human immune response. Based on the mapping of epitope-containing regions from oMGs: A, B, K and D, the experiment-based tetravalent chimeric vaccine is being developed to tigger anti-ABKD response. <ref>Christopher G. Earnhart, Eric L. Buckles, Richard T. Marconi. "Development of an OspC-based tetravalent, recombinant, chimeric vaccinogen that elicits bactericidal antibody against diverse Lyme disease spirochete strains, Vaccine." 25(3) 466-480 (2007). [http://dx.doi.org/10.1016/j.bbr.2011.03.031 DOI: 10.1016/j.vaccine.2006.07.052]</ref> Taking advantage of tetravalent ABKD construct, octavalent chimeric vaccine also known as OspC-A8.1, recognizing additional epitopes of oMGs: C, E, N and K, has been tested in mice. <ref name='protein'>PMID:17921702</ref>

== Antibodies to Osp A and Osp B and Lyme Disease ==

[[Image:Fab reigon.png|right|thumb|Digestion of an antibody by Papain separates the fab reigons from the antibody]]
A factor contributing to the severity of Lyme disease is its resistance to certain forms of complement-dependent immune response by the evasion of the [http://en.wikipedia.org/wiki/Alternative_complement_pathway alternative complement pathway] and the blocking of complement [http://en.wikipedia.org/wiki/Complement_component_3 C3].<ref>PMID:18080415</ref> This resistance increases the importance of the complement independent immune response when combating ''B. burgdorferi''. Certain fragment antigen binding regions ([http://en.wikipedia.org/wiki/Fragment_antigen-binding fab]) of IgG and IgM monoclonal antibodies (mAbs) are bactericidal even in the absence of complement. Binding of these fabs to their corresponding outer surface protein (OspA and OspB) of ''B. burgdoferi'' leads to the lysis of the bacteria.<ref name="connolly">PMID: 15864264</ref>

<h3>Fragment Antigen Binding (fab)</h3>
Fab consists of a [http://en.wikipedia.org/wiki/Immunoglobulin_heavy_chain heavy chain] and [http://en.wikipedia.org/wiki/Immunoglobulin_light_chain light chain] and each chain is composed of a variable and a constant region. The [http://en.wikipedia.org/wiki/Paratope paratope] is located in the N terminal of the variable region of the heavy and light chains of the fab. H6831 and CB2 are IgG mAbs that targets the C-terminal of OspB and LA-2 is an IgM mAb that targets the C-terminal of OspA.<ref>PMID:107164</ref>

<h3>OspB and H6831</h3>

<h4>Interaction between OspB and H6831</h4>
<table width='450' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1RJL' size='400' frame='true' align='center' scene='Studio:G1SecL01/1/10' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''OspB-H6831 Complex''' (<scene name='Studio:G1SecL01/1/10'>Initial Scene</scene>)<br>
<scene name='Studio:G1SecL01/1/11'>OspB</scene>''':''' <scene name='Studio:G1SecL01/1/31'>Loop 1</scene> <scene name='Studio:G1SecL01/1/32'>(w/ His 52)</scene> ·· <scene name='Studio:G1SecL01/1/33'>Loop 2</scene> ·· <scene name='Studio:G1SecL01/1/34'>Lysine 253</scene> <scene name='Studio:G1SecL01/1/35'>(CPK coloring)</scene> ·· <scene name='Studio:G1SecL01/1/36'>Loop 3</scene> <br>
<scene name='Studio:G1SecL01/1/12'>H6831</scene>''': '''
<scene name='Studio:G1SecL01/1/14'>Heavy Chain</scene> ·· <scene name='Studio:G1SecL01/1/13'>Light Chain</scene> ·· <scene name='Studio:G1SecL01/1/37'>Tyr-Trp-Glu-His</scene> <br> <scene name='Studio:G1SecL01/1/30'>OspB-H6831 Complex</scene>''': ''' <scene name='Studio:G1SecL01/1/38'>Residues 218-220</scene><br>
<scene name='Studio:G1SecL01/3/2'>OspB Unbound</scene>''':''' <scene name='Studio:G1SecL01/3/3'> Central β Sheet Strands 1-4 </scene>

</center></td></tr></table>
<scene name='Studio:G1SecL01/1/10'>Restore Original Scene</scene>

The <scene name='Studio:G1SecL01/1/30'>OspB-H6831 complex</scene> consist of two components, the outer surface protein <scene name='Studio:G1SecL01/1/11'>OspB</scene> and the <scene name='Studio:G1SecL01/1/12'>fab</scene>, which is subdivided into the <scene name='Studio:G1SecL01/1/14'>heavy chain</scene> and the <scene name='Studio:G1SecL01/1/13'>light chain</scene>. Most hydrogen bonds and electrostatic interactions that are responsible for the binding of H6831 to OspB are between the <scene name='Studio:G1SecL01/1/15'>three adjacent surface-exposed loops</scene> at the C-terminal of OspB and some <scene name='Studio:G1SecL01/1/37'>residues on the fab heavy chain</scene> that include tyrosine, tryptophan, glutamate, and histidine.<ref name=becker>PMID:15713683</ref>

The majority of hydrogen bonds and electrostatic interactions are between <scene name='Studio:G1SecL01/1/33'>Loop2</scene> (residues 250-254) and the fab heavy chain. <scene name='Studio:G1SecL01/1/34'>Lys 253</scene> in loop 2 of OspB has a necessary and major role due to its central position in the exposed loops. A mutation at its position abrogates the binding interaction and causes the resistance of the bacteria to the bactericidal effect of the fab. Lys 253 interacts with the two aromatic residues on the fab heavy chain, tyrosine and tryptophan. It also makes hydrogen bonds with the glutamate 50 in the heavy chain of the fab and forms an ionic bond. Carbonyl in <scene name='Studio:G1SecL01/1/31'>loop 1</scene> of the OspB interacts with <scene name='Studio:G1SecL01/1/32'>histidine 52</scene> in the fab heavy chain. <scene name='Studio:G1SecL01/1/36'>Loop 3</scene> of OspB interacts with fab light chain.<ref name=becker />

<h4>Structural changes to OspB in the complexed form</h4>
The binding of H6831 to OspB leads to some conformational changes in OspB compared to its <scene name='Studio:G1SecL01/3/2'> unbound form </scene>. [http://en.wikipedia.org/wiki/Crystallography Crystallography] has shown that the most significant difference is the loss of the <scene name='Studio:G1SecL01/3/3'>central β sheet strands 1-4 </scene>.<ref name=becker /> The loss of these β sheets may be due to conformational change as a result of the binding or a disorder that could have occurred during a crystallization of the complex. Both small positional shifts near the fab binding site and a few larger structural changes away from the binding site were observed. The largest shifts (7– 8 Å) correspond to the repositioning of a loop opposite the fab-binding site <scene name='Studio:G1SecL01/1/38'>at residues 218-220</scene>. In the free OspB structure, all regions that exhibit shifts are adjacent to the central sheet; in the OspB-H6831 complex they all shift toward, and slightly overlap the position of the missing sheet.

<h4>Bactericidal action</h4>
The fab binding destabilizes the [http://en.wikipedia.org/wiki/Bacterial_outer_membrane outer membrane] (OM) of B. burdorferi, with subsequent formation of [http://en.wikipedia.org/wiki/Spheroplast spheroplasts]. It has been observed that the bactericidal action, but not the binding, requires the presence of divalent cations (Mg<sup>2+</sup> and Ca<sup>2+</sup>), and
fab is unable to clear bacteria in the absence of these cations.<ref name=ding /> It is speculated that OspB-Cb2 (a fab similar to H6831) complexes could lead to the lysis of the cell by creating physical openings in the OM, allowing for rapid infusion of electrolytes and increasing the [http://en.wikipedia.org/wiki/Osmolarity osmolarity] of the [http://en.wikipedia.org/wiki/Periplasm periplasm].<ref name="escudero">PMID:9125579</ref>

<h3>OspA and LA-2</h3>

<table width='450' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='400' frame='true' align='center' scene='Studio:G1SecL01/2/1' />
</td></tr><tr><td bgcolor='#eeeeee'><center>'''OspA-LA2 Complex''' (<scene name='Studio:G1SecL01/2/1'>Initial Scene</scene>)<br>
<scene name='Studio:G1SecL01/2/1'>OspA-LA2 Complex</scene>''':''' <scene name='Studio:G1SecL01/2/2'>Three Loops</scene> '''··''' <scene name='Studio:G1SecL01/2/3'>Ser 206</scene>
</center></td></tr></table>

<h4>OspA</h4>
<scene name='Studio:G1SecL01/2/1'>Restore Original Scene</scene>

OspA is usually undetectable in the early stages of Lyme disease, and is down regulated when OspC is expressed.<ref name=kumaran>PMID:11230121</ref> OspA is 53% similar to OspB. Despite their similarity, OspB is susceptible to cleavage by exogenous [http://en.wikipedia.org/wiki/Protease proteases] both ''in vivo'' and ''in vitro'', whereas OspA is relatively resistant in both cases.<ref name=becker />

OspA is used in adhering to the tick's gut by binding with the tick receptor for OspA (TROSPA), a receptor necessary for the colonization of <i>Borrelia</i> inside the tick. High levels of both OspA and TROSPA are found prior to feeding, but are downregulated once the feeding process begins in order to initiate transmission into the host.<ref name="pal">PMID: 15537536</ref>
</p>

<h4>Interaction between OspA and LA-2</h4>
LA-2 is an IgM murine monoclonal antibody that interacts with <scene name='Studio:G1SecL01/2/2'> three exposed loops </scene> on the C-terminal of OspA. These interactions include eight direct [[hydrogen bonds]], four solvent-bridged hydrogen bonds, three ion pairs, and numerous van der Waals interactions.<ref name=ding />

<h4>Structural changes to OspA in the complexed form</h4>
Conformational changes upon the binding of OspA and LA-2 show that LA-2 recognition of OspA involves an induced fit mechanism where the conformations of loops 1-3 shift to optimize complementarity to the antigen-combining site.<ref name=ding /> The overall structure of the C-terminal of OspA is unchanged upon the binding of LA-2 with comparison to the free OspA. The maximum atomic shift is 4.7Å at the site of <scene name='Studio:G1SecL01/2/3'>Ser 206</scene>.<ref name=ding>PMID: 11183781</ref>

<h3>Medical Application</h3>

''B. burgdoferi'' is able to escape human immune response because the outer surface proteins, to which the immune system responds, are variable. The effectiveness of different Osp molecules as [http://en.wikipedia.org/wiki/Vaccine vaccines] can vary depending on their variability. OspC and OspB are highly polymorphic, with variability of OspC observed between strains collected from within a single geographical area.<ref name=kumaran>PMID:11230121</ref> Observed variations of OspB in ''B. burgdoferi'' (including its absence from the bacteria) are not accounted for by major [http://en.wikipedia.org/wiki/Dna DNA] arrangements or failure in [http://en.wikipedia.org/wiki/Transcription_(genetics) transcription] of the OspB gene. This indicates that the OspB gene may code for a variety of proteins, making OspB a poor candidate for use in vaccines.<ref>PMID:2668185</ref> OspA is the most conserved; of the three exposed loops, only loop 1 is variable while loops 2 and 3 are conserved. This makes OspA a more consistent antigen (compared to OspB and OspC) for the immune system to target and usable as a vaccine to Lyme disease.<ref name=ding />

== Osp A and Lyme Disease ==

<Structure load="1fj1" size="350" frame="true" align="right" name="OspA-secondary" caption="Outer surface protein A (OspA) ([[1fj1|1FJ1]], chain F) showing secondary structures." scene="Studio:G2SecL03/Ospa_default/5"/>
<p>
<b>Outer Surface Protein A (OspA)</b> is a major lipoprotein found on the surface of [http://en.wikipedia.org/wiki/Spirochaete spirochetes] from the genus [http://en.wikipedia.org/wiki/Borrelia <i>Borrelia</i>] and is comprised of 21 anti-parallel β-sheets and a single α-helix. OspA's expression is regulated at different points in time, from being downregulated during the tick's feeding process on its host to being upregulated in the host's cerebrospinal fluid (CSF) to induce inflammatory response, resulting in acute Lyme [http://en.wikipedia.org/wiki/Neuroborreliosis neuroborreliosis]. OspA has also been used as a vector in working towards the development of a vaccine for [http://en.wikipedia.org/wiki/Lyme_disease Lyme disease].
</p>
<p>
While feeding, OspA is downregulated in order to evade an immune response from the incoming host blood into the gut, releasing the <i>Borrelia</i> from the gut wall and migrating into the tick's salivary glands, thereby allowing it to enter the host through the bite. This is evidenced by the fact that patients with Lyme disease have been found to not possess OspA antibodies in the early stages of the disease.<ref name="connolly">PMID: 15864264</ref><ref name="rupprecht">PMID: 18097481</ref> OspA is the protein most related to acute Lyme neuroborreliosis (LNB), the neurological manifestations of Lyme disease.
</p>

<h3>OspA's Role in Invasion</h3>
<p>
Once inside the host, the <i>Borrelia</i> has a great number of mechanisms available to actively suppress the host's immune system response and neutralize its effector mechanisms, such as the expression of another outer surface protein, OspC, which prevents susceptibility to the host's [http://en.wikipedia.org/wiki/Innate_immune_system innate immunity] and [http://en.wikipedia.org/wiki/Complement_system complement systems]. Additionally, <i>Borrelia</i> is capable of suppressing many of its surface proteins to reduce its detectability, but can also utilize protective means by temporarily expressing them when needed.
</p>

<h4>Acute Lyme Neuroborreliosis (LNB)</h4>
<p>
Acute Lyme Neuroborreliosis (LNB) is part of the second stage of Lyme disease in which the spirochete invades the peripheral and central nervous systems (CNS). Symptoms of LNB include: meningoradiculitis with inflammation of the nerve roots and [http://en.wikipedia.org/wiki/Radicular_pain radiculitis] (Bannwarth’s syndrome), lymphocytic meningitis, and cranial and [http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001619/ peripheral neuritis]. In Europe, the strain predominantly found in the CSF of patients with Bannwarth's syndrome is <i>B. garinii</i>. However, in the United States, Bannwarth's syndrome is rare and the most common manifestations of Lyme neuroborreliosis is [http://en.wikipedia.org/wiki/Meningitis meningitis], caused by <i>B. burgdorferi</i>. The presence of OspA in the cerebrospinal fluid (CSF) is responsible for this complex inflammatory response in the brain that leads to the neuroborreliosis.<ref name="rupprecht">PMID: 18097481</ref>
</p>

<h4>Evasion and the Extracellular Matrix</h4>
<p>
The <i>Borrelia</i> are able to hide in the [http://en.wikipedia.org/wiki/Extracellular_matrix extracellular matrix], allowing it to survive by avoiding [http://en.wikipedia.org/wiki/Leukocytes leukocytes] circulating in the bloodstream. OspA can rapidly bind to plasminogen, which becomes [http://en.wikipedia.org/wiki/Plasmin plasmin] once activated, and degrades the extracellular matrix. By binding to plasminogen, <i>Borrelia</i> could be exploiting its function and utilizing it to invade the extracellular matrix. However, due to the fact that OspA is downregulated during feeding, and stays unexpressed, a different mechanism may be used instead. Additionally, <i>Borrelia</i> induces the local upregulation of matrix metalloproteinase-9, causing the digestion of the surrounding extracellular matrix. <i>Borrelia</i> can also bind to several proteins in the extracellular matrix, such as [http://en.wikipedia.org/wiki/Fibronectin fibronectin], [http://en.wikipedia.org/wiki/Integrins integrins] or [http://en.wikipedia.org/wiki/Decorin decorin], which can aid in the spread and survival of the spirochetes in these tissues.<ref name="rupprecht">PMID: 18097481</ref>
</p>

<h4>Migration Across the Blood-Brain Barrier</h4>
<p>
It is not fully understood how <i>Borrelia</i> get past the [http://en.wikipedia.org/wiki/Blood-brain_barrier blood-brain barrier], though some researchers suggest a paracellular route, which involves a process using transient tether-type associations, short-term dragging interactions, and stationary adhesion. There is evidence that <i>Borrelia</i> utilizes OspA in the transient tethering stage. The blood-brain barrier is composed of brain microvascular endothelial cells, astrocytes, a basement membrane, pericytes, and neurons. OspA is a major adherent molecule to brain microvascular cells by binding to the [[1aly|CD40]] receptors outside, which results in events that are typically seen when leukocytes cross the blood brain barrier.

Activation of CD40 receptors leads to the production of proinflammatory cytokines and enhanced expression of ICAM-1, E-selectin and VCAM-1, resulting in increased cell binding, and the formation of fenestrations due to increased vascular endothelial growth factor, and vascular permeability factor. OspA might be mimicking leukocytes in order to cross the blood-brain barrier. However not all strains of <i>Borrelia</i> can utilize OspA to do this, OspA only contributes about 70% to adherence, and other <i>Borrelia</i> proteins are also needed in this process. It has also been seen that OspA mediates the adhesion of <i>Borrelia</i> to murine neural and glial cell lines. <ref name="pulzova">PMID: 22355605</ref>
</p>

<h4>Role in Inflammation</h4>

[[Image:G2L03-OspA-mechanism.jpg|thumb|300px|Mechanism of the host inflammatory response to OspA]]

There are six steps involved in the host's inflammatory response to OspA: <ref name="rupprecht">PMID: 18097481</ref><br>
<ol>
<li>When the <i>Borrelia</i> enter the host’s CNS they encounter several different types of immune cells such as [http://en.wikipedia.org/wiki/Monocyte monocytes], [http://en.wikipedia.org/wiki/Macrophages macrophages], and [http://en.wikipedia.org/wiki/Dendritic_cells dendritic cells]. While in the CSF, outer surface protein A (OspA) is upregulated and it’s increased expression promotes recognition by a specific receptor on a monocyte.</li>

<li>The OspA-bound monocyte then releases proinflammatory [http://en.wikipedia.org/wiki/Cytokine cytokines] (i.e. [http://en.wikipedia.org/wiki/Interferon interferon]), as well as [http://en.wikipedia.org/wiki/Chemokine chemokines], such as [http://en.wikipedia.org/wiki/CXCL13 CXCL13]. In patients with LNB, there is an observed increase in the levels of these cytokines and chemokines in their CSF. The production of chemokines leads to the recruitment of other immune cells to the site of infection.</li>

<li>[http://en.wikipedia.org/wiki/B_lymphocyte B-lymphocytes] respond to the new concentration gradient of CXCL13 between the blood and CSF and migrate into the CSF.</li>

<li>B-lymphocytes undergo [http://en.wikipedia.org/wiki/Receptor-mediated_endocytosis receptor-mediated endocytosis], consuming the OspA antigens present in the CSF, thereby triggering its activation. The B-lymphocytes then are able to differentiate and mature into [http://en.wikipedia.org/wiki/Plasma_cells plasma cells].</li>

<li>The plasma cells create large quantities of anti-OspA antibodies specific to this strain of <i>Borrelia</i> and release them into the CSF.</li>

<li>The anti-OspA antibodies will then bind to the OspA on the spirochete’s membrane, thus killing the <i>Borrelia</i>.</li>
</ol>

<p>
This process is two-sided in the sense that the OspA aids in the pathogenesis of new symptoms (neuroborreliosis) through the chemokine’s actions, as well as initiating the signaling cascade to destroy itself.
</p>

<h3>OspA Vaccination</h3>

<table width='400' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='350' frame='true' align='center' name='complex' caption='Outer Surface Protein A (Osp A) in complex with the LA-2 Fab antibody ([[1fj1|1FJ1]]).' scene='Studio:G2SecL03/Ospafab-orig/3'/>
</td></tr><tr><td bgcolor='#eeeeee'><center><scene name='Studio:G2SecL03/Ospafab-orig/3' target="complex">Reset model</scene>,<scene name='Studio:G2SecL03/Ospafab-fab/3' target="complex">LA-2</scene> Fab antibody (Bluish regions indicate heavy chains (chains B & D of 1FJ1) and greenish regions indicate light chains (chains A & C of 1FJ1))<scene name='Studio:G2SecL03/Ospafab-ospa/3' target="complex">OspA</scene> proteins in complex with the LA-2 Fab antibody (chains E & F of 1FJ1)<scene name='Studio:G2SecL03/Ospafab-interaction/3' target="complex">Closeup</scene> of the OspA antigen : LA-2 Fab antibody interactions
</center></td></tr></table>

<p>
Risk of developing Lyme disease can be mitigated by staying clear of areas with populations of ticks, wearing proper attire to minimize easily bitten areas of the body, and using insect repellents containing [http://en.wikipedia.org/wiki/DEET DEET] (N,N-diethy-m-toluamide). However, another effective means for prevention could be possible by using an outer surface protein from <i>Borrelia</i> in the creation of a vaccine.<ref name="nigrovic">PMID: 16893489</ref>
</p>
<p>
The membrane composition of <i>Borrelia</i> is abundant in both OspA and OspB, and the two proteins share a 53% similarity in their primary sequences. Both OspA and OspB are expressed in the tick's gut and downregulated during feeding and aid in its survivability; however, OspA is overall less varied and reactive than OspB, which has greater variability.<ref name="becker">PMID: 15713683</ref> The relatively conserved sequence of OspA thus lends itself better to study and application toward the development of a vaccine for a broader range of <i>Borrelia</i> strains in the treatment of Lyme disease than that of OspB. The first vaccine used a purified recombinant form of OspA and functioned in blocking transmission of the spirochetes expressing OspA from tick to host during feeding, killing them while still attached to the tick's gut.<ref name="connolly">PMID: 15864264</ref><ref name="battisti">PMID: 18779341</ref> The vaccine, Lymerix, had shown 76% and 92% effectiveness in separate clinical trials in which patients were treated for two years following a three-dose schedule. However, the vaccination was suspended from use in 2002 when opponents claimed the [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG antibodies] for OspA were associated with the onset of severe chronic arthritis, as well as other side effects affecting immunity.<ref name="connolly">PMID: 15864264</ref><ref name="plotkin">PMID: 21217175</ref> This fact, in conjunction with the desire for a more widespread vaccine treating multiple strains of <i>Borrelia</i>, has spurred research towards a new vaccine.
</p>
<p>
To address the concerns of vaccine with broader protection, creation of a chimera, mixing the OspA of different strains of <i>Borrelia</i> would be ideal. Study of the epitope of <scene name='Studio:G2SecL03/Ospafab-ospa/3' target="complex">OspA</scene> and its <scene name='Studio:G2SecL03/Ospafab-interaction/3' target="complex">interactions</scene> with the murine monoclonal antibody <scene name='Studio:G2SecL03/Ospafab-fab/3' target="complex">LA-2</scene> have proved useful in determining effectiveness of a given vaccine trial as high levels of antibodies in test sera compete against LA-2 for binding with OspA. LA-2 makes direct contact with three exposed loops of the C-terminus of OspA. The recognition of OspA by LA-2 requires an induced fit mechanism where these three loops undergo conformational changes to optimize their interaction in the complex. <ref name="ding">PMID: 11183781</ref>
</p>

<h3>Structure of OspA</h3>

<table width='400' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='350' frame='true' align='center' name='OspA-manip' caption='Outer surface protein A (OspA) ([[1fj1|1FJ1]], chain F).' scene='Studio:G2SecL03/Ospa_3loopscartoon/3'/>
</td>
</tr>
<tr>
<td bgcolor='#eeeeee'>
<center>
<scene name='Studio:G2SecL03/Ospa_3loopscartoon/3' target="OspA-manip">Reset model</scene>,<br><scene name='Studio:G2SecL03/Ospa-3loops/4' target="OspA-manip">Three loops</scene> in C-terminus (close up)<scene name='Studio:G2SecL03/Ospa-loop1/1' target="OspA-manip">including Loop 1</scene> (residues 203-220)<scene name='Studio:G2SecL03/Ospa-loop2/1' target="OspA-manip">, Loop 2</scene> (residues 224-233)<scene name='Studio:G2SecL03/Ospa-loop3/1' target="OspA-manip">and Loop 3</scene> (residues 246-257)<scene name='Studio:G2SecL03/Ospa-3residues-nor/3' target="OspA-manip">Three residues</scene> in C-terminus (Ala208, Ala215 and Asn251 in <i>B. burgdorferi</i>; also hides R-groups). <scene name='Studio:G2SecL03/Ospa-3residues-r/2' target="OspA-manip">Display R-groups</scene> of Ala208, Ala215 and Asn251<scene name='Studio:G2SecL03/Ospa-ala208/1' target="OspA-manip">Alanine (Ala) 208</scene><scene name='Studio:G2SecL03/Ospa-ala215/1' target="OspA-manip">Alanine (Ala) 215</scene><scene name='Studio:G2SecL03/Ospa-asn251/1' target="OspA-manip">Asparagine (Asn) 251</scene><scene name='Studio:G2SecL03/Ospa-3loops3res/1' target="OspA-manip">Three loops and all three residues (with R-groups included)</scene>
</center>
</td>
</tr>
</table>

<p>
OspA is made up of 273 residues over 21 anti-parallel β-sheets and a single α-helix. It's folded conformation is divided into three main sections: a N-terminus "sandwich," a central region comprising of several β-sheets and a C-terminus "barrel" domain.<ref name="ding">PMID: 11183781</ref> The folded regions at its ends are connected by a single β-sheet layer in the middle, giving the protein the unique shape of a dumbell.<ref name="makabe">PMID: 16823038</ref>

There are <scene name='Studio:G2SecL03/Ospa-3loops/4' target="OspA-manip">three loops</scene> at the C-terminus of OspA that are important in binding with the LA-2 Fab antibody, whose interactions provide great insight into vaccine research and effectiveness. These three loops are linearly arranged and form protruding ridge at the C-terminus of OspA. Within these loops, there are <scene name='Studio:G2SecL03/Ospa-3residues-nor/3' target="OspA-manip">three residues</scene> <scene name='Studio:G2SecL03/Ospa-3residues-r/2' target="OspA-manip">(show residue R-groups)</scene> where there are distinct variations between the different strains of <i>Borrelia</i> and serve as potential targets for the creation of a broader vaccine.<ref name="ding">PMID: 11183781</ref> <scene name='Studio:G2SecL03/Ospa-3loops3res/1' target="OspA-manip">(display both the three loops and three residues together)</scene>
</p>
<p>
<scene name='Studio:G2SecL03/Ospa-loop1/1' target="OspA-manip">Loop 1</scene>, (residues 203-220), is important in showing variation amongst the different strains of <i>Borrelia</i> as well as being optimally conformed for binding without steric hindrance. <scene name='Studio:G2SecL03/Ospa-loop2/1' target="OspA-manip">Loop 2</scene> (residues 224-233) and <scene name='Studio:G2SecL03/Ospa-loop3/1' target="OspA-manip">Loop 3</scene> (residues 246-257) are more strongly conserved than Loop 1 but also help to show some variation amongst strains. The LA-2 Fab antibody readily recognizes OspA from <i>B. burgdorferi</i>, but does not recognize that from <i>B. afzelii</i> or <i>B. garinii</i>.

Between <i>B. burgdorferi</i> and <i>B. afzelii</i> genetic sequences are generally invariant, but two residues change between the species: <scene name='Studio:G2SecL03/Ospa-ala208/1' target="OspA-manip">Alanine (Ala) 208</scene> in <i>B. burgdorferi</i> is a Glutamine (Gln) in <i>B. afzelii</i>, and <scene name='Studio:G2SecL03/Ospa-asn251/1' target="OspA-manip">Asparagine (Asn) 251</scene> in <i>B. burgdorferi</i> is an Alanine (Ala) in <i>B. afzelii</i>. <i>B. garinii</i> has more variation and in addition to the previous two differences, having at least one more difference, where <scene name='Studio:G2SecL03/Ospa-ala215/1' target="OspA-manip">Alanine (Ala) 215</scene> in <i>B. burgdorferi</i> is a Lysine (Lys), and sometimes also has a deletion at <i>B. burgdorferi</i>’s Alanine 208. LA-2 and OspA of <i>B. burgdorferi</i> form a tight interface when binding, and the longer Glutamine (Gln) sidechain found in <i>B. afzelii</i> and <i>B. garinii</i> is more difficult to accommodate, causing less binding. A chimera that was weakly recognized by LA-2 was made with parts of loop 1 from <i>B. burgdorferi</i>, and loops 2 and 3 from <i>B. garinii</i>.<ref name="ding">PMID: 11183781</ref> Recently, a different kind of chimera has been made which combined the proximal region of <i>B. burgdorferi</i> and distal region of <i>B. afzelii</i>, and was able to successfully protect mice from both species.<ref name="livey">PMID: 21217174</ref>
</p>

== Osp B and Lyme Disease ==

<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="becker">PMID:15713683</ref>.

<h3>OspB Interaction with Fab of H6831</h3>
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="becker"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/7'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="becker"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="becker"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

<h4>Binding</h4>
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />
H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="becker"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="becker"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="becker"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref name="escudero">PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="becker"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain.

<h3>Potential Mechanism of Lysis</h3>

<h4>Potential Catalytic Triad</h4>

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]
The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/2'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease.

<h4>Potential Oxidative Mechanism</h4>
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement.

== VlsE and Lyme Disease ==

<table width='400' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><applet load='1l8w.pdb' size='400' frame='true' align='right' scene='Studio:G5SecL01/Main_image_vlse/1' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''Variable Major Protein (VMP)-like sequence Expressed''' ([[1l8w]]), resolution 2.3Å
[[Image:VLSE PRIMARY STRUCTURE4343.png|400px]]<br>
<nowiki>*</nowiki><small>'''This representation of VlsE illustrates the only crystal structure available on the [http://www.rcsb.org/pdb/explore/explore.do?structureId=1l8w PDB site]. There are 10<sup>30</sup> possible combinations of the VR.'''</small>
<br></center></td></tr></table>

<h3>Structural Overview</h3>

Variable Major Protein (VMP)-like sequence Expressed ('''VlsE''') is a surface lipoprotein of ''Borrelia burgdorferi''. It undergoes [http://en.wikipedia.org/wiki/Antigenic_variation antigenic variation] seemingly important in evasion of the host’s immune system. In addition, the protein is used for Lyme disease diagnosis. It is composed of four similar subunits each possessing two invariable domains and one variable domain.<ref name="A">PMID:10569796</ref> The variable domain contains six variable regions (VR<sub>1</sub>-VR<sub>6</sub>), and six invariable regions (IR<sub>1</sub>-IR<sub>6</sub>). Research suggests that the protein may exist as a dimer where each monomeric C & N termini neighbor each other forming the membrane proximal portion of the protein, and the variable regions form the membrane distal portion.<ref name="B">PMID:11923306</ref> <ref name="C">PMID:11716485</ref> The invariable regions are largely embedded in the protein and remain relatively unchanged within the host and across strains. The variable regions encompass 37% of the VlsE’s exposed surface area despite comprising only 25% of the protein.<ref name="A" /> <ref name="B" /> However, 50% of the VR surface area is exposed while IR<sub>6</sub>, a strong [http://en.wikipedia.org/wiki/Antigen antigen], exposes just 13.7% of its surface area. This leaves only <scene name='Studio:G5SecL01/Ir_6_4_residues/1'>four amino residues</scene> of the antigenic IR<sub>6</sub> unprotected: lysine-276, glutamine-279, lysine-291, and lysine-294. Thus, it is almost entirely embedded in the protein and <scene name='Studio:G5SecL01/Ir6_embedded/1'>sheilded by the variable regions </scene>.<ref name="B" />

<h3>Antigenicity</h3>

The variable regions undergo a recombination event stimulated by the host’s cytokines and absence of those cytokines results in a decreased bacterial burden.<ref name="D">PMID:11544329</ref> This leads to variation with an estimated 10<sup>30</sup> possible combinations, far exceeding the number of antibodies found in the human immune system. While the VR does exhibit antigenicity, this recombination makes it unlikely that a sufficient amount of a single VR variation will be present in large enough supply to lead to an immunodominant variable region.<ref name="E">PMID:10553085</ref> IR<sub>6</sub>, however, exhibits immunodominance while IR<sub>1</sub>-IR<sub>5</sub> are primarily nonantigenic in humans. Thus, shielding of the immunodominant IR<sub>6</sub> by VR regions not subject to antibody response allows for IR<sub>6</sub> to elicit an immune response while remaining inaccessible to antibody binding. Research suggests that the 26 amino residues of <scene name='Studio:G5SecL01/Ir6_with_epitope/1'>IR6</scene> may function as a single epitope with a central alpha helical core.<ref name="B" /> <ref name="D" /> <ref name="F">PMID:10722641</ref>

<h3>Function in Immune System Evasion</h3>

VlsE is essential to the persistence and virulence of Lyme disease and is upregulated under humoral immune pressure.<ref name="G">PMID:17714442</ref> <ref name="H">PMID:15385475</ref> While the exact mechanism for immune evasion remains unknown, several theories have been put forth. One popular theory maintains that VlsE masks other surface antigens by coating the surface of the bacteria, thereby sterically blocking the antigens from antibody binding. This is similar to other pathogens with variable regions, such as [http://en.wikipedia.org/wiki/Trypanosoma_brucei Trypanosoma brucei], the protozoa responsible for African sleeping sickness and [http://en.wikipedia.org/wiki/Neisseria_gonorrhoeae Neisseria gonorrhea], the bacterial cause of gonorrhea. However, recent studies have cast doubt on this theory. An alternate theory provides that VlsE directly stimulates B cell antibody production independent of T-cells. The robust response elicited is thought to override antibody production against other antigens.<ref name="G" />
<br>

<table width='400' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><applet load='1l8w.pdb' size='400' frame='true' align='right' scene='Studio:G5SecL01/Chart_main/4' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''VlsE''' ([[1l8w]]), resolution 2.3Å (<scene name='Studio:G5SecL01/Chart_main/4'>Original</scene>). <br>
Highlight: <scene name='Studio:G5SecL01/Chart_main/3'>IR6</scene>, <scene name='Studio:G5SecL01/Chart_vr_seagreen/1'>VR</scene>, <scene name='Studio:G5SecL01/Chart_ir1_yellow/1'>IR1</scene>, <scene name='Studio:G5SecL01/Chart_ir2_yellow/1'>IR2</scene>, <scene name='Studio:G5SecL01/Chart_ir3_yellow/1'>IR3</scene>, <scene name='Studio:G5SecL01/Chart_ir4_yellow/1'>IR4</scene>, <scene name='Studio:G5SecL01/Chart_ir5_yellow/1'>IR5</scene>. <br>
<br></center></td></tr></table>

<h3>C<sub>6</sub> Diagnostic Testing</h3>

Throughout the course of the disease, IR<sub>6</sub> produces a strong antibody response that can be identified from early to late phases. Applications in diagnostic testing have been identified as a result of this strong immune response and IR<sub>6</sub>’s relative invariability across strains.<ref name="I">PMID:10565920</ref> <ref name="F" /> A C<sub>6</sub> [http://en.wikipedia.org/wiki/ELISA ELISA] test has been developed which uses a 26 amino acid synthetic peptide, C<sub>6</sub>, containing the IR<sub>6</sub> sequence. Results show 99% specificity and 100% precision with high sensitivity. In fact, OspA vaccination does not influence C6 specificity; therefore, C<sub>6</sub> ELISA tests are valuable diagnostic tools.<ref name="I" /> The CDC currently recommends a [http://www.cdc.gov/lyme/healthcare/clinician_twotier.html two-step test] incorporating first a polyvalent, whole-cell [http://en.wikipedia.org/wiki/Sonicate sonicate] (WCS) [http://en.wikipedia.org/wiki/Immunofluorescence_assay immunofluorescent assay]. If results are positive, this is followed by IgG and IgM WCS [http://en.wikipedia.org/wiki/Western_blot Western blots] to eliminate false positives.<ref name="J">PMID:21865190</ref> Therefore, this one-step ELISA test presents an accurate and economical alternative to the current two-step model.<ref name="I" />

<br><br><br><br><br><br><br><br><br><br><br><br><br>
==References==

<references />

==Teaching at Stony Brook University==

This Proteopedia page is the product of a new introductory biology laboratory started in the spring of 2012 at Stony Brook University. Undergraduates model and print tactile 3-dimensional proteins involved in Lyme disease in order to understand and interpret contemporary structural biology research. The best student-authored summaries from spring and summer 2012 were selected for this Proteopedia page, thereby connecting students to scientists and facilitating further research experiences.<br>

Author contributions:<br>
Osp C: Irene Chen, Khine Tun<br>
Antibodies to Osp A and B: Safa Abdelhakim, Alexandros Konstantinidis, Philip J. Pipitone, Christopher Smilios<br>
Osp A: Jenny Kim Kim, Cara Lin, Andrea Mullen, Kimberly Slade<br>
Osp B: Olivia Cheng, Stephanie Maung, Ying Zhao<br>
VlsE: Frank J. Albergo, Rachel Cirineo, Tanya Turkewitz<br>
Editors, teachers: Jeff Ecklund, Joan M. Miyazaki, Christopher Morales, Carol Nicosia, Deborah A. Spikes, Raymond Suhandynata, La Zhong<br>
Technical support: Nancy A. Black, Jameson T. Crowley<br>
Collaborating research scientists, editors: Jorge L. Benach, Timothy J. LaRocca<br>
Course co-developer, writer, editor: Niamh B. O'Hara<br>
Course director, course co-developer, writer, editor: Marvin H. O'Neal III<br>
Supported by: Howard Hughes Medical Institute 52006940

==Proteopedia Page Authors==
[http://proteopedia.org/wiki/index.php/User:Safa_Abdelhakim Safa Abdelhakim],
[http://proteopedia.org/wiki/index.php/User:Frank_J._Albergo Frank J. Albergo],
[http://proteopedia.org/wiki/index.php/User:Irene_Chen Irene Chen],
[http://proteopedia.org/wiki/index.php/User:Olivia_Cheng Olivia Cheng],
[http://proteopedia.org/wiki/index.php/User:Rachel_Cirineo Rachel Cirineo],
[http://proteopedia.org/wiki/index.php/User:Jenny_Kim_Kim Jenny Kim Kim],
[http://proteopedia.org/wiki/index.php/User:Alexandros_Konstantinidis Alexandros Konstantinidis],
[http://proteopedia.org/wiki/index.php/User:Cara_Lin Cara Lin],
[http://proteopedia.org/wiki/index.php/User:Stephanie_Maung Stephanie Maung],
[http://proteopedia.org/wiki/index.php/User:Christopher_Morales Christopher Morales],
[http://proteopedia.org/wiki/index.php/User:Andrea_Mullen Andrea Mullen],
[http://proteopedia.org/wiki/index.php/User:Niamh_O'Hara Niamh B. O'Hara],
[http://proteopedia.org/wiki/index.php/User:Marvin_O'Neal Marvin H. O'Neal III],
[http://proteopedia.org/wiki/index.php/User:Philip_J._Pipitone Philip J. Pipitone],
[http://proteopedia.org/wiki/index.php/User:Kimberly_Slade Kimberly Slade],
[http://proteopedia.org/wiki/index.php/User:Christopher_Smilios Christopher Smilios],
[http://proteopedia.org/wiki/index.php/User:Raymond_Suhandynata Raymond Suhandynata],
[http://proteopedia.org/wiki/index.php/User:Khine_Tun Khine Tun],
[http://proteopedia.org/wiki/index.php/User:Tanya_Turkewitz Tanya Turkewitz],
[http://proteopedia.org/wiki/index.php/User:Ying_Zhao Ying Zhao],
[http://proteopedia.org/wiki/index.php/User:La_Zhong La Zhong].

Highlighted Proteins of Lyme Disease

2012-09-25T19:07:08Z

Marvin O'Neal:

<font size='4'>Highlighted Proteins of Lyme Disease</font size>
<p>
[http://en.wikipedia.org/wiki/Lyme_disease Lyme disease] is caused by three species of bacteria belonging to the <i>Borrelia</i> genus, with <i>Borrelia burgdorferi</i> being the most common in the US. The bacteria are transmitted via hard-bodied ticks of the [http://en.wikipedia.org/wiki/Ixodidae <i>Ixodidae</i>] family. <i>Borrelia</i> spirochetes are motile, helical bacteria that have many lipoproteins exposed on the surfaces of their membranes. Two predominant groups of surface lipoproteins are classified as the outer surface proteins (Osps) and the variable major protein-like sequence expressed (VlsE). Both groups of proteins play important roles in pathogenesis, the the life cycle of <i>Borrelia</i>, and eliciting an immune response from the host (Figure 1).<ref name="connolly">PMID: 15864264</ref>
</p>
<p>
In a introductory biology course at Stony Brook University, undergraduates are modeling and exploring <i>Borrelia burgdorferi</i> surface proteins, as well as host produced antibodies to these proteins. This Proteopedia page is the product of their efforts, with a focus on highlighted proteins from five categories: [[#Osp C and Lyme Disease|Osp C]], [[#Antibodies to Osp A and Osp B and Lyme Disease|antibodies to Osp A and Osp B]], [[#Osp A and Lyme Disease|Osp A]], [[#Osp B and Lyme Disease|Osp B]], and [[#VlsE and Lyme Disease|VlsE]].
</p>
<p>
The goal of this Proteopedia page is to describe Lyme disease from a structural biology perspective. What do <i>Borrelia burgdorferi</i> outer surface proteins look like? How does the structure/function of these proteins relate to the infection cycle of <i>Borrelia burgdorferi</i>? What are the structural targets of the human immune system and how have these targets evolved? What are the ideal structural targets for a vaccine to protect against Lyme disease?
</P>
[[Image:BorreliaGeneExpressionCycle.png|500px|right|thumb|Borrelia Gene Expression Over Life Cycle.]]

== Osp C and Lyme Disease ==

<font size='4'>Imporance of OspC in Lyme disease</font size>
[[Image:Spirochetes in infected unfed and feeding nymph.png|300px|right|thumb|Migration of infected nymph from midgut to salivary glands]]

OspC, one of the major outer surface proteins of ''B. burgdoferi'', plays a pivotal role in transmission of ''B. burgdoferi'' from the tick vector to mammalian host. The protein gets upregulated when the tick feeds, allowing for the ''B. burgdoferi'' to adhere to the tick's saliva and move to the tick's mouth and into the host.<ref>PMID:14981110</ref> The upregulation of OspC is accompanied by a downregulation of OspA and OspB, which is thought to be induced by changes in environmental temperature and pH. <ref>PMID:14981110</ref>

OspC is a highly variable protein and strains of ''B. burgdoferi'' are classified according to the sequence of the OspC locus into 19 outer surface major groups (oMGs), denoted by type A to S, only four of which are invasive (disease causing).<ref name=kumaran>PMID:11230121</ref> Polymorphism of OspC and abundance of invasive strains in a population of ''B. burgdoferi''are driven by ecological factors, such as host mammalian community composition, and is a determinant of human Lyme disease risk<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>.

Researchers are attempting to take advantage of the presence of OspC on Borrelia's surface, while the bacteria is in the host, to develop an OspC-based vaccine. However, development of OspC-based vaccination has presented difficulties due to the highly variable nature of OspC. <ref name='protein'>PMID:17921702</ref>

<h3>Exploring the Structure of OspC</h3>{{STRUCTURE_1ggq| PDB=1ggq | SCENE=Studio:G4SecL04/Dimer_with_mg/1 }}
The model presented to the right is the B31 strain (residues 38-201), which is also known as oMG A. This is one of four invasive oMGs that are responsible for systematic Lyme disease. In crystal structure, OspC exists as a dimer with the coordination of divalent ion, which is modeled as a magnesium ion. Each subunit is predominantly helical, consisting of five parallel
<scene name='Studio:G4SecL04/Helix_blue_in_ribbon/1'> α-helices </scene>
, two short antiparallel
<scene name='Studio:G4SecL04/Sheet_yellow_in_ribbon/1'> β-sheets</scene>
and six
<scene name='Studio:G4SecL04/Random_coils/2'>random coils</scene>
. The '''N and C termini''' at the membrane proximal end of two long alpha helices,
<scene name='Studio:G4SecL04/Alpha_1_with_pointer/1'>α1</scene> (residues 38-76) and
<scene name='Studio:G4SecL04/Alpha_5_with_pointer/1'>α5</scene> (residues 170-201) are in close proximity to each other. At the membrane distal end, there are three remaining alpha helices,
<scene name='Studio:G4SecL04/Alpha_2_with_pointer/1'>α2</scene> (residues 95-112),
<scene name='Studio:G4SecL04/Alpha_3_with_pointer/1'>α3</scene> (residues 121-145), including a short
<scene name='Studio:G4SecL04/Alpha_4_with_pointer/1'>α4</scene> (residues 152-159). At the end of membrane surface, the connection between helices α1 and α2 forms two short anti-parallel β-strands,
<scene name='Studio:G4SecL04/Beta_1_with_pointer/2'>β1</scene> (residues 79-80),and
<scene name='Studio:G4SecL04/Beta_2_with_pointer/1'>β2</scene> (residues 88-89) are formed.

While most of the OspC locus is highly variable, the sequence alignment of all oMGs reveals that towards the membrane proximal end, the surface-exposed residues on α1 and α5 are highly <scene name='Studio:G4SecL04/Conserved_region/1'>conserved</scene>, resulting in a positively charged surface. Other than those on helices, α1 and α5, the surface-exposed residues on the remaining regions of OspC molecule are variable.<ref name= variable>PMID: 11139584 </ref>

{{Template:ColorKey_ConSurf_NoYellow}}

<h3>OspC Structure and Antigenicity</h3>

At the membrane distal region, the six loop regions, including two β-strands illustrates the
<scene name='Studio:G4SecL04/Most_antigenic_site/1'>most antigenic sites</scene>
of OspC due to the presence of variable surface-exposed residues among OspC isolates. <ref>Earnhart C, LeBlanc D, Alix K, Desrosiers D, Radolf J, and Marconi R. 2010. Identification of residues within ligand-binding domain 1 (LBD1) of the ''Borrelia burgdorferi'' OspC protein required for function in the mammalian environment. Molecular Microbiology 76(2): 393-408. [http://dx.crossref.org/10.1111%2Fj.1365-2958.2010.07103.x DOI: 10.1111/j.1365-2958.2010.07103.x]</ref> However, among these variable regions, the outer surface-exposed residues connecting the helices α1 and α2, forming the loops,
<scene name='Studio:G4SecL04/L1/4'>L1</scene> (residues 74-78),
<scene name='Studio:G4SecL04/L2/3'>L2</scene> (residues 81-87),
<scene name='Studio:G4SecL04/L3/3'>L3</scene> (residues 90-93)and two short beta strands, β1 and β2, and also
<scene name='Studio:G4SecL04/L5/3'>L5</scene> (residues 146-150)
are more highly variable than those present in the loops,
<scene name='Studio:G4SecL04/L4/2'>L4</scene> (residues 115-119)and
<scene name='Studio:G4SecL04/L6/3'>L6</scene> (residues 161-169). Consequently, the surface potential of red region that projects away from the membrane is negatively charged and mainly involved in the protein-protein or protein-ligand interactions.<ref name= variable>PMID: 11139584 </ref> Only four types of oMGs (A, B, I and K), whose surface potential in red region is highly negative relative to non-invasive one plays a major role in pathogenesis of human Lyme disease. <ref name=kumaran>PMID:11230121</ref>The residue, <scene name='Studio:G4SecL04/His_82/1'>His82</scene>, located on the red region at the membrane distal end is unique that the replacement of other residues except His82, Lys82, Gln82, which are present only in four invasive oMGs enhances the possibility of turning invasive strains to non-invasive one. Thus, the stronger the electrostatic potential on red region, the higher the chance for OspC to bind with positively charged host ligands. Therefore, the alternation of an amino acid residue at the 82nd position on red region not only demonstrates OspC polymorphism, but also points out the probability for turning invasive strains to non-invasive strains.<ref name=kumaran>PMID:11230121</ref>

<h3>Lyme Disease and Ecology</h3>

[[Image:Life cycle of tick.png|300px|right|thumb|Life cycle of tick.[[http://www.cdc.gov/ticks/life_cycle_and_hosts.html]]]]

The number of reported cases of Lyme disease is increasing annually in highly focused geographic locations of the United States (CITE). The occurrence of Lyme disease is dependent upon the abundance of ticks infected with ''B. burgdorferi'' in natural ecosystems. Ticks are born without ''B. burgdorferi'' and acquire the bacteria while feeding on the blood of natural reservoir hosts such as mice, squirrels, shrews and other small vertebrates (Figure XX). After growth and development, the infected nymphal ticks can transmit ''B. burgdorferi'' to incidental vertebrates, including humans. The ecological interaction between the competence of reservoir hosts and the ticks is an underlying measure of human Lyme disease risk.<ref>PMID: 12525705</ref>

<h3>Ecological factors responsible for human Lyme disease risk</h3>

* '''Vertebrate Community Composition'''<ref>PMID:15514047</ref>: Two types of environment that the vertebrate hosts reside, which is also called vertebrate host density are interspecific community, which involves organisms of different species and intraspecific community, which is composed of organisms of same species. The hosts living in the community within different species or same species strongly affects the proportion of infected nymphal tick that can cause human Lyme disease.
* '''Distribution frequency of particular oMGs'''<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>: After taking blood meal from their hosts, the proportion of host-seeking nymphs infected with each oMG differs among oMGs. As only four types of oMGs (A, B, I and K) are responsible for systemic human Lyme disease, the host-seeking nymphs that have high distribution frequency of four invasive oMGs is one of the standard measures of human Lyme disease risk.
* '''Transmission Probability'''<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>: The transmission probability of each oMGs from individual species differs. The higher the transmission probability of a particular oMG from vertebrate host, the higher the chance of carrying that particular oMG by the ticks after receiving blood meal from their hosts is. Thus, it is one of the parameters that contributes the prevalence of human Lyme disease.

<h3>Using Ecological Models to Predict Lyme Disease Risk</h3>

[[Image:Lyme_Disease_Risk_Map.gif|300px|right|thumb|Map illustrating prevalence of Lyme disease in the Untied States by CDC.[[http://www.cdc.gov/mmwr/preview/mmwrhtml/rr4807a2.htm]]]]

Conceptual and mathematical models have been developed by researchers to characterize the ecological interaction between vertebrate host community and distribution frequency of invasive oMGs and predict the cases of human Lyme disease. In one model, the principal natural reservoir host used in the model for the epidemic of Lyme disease in northeastern and central United States is the presence of the white-footed mice (''Peromyscus leucopus'') population, which has both high frequency distribution in all four human infectious oMGs and high transmission probabilities of oMGs A, B, I and K.<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref> Ticks are least likely to parasitize inefficient reservoir hosts, thereby increasing high infection prevalence in the tick population, which enhances the risk of exposure of Lyme disease in humans. Many studies have found support for this "dilution-effect model" which proposes that maintaining high diversity of vertebrate host community may dilute the power of one host, such as the white-footed mouse by increasing the degree of specialization of ticks on inefficient hosts. This model strongly demonstrates the relationship between species diversity in the community of hosts and the risk of human exposure to Lyme disease. These ecological driving forces described in the model are useful tools in predicting the prevalence and risk of human Lyme disease.

<h3>OspC-based vaccine against Lyme disease</h3>

An OspC-based vaccine against Lyme disease is currently being developed. Because of the variability of OspC, the recombinant OspC vaccine, targeting the antigenic site of one specific OspC type is ineffective for ''B. burgdorferi'' with different OspC types. Therefore, the development of vaccine that recognizes the antigenic determinant on the variable regions of multiple OspC types is required in order to effectively activate human immune response. Based on the mapping of epitope-containing regions from oMGs: A, B, K and D, the experiment-based tetravalent chimeric vaccine is being developed to tigger anti-ABKD response. <ref>Christopher G. Earnhart, Eric L. Buckles, Richard T. Marconi. "Development of an OspC-based tetravalent, recombinant, chimeric vaccinogen that elicits bactericidal antibody against diverse Lyme disease spirochete strains, Vaccine." 25(3) 466-480 (2007). [http://dx.doi.org/10.1016/j.bbr.2011.03.031 DOI: 10.1016/j.vaccine.2006.07.052]</ref> Taking advantage of tetravalent ABKD construct, octavalent chimeric vaccine also known as OspC-A8.1, recognizing additional epitopes of oMGs: C, E, N and K, has been tested in mice. <ref name='protein'>PMID:17921702</ref>

== Antibodies to Osp A and Osp B and Lyme Disease ==

[[Image:Fab reigon.png|right|thumb|Digestion of an antibody by Papain separates the fab reigons from the antibody]]
A factor contributing to the severity of Lyme disease is its resistance to certain forms of complement-dependent immune response by the evasion of the [http://en.wikipedia.org/wiki/Alternative_complement_pathway alternative complement pathway] and the blocking of complement [http://en.wikipedia.org/wiki/Complement_component_3 C3].<ref>PMID:18080415</ref> This resistance increases the importance of the complement independent immune response when combating ''B. burgdorferi''. Certain fragment antigen binding regions ([http://en.wikipedia.org/wiki/Fragment_antigen-binding fab]) of IgG and IgM monoclonal antibodies (mAbs) are bactericidal even in the absence of complement. Binding of these fabs to their corresponding outer surface protein (OspA and OspB) of ''B. burgdoferi'' leads to the lysis of the bacteria.<ref name="connolly">PMID: 15864264</ref>

<h3>Fragment Antigen Binding (fab)</h3>
Fab consists of a [http://en.wikipedia.org/wiki/Immunoglobulin_heavy_chain heavy chain] and [http://en.wikipedia.org/wiki/Immunoglobulin_light_chain light chain] and each chain is composed of a variable and a constant region. The [http://en.wikipedia.org/wiki/Paratope paratope] is located in the N terminal of the variable region of the heavy and light chains of the fab. H6831 and CB2 are IgG mAbs that targets the C-terminal of OspB and LA-2 is an IgM mAb that targets the C-terminal of OspA.<ref>PMID:107164</ref>

<h3>OspB and H6831</h3>

<h4>Interaction between OspB and H6831</h4>
<table width='450' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1RJL' size='400' frame='true' align='center' scene='Studio:G1SecL01/1/10' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''OspB-H6831 Complex''' (<scene name='Studio:G1SecL01/1/10'>Initial Scene</scene>)<br>
<scene name='Studio:G1SecL01/1/11'>OspB</scene>''':''' <scene name='Studio:G1SecL01/1/31'>Loop 1</scene> <scene name='Studio:G1SecL01/1/32'>(w/ His 52)</scene> ·· <scene name='Studio:G1SecL01/1/33'>Loop 2</scene> ·· <scene name='Studio:G1SecL01/1/34'>Lysine 253</scene> <scene name='Studio:G1SecL01/1/35'>(CPK coloring)</scene> ·· <scene name='Studio:G1SecL01/1/36'>Loop 3</scene> <br>
<scene name='Studio:G1SecL01/1/12'>H6831</scene>''': '''
<scene name='Studio:G1SecL01/1/14'>Heavy Chain</scene> ·· <scene name='Studio:G1SecL01/1/13'>Light Chain</scene> ·· <scene name='Studio:G1SecL01/1/37'>Tyr-Trp-Glu-His</scene> <br> <scene name='Studio:G1SecL01/1/30'>OspB-H6831 Complex</scene>''': ''' <scene name='Studio:G1SecL01/1/38'>Residues 218-220</scene><br>
<scene name='Studio:G1SecL01/3/2'>OspB Unbound</scene>''':''' <scene name='Studio:G1SecL01/3/3'> Central β Sheet Strands 1-4 </scene>

</center></td></tr></table>
<scene name='Studio:G1SecL01/1/10'>Restore Original Scene</scene>

The <scene name='Studio:G1SecL01/1/30'>OspB-H6831 complex</scene> consist of two components, the outer surface protein <scene name='Studio:G1SecL01/1/11'>OspB</scene> and the <scene name='Studio:G1SecL01/1/12'>fab</scene>, which is subdivided into the <scene name='Studio:G1SecL01/1/14'>heavy chain</scene> and the <scene name='Studio:G1SecL01/1/13'>light chain</scene>. Most hydrogen bonds and electrostatic interactions that are responsible for the binding of H6831 to OspB are between the <scene name='Studio:G1SecL01/1/15'>three adjacent surface-exposed loops</scene> at the C-terminal of OspB and some <scene name='Studio:G1SecL01/1/37'>residues on the fab heavy chain</scene> that include tyrosine, tryptophan, glutamate, and histidine.<ref name=becker>PMID:15713683</ref>

The majority of hydrogen bonds and electrostatic interactions are between <scene name='Studio:G1SecL01/1/33'>Loop2</scene> (residues 250-254) and the fab heavy chain. <scene name='Studio:G1SecL01/1/34'>Lys 253</scene> in loop 2 of OspB has a necessary and major role due to its central position in the exposed loops. A mutation at its position abrogates the binding interaction and causes the resistance of the bacteria to the bactericidal effect of the fab. Lys 253 interacts with the two aromatic residues on the fab heavy chain, tyrosine and tryptophan. It also makes hydrogen bonds with the glutamate 50 in the heavy chain of the fab and forms an ionic bond. Carbonyl in <scene name='Studio:G1SecL01/1/31'>loop 1</scene> of the OspB interacts with <scene name='Studio:G1SecL01/1/32'>histidine 52</scene> in the fab heavy chain. <scene name='Studio:G1SecL01/1/36'>Loop 3</scene> of OspB interacts with fab light chain.<ref name=becker />

<h4>Structural changes to OspB in the complexed form</h4>
The binding of H6831 to OspB leads to some conformational changes in OspB compared to its <scene name='Studio:G1SecL01/3/2'> unbound form </scene>. [http://en.wikipedia.org/wiki/Crystallography Crystallography] has shown that the most significant difference is the loss of the <scene name='Studio:G1SecL01/3/3'>central β sheet strands 1-4 </scene>.<ref name=becker /> The loss of these β sheets may be due to conformational change as a result of the binding or a disorder that could have occurred during a crystallization of the complex. Both small positional shifts near the fab binding site and a few larger structural changes away from the binding site were observed. The largest shifts (7– 8 Å) correspond to the repositioning of a loop opposite the fab-binding site <scene name='Studio:G1SecL01/1/38'>at residues 218-220</scene>. In the free OspB structure, all regions that exhibit shifts are adjacent to the central sheet; in the OspB-H6831 complex they all shift toward, and slightly overlap the position of the missing sheet.

<h4>Bactericidal action</h4>
The fab binding destabilizes the [http://en.wikipedia.org/wiki/Bacterial_outer_membrane outer membrane] (OM) of B. burdorferi, with subsequent formation of [http://en.wikipedia.org/wiki/Spheroplast spheroplasts]. It has been observed that the bactericidal action, but not the binding, requires the presence of divalent cations (Mg<sup>2+</sup> and Ca<sup>2+</sup>), and
fab is unable to clear bacteria in the absence of these cations.<ref name=ding /> It is speculated that OspB-Cb2 (a fab similar to H6831) complexes could lead to the lysis of the cell by creating physical openings in the OM, allowing for rapid infusion of electrolytes and increasing the [http://en.wikipedia.org/wiki/Osmolarity osmolarity] of the [http://en.wikipedia.org/wiki/Periplasm periplasm].<ref name="escudero">PMID:9125579</ref>

<h3>OspA and LA-2</h3>

<table width='450' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='400' frame='true' align='center' scene='Studio:G1SecL01/2/1' />
</td></tr><tr><td bgcolor='#eeeeee'><center>'''OspA-LA2 Complex''' (<scene name='Studio:G1SecL01/2/1'>Initial Scene</scene>)<br>
<scene name='Studio:G1SecL01/2/1'>OspA-LA2 Complex</scene>''':''' <scene name='Studio:G1SecL01/2/2'>Three Loops</scene> '''··''' <scene name='Studio:G1SecL01/2/3'>Ser 206</scene>
</center></td></tr></table>

<h4>OspA</h4>
<scene name='Studio:G1SecL01/2/1'>Restore Original Scene</scene>

OspA is usually undetectable in the early stages of Lyme disease, and is down regulated when OspC is expressed.<ref name=kumaran>PMID:11230121</ref> OspA is 53% similar to OspB. Despite their similarity, OspB is susceptible to cleavage by exogenous [http://en.wikipedia.org/wiki/Protease proteases] both ''in vivo'' and ''in vitro'', whereas OspA is relatively resistant in both cases.<ref name=becker />

OspA is used in adhering to the tick's gut by binding with the tick receptor for OspA (TROSPA), a receptor necessary for the colonization of <i>Borrelia</i> inside the tick. High levels of both OspA and TROSPA are found prior to feeding, but are downregulated once the feeding process begins in order to initiate transmission into the host.<ref name="pal">PMID: 15537536</ref>
</p>

<h4>Interaction between OspA and LA-2</h4>
LA-2 is an IgM murine monoclonal antibody that interacts with <scene name='Studio:G1SecL01/2/2'> three exposed loops </scene> on the C-terminal of OspA. These interactions include eight direct [[hydrogen bonds]], four solvent-bridged hydrogen bonds, three ion pairs, and numerous van der Waals interactions.<ref name=ding />

<h4>Structural changes to OspA in the complexed form</h4>
Conformational changes upon the binding of OspA and LA-2 show that LA-2 recognition of OspA involves an induced fit mechanism where the conformations of loops 1-3 shift to optimize complementarity to the antigen-combining site.<ref name=ding /> The overall structure of the C-terminal of OspA is unchanged upon the binding of LA-2 with comparison to the free OspA. The maximum atomic shift is 4.7Å at the site of <scene name='Studio:G1SecL01/2/3'>Ser 206</scene>.<ref name=ding>PMID: 11183781</ref>

<h3>Medical Application</h3>

''B. burgdoferi'' is able to escape human immune response because the outer surface proteins, to which the immune system responds, are variable. The effectiveness of different Osp molecules as [http://en.wikipedia.org/wiki/Vaccine vaccines] can vary depending on their variability. OspC and OspB are highly polymorphic, with variability of OspC observed between strains collected from within a single geographical area.<ref name=kumaran>PMID:11230121</ref> Observed variations of OspB in ''B. burgdoferi'' (including its absence from the bacteria) are not accounted for by major [http://en.wikipedia.org/wiki/Dna DNA] arrangements or failure in [http://en.wikipedia.org/wiki/Transcription_(genetics) transcription] of the OspB gene. This indicates that the OspB gene may code for a variety of proteins, making OspB a poor candidate for use in vaccines.<ref>PMID:2668185</ref> OspA is the most conserved; of the three exposed loops, only loop 1 is variable while loops 2 and 3 are conserved. This makes OspA a more consistent antigen (compared to OspB and OspC) for the immune system to target and usable as a vaccine to Lyme disease.<ref name=ding />

== Osp A and Lyme Disease ==

<Structure load="1fj1" size="350" frame="true" align="right" name="OspA-secondary" caption="Outer surface protein A (OspA) ([[1fj1|1FJ1]], chain F) showing secondary structures." scene="Studio:G2SecL03/Ospa_default/5"/>
<p>
<b>Outer Surface Protein A (OspA)</b> is a major lipoprotein found on the surface of [http://en.wikipedia.org/wiki/Spirochaete spirochetes] from the genus [http://en.wikipedia.org/wiki/Borrelia <i>Borrelia</i>] and is comprised of 21 anti-parallel β-sheets and a single α-helix. OspA's expression is regulated at different points in time, from being downregulated during the tick's feeding process on its host to being upregulated in the host's cerebrospinal fluid (CSF) to induce inflammatory response, resulting in acute Lyme [http://en.wikipedia.org/wiki/Neuroborreliosis neuroborreliosis]. OspA has also been used as a vector in working towards the development of a vaccine for [http://en.wikipedia.org/wiki/Lyme_disease Lyme disease].
</p>
<p>
While feeding, OspA is downregulated in order to evade an immune response from the incoming host blood into the gut, releasing the <i>Borrelia</i> from the gut wall and migrating into the tick's salivary glands, thereby allowing it to enter the host through the bite. This is evidenced by the fact that patients with Lyme disease have been found to not possess OspA antibodies in the early stages of the disease.<ref name="connolly">PMID: 15864264</ref><ref name="rupprecht">PMID: 18097481</ref> OspA is the protein most related to acute Lyme neuroborreliosis (LNB), the neurological manifestations of Lyme disease.
</p>

<h3>OspA's Role in Invasion</h3>
<p>
Once inside the host, the <i>Borrelia</i> has a great number of mechanisms available to actively suppress the host's immune system response and neutralize its effector mechanisms, such as the expression of another outer surface protein, OspC, which prevents susceptibility to the host's [http://en.wikipedia.org/wiki/Innate_immune_system innate immunity] and [http://en.wikipedia.org/wiki/Complement_system complement systems]. Additionally, <i>Borrelia</i> is capable of suppressing many of its surface proteins to reduce its detectability, but can also utilize protective means by temporarily expressing them when needed.
</p>

<h4>Acute Lyme Neuroborreliosis (LNB)</h4>
<p>
Acute Lyme Neuroborreliosis (LNB) is part of the second stage of Lyme disease in which the spirochete invades the peripheral and central nervous systems (CNS). Symptoms of LNB include: meningoradiculitis with inflammation of the nerve roots and [http://en.wikipedia.org/wiki/Radicular_pain radiculitis] (Bannwarth’s syndrome), lymphocytic meningitis, and cranial and [http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001619/ peripheral neuritis]. In Europe, the strain predominantly found in the CSF of patients with Bannwarth's syndrome is <i>B. garinii</i>. However, in the United States, Bannwarth's syndrome is rare and the most common manifestations of Lyme neuroborreliosis is [http://en.wikipedia.org/wiki/Meningitis meningitis], caused by <i>B. burgdorferi</i>. The presence of OspA in the cerebrospinal fluid (CSF) is responsible for this complex inflammatory response in the brain that leads to the neuroborreliosis.<ref name="rupprecht">PMID: 18097481</ref>
</p>

<h4>Evasion and the Extracellular Matrix</h4>
<p>
The <i>Borrelia</i> are able to hide in the [http://en.wikipedia.org/wiki/Extracellular_matrix extracellular matrix], allowing it to survive by avoiding [http://en.wikipedia.org/wiki/Leukocytes leukocytes] circulating in the bloodstream. OspA can rapidly bind to plasminogen, which becomes [http://en.wikipedia.org/wiki/Plasmin plasmin] once activated, and degrades the extracellular matrix. By binding to plasminogen, <i>Borrelia</i> could be exploiting its function and utilizing it to invade the extracellular matrix. However, due to the fact that OspA is downregulated during feeding, and stays unexpressed, a different mechanism may be used instead. Additionally, <i>Borrelia</i> induces the local upregulation of matrix metalloproteinase-9, causing the digestion of the surrounding extracellular matrix. <i>Borrelia</i> can also bind to several proteins in the extracellular matrix, such as [http://en.wikipedia.org/wiki/Fibronectin fibronectin], [http://en.wikipedia.org/wiki/Integrins integrins] or [http://en.wikipedia.org/wiki/Decorin decorin], which can aid in the spread and survival of the spirochetes in these tissues.<ref name="rupprecht">PMID: 18097481</ref>
</p>

<h4>Migration Across the Blood-Brain Barrier</h4>
<p>
It is not fully understood how <i>Borrelia</i> get past the [http://en.wikipedia.org/wiki/Blood-brain_barrier blood-brain barrier], though some researchers suggest a paracellular route, which involves a process using transient tether-type associations, short-term dragging interactions, and stationary adhesion. There is evidence that <i>Borrelia</i> utilizes OspA in the transient tethering stage. The blood-brain barrier is composed of brain microvascular endothelial cells, astrocytes, a basement membrane, pericytes, and neurons. OspA is a major adherent molecule to brain microvascular cells by binding to the [[1aly|CD40]] receptors outside, which results in events that are typically seen when leukocytes cross the blood brain barrier.

Activation of CD40 receptors leads to the production of proinflammatory cytokines and enhanced expression of ICAM-1, E-selectin and VCAM-1, resulting in increased cell binding, and the formation of fenestrations due to increased vascular endothelial growth factor, and vascular permeability factor. OspA might be mimicking leukocytes in order to cross the blood-brain barrier. However not all strains of <i>Borrelia</i> can utilize OspA to do this, OspA only contributes about 70% to adherence, and other <i>Borrelia</i> proteins are also needed in this process. It has also been seen that OspA mediates the adhesion of <i>Borrelia</i> to murine neural and glial cell lines. <ref name="pulzova">PMID: 22355605</ref>
</p>

<h4>Role in Inflammation</h4>

[[Image:G2L03-OspA-mechanism.jpg|thumb|300px|Mechanism of the host inflammatory response to OspA]]

There are six steps involved in the host's inflammatory response to OspA: <ref name="rupprecht">PMID: 18097481</ref><br>
<ol>
<li>When the <i>Borrelia</i> enter the host’s CNS they encounter several different types of immune cells such as [http://en.wikipedia.org/wiki/Monocyte monocytes], [http://en.wikipedia.org/wiki/Macrophages macrophages], and [http://en.wikipedia.org/wiki/Dendritic_cells dendritic cells]. While in the CSF, outer surface protein A (OspA) is upregulated and it’s increased expression promotes recognition by a specific receptor on a monocyte.</li>

<li>The OspA-bound monocyte then releases proinflammatory [http://en.wikipedia.org/wiki/Cytokine cytokines] (i.e. [http://en.wikipedia.org/wiki/Interferon interferon]), as well as [http://en.wikipedia.org/wiki/Chemokine chemokines], such as [http://en.wikipedia.org/wiki/CXCL13 CXCL13]. In patients with LNB, there is an observed increase in the levels of these cytokines and chemokines in their CSF. The production of chemokines leads to the recruitment of other immune cells to the site of infection.</li>

<li>[http://en.wikipedia.org/wiki/B_lymphocyte B-lymphocytes] respond to the new concentration gradient of CXCL13 between the blood and CSF and migrate into the CSF.</li>

<li>B-lymphocytes undergo [http://en.wikipedia.org/wiki/Receptor-mediated_endocytosis receptor-mediated endocytosis], consuming the OspA antigens present in the CSF, thereby triggering its activation. The B-lymphocytes then are able to differentiate and mature into [http://en.wikipedia.org/wiki/Plasma_cells plasma cells].</li>

<li>The plasma cells create large quantities of anti-OspA antibodies specific to this strain of <i>Borrelia</i> and release them into the CSF.</li>

<li>The anti-OspA antibodies will then bind to the OspA on the spirochete’s membrane, thus killing the <i>Borrelia</i>.</li>
</ol>

<p>
This process is two-sided in the sense that the OspA aids in the pathogenesis of new symptoms (neuroborreliosis) through the chemokine’s actions, as well as initiating the signaling cascade to destroy itself.
</p>

<h3>OspA Vaccination</h3>

<table width='400' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='350' frame='true' align='center' name='complex' caption='Outer Surface Protein A (Osp A) in complex with the LA-2 Fab antibody ([[1fj1|1FJ1]]).' scene='Studio:G2SecL03/Ospafab-orig/3'/>
</td></tr><tr><td bgcolor='#eeeeee'><center><scene name='Studio:G2SecL03/Ospafab-orig/3' target="complex">Reset model</scene>,<scene name='Studio:G2SecL03/Ospafab-fab/3' target="complex">LA-2</scene> Fab antibody (Bluish regions indicate heavy chains (chains B & D of 1FJ1) and greenish regions indicate light chains (chains A & C of 1FJ1))<scene name='Studio:G2SecL03/Ospafab-ospa/3' target="complex">OspA</scene> proteins in complex with the LA-2 Fab antibody (chains E & F of 1FJ1)<scene name='Studio:G2SecL03/Ospafab-interaction/3' target="complex">Closeup</scene> of the OspA antigen : LA-2 Fab antibody interactions
</center></td></tr></table>

<p>
Risk of developing Lyme disease can be mitigated by staying clear of areas with populations of ticks, wearing proper attire to minimize easily bitten areas of the body, and using insect repellents containing [http://en.wikipedia.org/wiki/DEET DEET] (N,N-diethy-m-toluamide). However, another effective means for prevention could be possible by using an outer surface protein from <i>Borrelia</i> in the creation of a vaccine.<ref name="nigrovic">PMID: 16893489</ref>
</p>
<p>
The membrane composition of <i>Borrelia</i> is abundant in both OspA and OspB, and the two proteins share a 53% similarity in their primary sequences. Both OspA and OspB are expressed in the tick's gut and downregulated during feeding and aid in its survivability; however, OspA is overall less varied and reactive than OspB, which has greater variability.<ref name="becker">PMID: 15713683</ref> The relatively conserved sequence of OspA thus lends itself better to study and application toward the development of a vaccine for a broader range of <i>Borrelia</i> strains in the treatment of Lyme disease than that of OspB. The first vaccine used a purified recombinant form of OspA and functioned in blocking transmission of the spirochetes expressing OspA from tick to host during feeding, killing them while still attached to the tick's gut.<ref name="connolly">PMID: 15864264</ref><ref name="battisti">PMID: 18779341</ref> The vaccine, Lymerix, had shown 76% and 92% effectiveness in separate clinical trials in which patients were treated for two years following a three-dose schedule. However, the vaccination was suspended from use in 2002 when opponents claimed the [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG antibodies] for OspA were associated with the onset of severe chronic arthritis, as well as other side effects affecting immunity.<ref name="connolly">PMID: 15864264</ref><ref name="plotkin">PMID: 21217175</ref> This fact, in conjunction with the desire for a more widespread vaccine treating multiple strains of <i>Borrelia</i>, has spurred research towards a new vaccine.
</p>
<p>
To address the concerns of vaccine with broader protection, creation of a chimera, mixing the OspA of different strains of <i>Borrelia</i> would be ideal. Study of the epitope of <scene name='Studio:G2SecL03/Ospafab-ospa/3' target="complex">OspA</scene> and its <scene name='Studio:G2SecL03/Ospafab-interaction/3' target="complex">interactions</scene> with the murine monoclonal antibody <scene name='Studio:G2SecL03/Ospafab-fab/3' target="complex">LA-2</scene> have proved useful in determining effectiveness of a given vaccine trial as high levels of antibodies in test sera compete against LA-2 for binding with OspA. LA-2 makes direct contact with three exposed loops of the C-terminus of OspA. The recognition of OspA by LA-2 requires an induced fit mechanism where these three loops undergo conformational changes to optimize their interaction in the complex. <ref name="ding">PMID: 11183781</ref>
</p>

<h3>Structure of OspA</h3>

<table width='400' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='350' frame='true' align='center' name='OspA-manip' caption='Outer surface protein A (OspA) ([[1fj1|1FJ1]], chain F).' scene='Studio:G2SecL03/Ospa_3loopscartoon/3'/>
</td>
</tr>
<tr>
<td bgcolor='#eeeeee'>
<center>
<scene name='Studio:G2SecL03/Ospa_3loopscartoon/3' target="OspA-manip">Reset model</scene>,<br><scene name='Studio:G2SecL03/Ospa-3loops/4' target="OspA-manip">Three loops</scene> in C-terminus (close up)<scene name='Studio:G2SecL03/Ospa-loop1/1' target="OspA-manip">including Loop 1</scene> (residues 203-220)<scene name='Studio:G2SecL03/Ospa-loop2/1' target="OspA-manip">, Loop 2</scene> (residues 224-233)<scene name='Studio:G2SecL03/Ospa-loop3/1' target="OspA-manip">and Loop 3</scene> (residues 246-257)<scene name='Studio:G2SecL03/Ospa-3residues-nor/3' target="OspA-manip">Three residues</scene> in C-terminus (Ala208, Ala215 and Asn251 in <i>B. burgdorferi</i>; also hides R-groups). <scene name='Studio:G2SecL03/Ospa-3residues-r/2' target="OspA-manip">Display R-groups</scene> of Ala208, Ala215 and Asn251<scene name='Studio:G2SecL03/Ospa-ala208/1' target="OspA-manip">Alanine (Ala) 208</scene><scene name='Studio:G2SecL03/Ospa-ala215/1' target="OspA-manip">Alanine (Ala) 215</scene><scene name='Studio:G2SecL03/Ospa-asn251/1' target="OspA-manip">Asparagine (Asn) 251</scene><scene name='Studio:G2SecL03/Ospa-3loops3res/1' target="OspA-manip">Three loops and all three residues (with R-groups included)</scene>
</center>
</td>
</tr>
</table>

<p>
OspA is made up of 273 residues over 21 anti-parallel β-sheets and a single α-helix. It's folded conformation is divided into three main sections: a N-terminus "sandwich," a central region comprising of several β-sheets and a C-terminus "barrel" domain.<ref name="ding">PMID: 11183781</ref> The folded regions at its ends are connected by a single β-sheet layer in the middle, giving the protein the unique shape of a dumbell.<ref name="makabe">PMID: 16823038</ref>

There are <scene name='Studio:G2SecL03/Ospa-3loops/4' target="OspA-manip">three loops</scene> at the C-terminus of OspA that are important in binding with the LA-2 Fab antibody, whose interactions provide great insight into vaccine research and effectiveness. These three loops are linearly arranged and form protruding ridge at the C-terminus of OspA. Within these loops, there are <scene name='Studio:G2SecL03/Ospa-3residues-nor/3' target="OspA-manip">three residues</scene> <scene name='Studio:G2SecL03/Ospa-3residues-r/2' target="OspA-manip">(show residue R-groups)</scene> where there are distinct variations between the different strains of <i>Borrelia</i> and serve as potential targets for the creation of a broader vaccine.<ref name="ding">PMID: 11183781</ref> <scene name='Studio:G2SecL03/Ospa-3loops3res/1' target="OspA-manip">(display both the three loops and three residues together)</scene>
</p>
<p>
<scene name='Studio:G2SecL03/Ospa-loop1/1' target="OspA-manip">Loop 1</scene>, (residues 203-220), is important in showing variation amongst the different strains of <i>Borrelia</i> as well as being optimally conformed for binding without steric hindrance. <scene name='Studio:G2SecL03/Ospa-loop2/1' target="OspA-manip">Loop 2</scene> (residues 224-233) and <scene name='Studio:G2SecL03/Ospa-loop3/1' target="OspA-manip">Loop 3</scene> (residues 246-257) are more strongly conserved than Loop 1 but also help to show some variation amongst strains. The LA-2 Fab antibody readily recognizes OspA from <i>B. burgdorferi</i>, but does not recognize that from <i>B. afzelii</i> or <i>B. garinii</i>.

Between <i>B. burgdorferi</i> and <i>B. afzelii</i> genetic sequences are generally invariant, but two residues change between the species: <scene name='Studio:G2SecL03/Ospa-ala208/1' target="OspA-manip">Alanine (Ala) 208</scene> in <i>B. burgdorferi</i> is a Glutamine (Gln) in <i>B. afzelii</i>, and <scene name='Studio:G2SecL03/Ospa-asn251/1' target="OspA-manip">Asparagine (Asn) 251</scene> in <i>B. burgdorferi</i> is an Alanine (Ala) in <i>B. afzelii</i>. <i>B. garinii</i> has more variation and in addition to the previous two differences, having at least one more difference, where <scene name='Studio:G2SecL03/Ospa-ala215/1' target="OspA-manip">Alanine (Ala) 215</scene> in <i>B. burgdorferi</i> is a Lysine (Lys), and sometimes also has a deletion at <i>B. burgdorferi</i>’s Alanine 208. LA-2 and OspA of <i>B. burgdorferi</i> form a tight interface when binding, and the longer Glutamine (Gln) sidechain found in <i>B. afzelii</i> and <i>B. garinii</i> is more difficult to accommodate, causing less binding. A chimera that was weakly recognized by LA-2 was made with parts of loop 1 from <i>B. burgdorferi</i>, and loops 2 and 3 from <i>B. garinii</i>.<ref name="ding">PMID: 11183781</ref> Recently, a different kind of chimera has been made which combined the proximal region of <i>B. burgdorferi</i> and distal region of <i>B. afzelii</i>, and was able to successfully protect mice from both species.<ref name="livey">PMID: 21217174</ref>
</p>

== Osp B and Lyme Disease ==

<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="becker">PMID:15713683</ref>.

<h3>OspB Interaction with Fab of H6831</h3>
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="becker"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/7'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="becker"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="becker"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

<h4>Binding</h4>
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />
H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="becker"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="becker"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="becker"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref name="escudero">PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="becker"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain.

<h3>Potential Mechanism of Lysis</h3>

<h4>Potential Catalytic Triad</h4>

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]
The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/2'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease.

<h4>Potential Oxidative Mechanism</h4>
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement.

== VlsE and Lyme Disease ==

<table width='400' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><applet load='1l8w.pdb' size='400' frame='true' align='right' scene='Studio:G5SecL01/Main_image_vlse/1' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''Variable Major Protein (VMP)-like sequence Expressed''' ([[1l8w]]), resolution 2.3Å
[[Image:VLSE PRIMARY STRUCTURE4343.png|400px]]<br>
<nowiki>*</nowiki><small>'''This representation of VlsE illustrates the only crystal structure available on the [http://www.rcsb.org/pdb/explore/explore.do?structureId=1l8w PDB site]. There are 10<sup>30</sup> possible combinations of the VR.'''</small>
<br></center></td></tr></table>

<h3>Structural Overview</h3>

Variable Major Protein (VMP)-like sequence Expressed ('''VlsE''') is a surface lipoprotein of ''Borrelia burgdorferi''. It undergoes [http://en.wikipedia.org/wiki/Antigenic_variation antigenic variation] seemingly important in evasion of the host’s immune system. In addition, the protein is used for Lyme disease diagnosis. It is composed of four similar subunits each possessing two invariable domains and one variable domain.<ref name="A">PMID:10569796</ref> The variable domain contains six variable regions (VR<sub>1</sub>-VR<sub>6</sub>), and six invariable regions (IR<sub>1</sub>-IR<sub>6</sub>). Research suggests that the protein may exist as a dimer where each monomeric C & N termini neighbor each other forming the membrane proximal portion of the protein, and the variable regions form the membrane distal portion.<ref name="B">PMID:11923306</ref> <ref name="C">PMID:11716485</ref> The invariable regions are largely embedded in the protein and remain relatively unchanged within the host and across strains. The variable regions encompass 37% of the VlsE’s exposed surface area despite comprising only 25% of the protein.<ref name="A" /> <ref name="B" /> However, 50% of the VR surface area is exposed while IR<sub>6</sub>, a strong [http://en.wikipedia.org/wiki/Antigen antigen], exposes just 13.7% of its surface area. This leaves only <scene name='Studio:G5SecL01/Ir_6_4_residues/1'>four amino residues</scene> of the antigenic IR<sub>6</sub> unprotected: lysine-276, glutamine-279, lysine-291, and lysine-294. Thus, it is almost entirely embedded in the protein and <scene name='Studio:G5SecL01/Ir6_embedded/1'>sheilded by the variable regions </scene>.<ref name="B" />

<h3>Antigenicity</h3>

The variable regions undergo a recombination event stimulated by the host’s cytokines and absence of those cytokines results in a decreased bacterial burden.<ref name="D">PMID:11544329</ref> This leads to variation with an estimated 10<sup>30</sup> possible combinations, far exceeding the number of antibodies found in the human immune system. While the VR does exhibit antigenicity, this recombination makes it unlikely that a sufficient amount of a single VR variation will be present in large enough supply to lead to an immunodominant variable region.<ref name="E">PMID:10553085</ref> IR<sub>6</sub>, however, exhibits immunodominance while IR<sub>1</sub>-IR<sub>5</sub> are primarily nonantigenic in humans. Thus, shielding of the immunodominant IR<sub>6</sub> by VR regions not subject to antibody response allows for IR<sub>6</sub> to elicit an immune response while remaining inaccessible to antibody binding. Research suggests that the 26 amino residues of <scene name='Studio:G5SecL01/Ir6_with_epitope/1'>IR6</scene> may function as a single epitope with a central alpha helical core.<ref name="B" /> <ref name="D" /> <ref name="F">PMID:10722641</ref>

<h3>Function in Immune System Evasion</h3>

VlsE is essential to the persistence and virulence of Lyme disease and is upregulated under humoral immune pressure.<ref name="G">PMID:17714442</ref> <ref name="H">PMID:15385475</ref> While the exact mechanism for immune evasion remains unknown, several theories have been put forth. One popular theory maintains that VlsE masks other surface antigens by coating the surface of the bacteria, thereby sterically blocking the antigens from antibody binding. This is similar to other pathogens with variable regions, such as [http://en.wikipedia.org/wiki/Trypanosoma_brucei Trypanosoma brucei], the protozoa responsible for African sleeping sickness and [http://en.wikipedia.org/wiki/Neisseria_gonorrhoeae Neisseria gonorrhea], the bacterial cause of gonorrhea. However, recent studies have cast doubt on this theory. An alternate theory provides that VlsE directly stimulates B cell antibody production independent of T-cells. The robust response elicited is thought to override antibody production against other antigens.<ref name="G" />
<br>

<table width='400' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><applet load='1l8w.pdb' size='400' frame='true' align='right' scene='Studio:G5SecL01/Chart_main/4' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''VlsE''' ([[1l8w]]), resolution 2.3Å (<scene name='Studio:G5SecL01/Chart_main/4'>Original</scene>). <br>
Highlight: <scene name='Studio:G5SecL01/Chart_main/3'>IR6</scene>, <scene name='Studio:G5SecL01/Chart_vr_seagreen/1'>VR</scene>, <scene name='Studio:G5SecL01/Chart_ir1_yellow/1'>IR1</scene>, <scene name='Studio:G5SecL01/Chart_ir2_yellow/1'>IR2</scene>, <scene name='Studio:G5SecL01/Chart_ir3_yellow/1'>IR3</scene>, <scene name='Studio:G5SecL01/Chart_ir4_yellow/1'>IR4</scene>, <scene name='Studio:G5SecL01/Chart_ir5_yellow/1'>IR5</scene>. <br>
<br></center></td></tr></table>

<h3>C<sub>6</sub> Diagnostic Testing</h3>

Throughout the course of the disease, IR<sub>6</sub> produces a strong antibody response that can be identified from early to late phases. Applications in diagnostic testing have been identified as a result of this strong immune response and IR<sub>6</sub>’s relative invariability across strains.<ref name="I">PMID:10565920</ref> <ref name="F" /> A C<sub>6</sub> [http://en.wikipedia.org/wiki/ELISA ELISA] test has been developed which uses a 26 amino acid synthetic peptide, C<sub>6</sub>, containing the IR<sub>6</sub> sequence. Results show 99% specificity and 100% precision with high sensitivity. In fact, OspA vaccination does not influence C6 specificity; therefore, C<sub>6</sub> ELISA tests are valuable diagnostic tools.<ref name="I" /> The CDC currently recommends a [http://www.cdc.gov/lyme/healthcare/clinician_twotier.html two-step test] incorporating first a polyvalent, whole-cell [http://en.wikipedia.org/wiki/Sonicate sonicate] (WCS) [http://en.wikipedia.org/wiki/Immunofluorescence_assay immunofluorescent assay]. If results are positive, this is followed by IgG and IgM WCS [http://en.wikipedia.org/wiki/Western_blot Western blots] to eliminate false positives.<ref name="J">PMID:21865190</ref> Therefore, this one-step ELISA test presents an accurate and economical alternative to the current two-step model.<ref name="I" />

<br><br><br><br><br><br><br><br><br><br><br><br><br>
==References==

<references />

==Teaching at Stony Brook University==

This Proteopedia page is the product of a new introductory biology laboratory started in the spring of 2012 at Stony Brook University. Undergraduates model and print tactile 3-dimensional proteins involved in Lyme disease in order to understand and interpret contemporary structural biology research. The best student-authored summaries from spring and summer 2012 were selected for this Proteopedia page, thereby connecting students to scientists and facilitating further research experiences.<br>

Author contributions:<br>
Osp C: Irene Chen, Khine Tun<br>
Antibodies to Osp A and B: Safa Abdelhakim, Alexandros Konstantinidis, Philip J. Pipitone, Christopher Smilios<br>
Osp A: Jenny Kim Kim, Cara Lin, Andrea Mullen, Kimberly Slade<br>
Osp B: Olivia Cheng, Stephanie Maung, Ying Zhao<br>
VlsE: Frank J. Albergo, Rachel Cirineo, Tanya Turkewitz<br>
Editors, teachers: Jeff Ecklund, Joan M. Miyazaki, Christopher Morales, Carol Nicosia, Deborah A. Spikes, Raymond Suhandynata, La Zhong<br>
Technical support: Nancy A. Black, Jameson T. Crowley<br>
Collaborating research scientists, editors: Jorge L. Benach, Timothy J. LaRocca<br>
Course co-developer, writer, editor: Niamh B. O'Hara<br>
Course director, course co-developer, writer, editor: Marvin H. O'Neal III<br>
Supported by: Howard Hughes Medical Institute 52006940

==Proteopedia Page Authors==
[http://proteopedia.org/wiki/index.php/User:Safa_Abdelhakim Safa Abdelhakim],
[http://proteopedia.org/wiki/index.php/User:Frank_J._Albergo Frank J. Albergo],
[http://proteopedia.org/wiki/index.php/User:Irene_Chen Irene Chen],
[http://proteopedia.org/wiki/index.php/User:Olivia_Cheng Olivia Cheng],
[http://proteopedia.org/wiki/index.php/User:Rachel_Cirineo Rachel Cirineo],
[http://proteopedia.org/wiki/index.php/User:Jenny_Kim_Kim Jenny Kim Kim],
[http://proteopedia.org/wiki/index.php/User:Alexandros_Konstantinidis Alexandros Konstantinidis],
[http://proteopedia.org/wiki/index.php/User:Cara_Lin Cara Lin],
[http://proteopedia.org/wiki/index.php/User:Stephanie_Maung Stephanie Maung],
[http://proteopedia.org/wiki/index.php/User:Christopher_Morales Christopher Morales],
[http://proteopedia.org/wiki/index.php/User:Andrea_Mullen Andrea Mullen],
[http://proteopedia.org/wiki/index.php/User:Niamh_O'Hara Niamh B. O'Hara],
[http://proteopedia.org/wiki/index.php/User:Marvin_O'Neal Marvin H. O'Neal III],
[http://proteopedia.org/wiki/index.php/User:Philip_J._Pipitone Philip J. Pipitone],
[http://proteopedia.org/wiki/index.php/User:Kimberly_Slade Kimberly Slade],
[http://proteopedia.org/wiki/index.php/User:Christopher_Smilios Christopher Smilios],
[http://proteopedia.org/wiki/index.php/User:Raymond_Suhandynata Raymond Suhandynata],
[http://proteopedia.org/wiki/index.php/User:Khine_Tun Khine Tun],
[http://proteopedia.org/wiki/index.php/User:Tanya_Turkewitz Tanya Turkewitz],
[http://proteopedia.org/wiki/index.php/User:Ying_Zhao Ying Zhao],
[http://proteopedia.org/wiki/index.php/User:La_Zhong La Zhong].

Highlighted Proteins of Lyme Disease

2012-09-25T19:06:23Z

Marvin O'Neal:

<font size='4'>Highlighted Proteins of Lyme Disease</font size>
<p>
[http://en.wikipedia.org/wiki/Lyme_disease Lyme disease] is caused by three species of bacteria belonging to the <i>Borrelia</i> genus, with <i>Borrelia burgdorferi</i> being the most common in the US. The bacteria are transmitted via hard-bodied ticks of the [http://en.wikipedia.org/wiki/Ixodidae <i>Ixodidae</i>] family. <i>Borrelia</i> spirochetes are motile, helical bacteria that have many lipoproteins exposed on the surfaces of their membranes. Two predominant groups of surface lipoproteins are classified as the outer surface proteins (Osps) and the variable major protein-like sequence expressed (VlsE). Both groups of proteins play important roles in pathogenesis, the the life cycle of <i>Borrelia</i>, and eliciting an immune response from the host (Figure 1).<ref name="connolly">PMID: 15864264</ref>
</p>
<p>
In a introductory biology course at Stony Brook University, undergraduates are modeling and exploring <i>Borrelia burgdorferi</i> surface proteins, as well as host produced antibodies to these proteins. This Proteopedia page is the product of their efforts, with a focus on highlighted proteins from five categories: [[#Osp C and Lyme Disease|Osp C]], [[#Antibodies to Osp A and Osp B and Lyme Disease|antibodies to Osp A and Osp B]], [[#Osp A and Lyme Disease|Osp A]], [[#Osp B and Lyme Disease|Osp B]], and [[#VlsE and Lyme Disease|VlsE]].
</p>
<p>
The goal of this Proteopedia page is to describe Lyme disease from a structural biology perspective. What do <i>Borrelia burgdorferi</i> outer surface proteins look like? How does the structure/function of these proteins relate to the infection cycle of <i>Borrelia burgdorferi</i>? What are the structural targets of the human immune system and how have these targets evolved? What are the ideal structural targets for a vaccine to protect against Lyme disease?
</P>
[[Image:BorreliaGeneExpressionCycle.png|500px|right|thumb|Borrelia Gene Expression Over Life Cycle.]]

== Osp C and Lyme Disease ==

<font size='4'>Imporance of OspC in Lyme disease</font size>
[[Image:Spirochetes in infected unfed and feeding nymph.png|300px|right|thumb|Migration of infected nymph from midgut to salivary glands]]

OspC, one of the major outer surface proteins of ''B. burgdoferi'', plays a pivotal role in transmission of ''B. burgdoferi'' from the tick vector to mammalian host. The protein gets upregulated when the tick feeds, allowing for the ''B. burgdoferi'' to adhere to the tick's saliva and move to the tick's mouth and into the host.<ref>PMID:14981110</ref> The upregulation of OspC is accompanied by a downregulation of OspA and OspB, which is thought to be induced by changes in environmental temperature and pH. <ref>PMID:14981110</ref>

OspC is a highly variable protein and strains of ''B. burgdoferi'' are classified according to the sequence of the OspC locus into 19 outer surface major groups (oMGs), denoted by type A to S, only four of which are invasive (disease causing).<ref name=kumaran>PMID:11230121</ref> Polymorphism of OspC and abundance of invasive strains in a population of ''B. burgdoferi''are driven by ecological factors, such as host mammalian community composition, and is a determinant of human Lyme disease risk<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>.

Researchers are attempting to take advantage of the presence of OspC on Borrelia's surface, while the bacteria is in the host, to develop an OspC-based vaccine. However, development of OspC-based vaccination has presented difficulties due to the highly variable nature of OspC. <ref name='protein'>PMID:17921702</ref>

<h3>Exploring the Structure of OspC</h3>
{{STRUCTURE_1ggq| PDB=1ggq | SCENE=Studio:G4SecL04/Dimer_with_mg/1 }}
The model presented to the right is the B31 strain (residues 38-201), which is also known as oMG A. This is one of four invasive oMGs that are responsible for systematic Lyme disease. In crystal structure, OspC exists as a dimer with the coordination of divalent ion, which is modeled as a magnesium ion. Each subunit is predominantly helical, consisting of five parallel
<scene name='Studio:G4SecL04/Helix_blue_in_ribbon/1'> α-helices </scene>
, two short antiparallel
<scene name='Studio:G4SecL04/Sheet_yellow_in_ribbon/1'> β-sheets</scene>
and six
<scene name='Studio:G4SecL04/Random_coils/2'>random coils</scene>
. The '''N and C termini''' at the membrane proximal end of two long alpha helices,
<scene name='Studio:G4SecL04/Alpha_1_with_pointer/1'>α1</scene> (residues 38-76) and
<scene name='Studio:G4SecL04/Alpha_5_with_pointer/1'>α5</scene> (residues 170-201) are in close proximity to each other. At the membrane distal end, there are three remaining alpha helices,
<scene name='Studio:G4SecL04/Alpha_2_with_pointer/1'>α2</scene> (residues 95-112),
<scene name='Studio:G4SecL04/Alpha_3_with_pointer/1'>α3</scene> (residues 121-145), including a short
<scene name='Studio:G4SecL04/Alpha_4_with_pointer/1'>α4</scene> (residues 152-159). At the end of membrane surface, the connection between helices α1 and α2 forms two short anti-parallel β-strands,
<scene name='Studio:G4SecL04/Beta_1_with_pointer/2'>β1</scene> (residues 79-80),and
<scene name='Studio:G4SecL04/Beta_2_with_pointer/1'>β2</scene> (residues 88-89) are formed.

While most of the OspC locus is highly variable, the sequence alignment of all oMGs reveals that towards the membrane proximal end, the surface-exposed residues on α1 and α5 are highly <scene name='Studio:G4SecL04/Conserved_region/1'>conserved</scene>, resulting in a positively charged surface. Other than those on helices, α1 and α5, the surface-exposed residues on the remaining regions of OspC molecule are variable.<ref name= variable>PMID: 11139584 </ref>

{{Template:ColorKey_ConSurf_NoYellow}}

<h3>OspC Structure and Antigenicity</h3>

At the membrane distal region, the six loop regions, including two β-strands illustrates the
<scene name='Studio:G4SecL04/Most_antigenic_site/1'>most antigenic sites</scene>
of OspC due to the presence of variable surface-exposed residues among OspC isolates. <ref>Earnhart C, LeBlanc D, Alix K, Desrosiers D, Radolf J, and Marconi R. 2010. Identification of residues within ligand-binding domain 1 (LBD1) of the ''Borrelia burgdorferi'' OspC protein required for function in the mammalian environment. Molecular Microbiology 76(2): 393-408. [http://dx.crossref.org/10.1111%2Fj.1365-2958.2010.07103.x DOI: 10.1111/j.1365-2958.2010.07103.x]</ref> However, among these variable regions, the outer surface-exposed residues connecting the helices α1 and α2, forming the loops,
<scene name='Studio:G4SecL04/L1/4'>L1</scene> (residues 74-78),
<scene name='Studio:G4SecL04/L2/3'>L2</scene> (residues 81-87),
<scene name='Studio:G4SecL04/L3/3'>L3</scene> (residues 90-93)and two short beta strands, β1 and β2, and also
<scene name='Studio:G4SecL04/L5/3'>L5</scene> (residues 146-150)
are more highly variable than those present in the loops,
<scene name='Studio:G4SecL04/L4/2'>L4</scene> (residues 115-119)and
<scene name='Studio:G4SecL04/L6/3'>L6</scene> (residues 161-169). Consequently, the surface potential of red region that projects away from the membrane is negatively charged and mainly involved in the protein-protein or protein-ligand interactions.<ref name= variable>PMID: 11139584 </ref> Only four types of oMGs (A, B, I and K), whose surface potential in red region is highly negative relative to non-invasive one plays a major role in pathogenesis of human Lyme disease. <ref name=kumaran>PMID:11230121</ref>The residue, <scene name='Studio:G4SecL04/His_82/1'>His82</scene>, located on the red region at the membrane distal end is unique that the replacement of other residues except His82, Lys82, Gln82, which are present only in four invasive oMGs enhances the possibility of turning invasive strains to non-invasive one. Thus, the stronger the electrostatic potential on red region, the higher the chance for OspC to bind with positively charged host ligands. Therefore, the alternation of an amino acid residue at the 82nd position on red region not only demonstrates OspC polymorphism, but also points out the probability for turning invasive strains to non-invasive strains.<ref name=kumaran>PMID:11230121</ref>

<h3>Lyme Disease and Ecology</h3>

[[Image:Life cycle of tick.png|300px|right|thumb|Life cycle of tick.[[http://www.cdc.gov/ticks/life_cycle_and_hosts.html]]]]

The number of reported cases of Lyme disease is increasing annually in highly focused geographic locations of the United States (CITE). The occurrence of Lyme disease is dependent upon the abundance of ticks infected with ''B. burgdorferi'' in natural ecosystems. Ticks are born without ''B. burgdorferi'' and acquire the bacteria while feeding on the blood of natural reservoir hosts such as mice, squirrels, shrews and other small vertebrates (Figure XX). After growth and development, the infected nymphal ticks can transmit ''B. burgdorferi'' to incidental vertebrates, including humans. The ecological interaction between the competence of reservoir hosts and the ticks is an underlying measure of human Lyme disease risk.<ref>PMID: 12525705</ref>

<h3>Ecological factors responsible for human Lyme disease risk</h3>

* '''Vertebrate Community Composition'''<ref>PMID:15514047</ref>: Two types of environment that the vertebrate hosts reside, which is also called vertebrate host density are interspecific community, which involves organisms of different species and intraspecific community, which is composed of organisms of same species. The hosts living in the community within different species or same species strongly affects the proportion of infected nymphal tick that can cause human Lyme disease.
* '''Distribution frequency of particular oMGs'''<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>: After taking blood meal from their hosts, the proportion of host-seeking nymphs infected with each oMG differs among oMGs. As only four types of oMGs (A, B, I and K) are responsible for systemic human Lyme disease, the host-seeking nymphs that have high distribution frequency of four invasive oMGs is one of the standard measures of human Lyme disease risk.
* '''Transmission Probability'''<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref>: The transmission probability of each oMGs from individual species differs. The higher the transmission probability of a particular oMG from vertebrate host, the higher the chance of carrying that particular oMG by the ticks after receiving blood meal from their hosts is. Thus, it is one of the parameters that contributes the prevalence of human Lyme disease.

<h3>Using Ecological Models to Predict Lyme Disease Risk</h3>

[[Image:Lyme_Disease_Risk_Map.gif|300px|right|thumb|Map illustrating prevalence of Lyme disease in the Untied States by CDC.[[http://www.cdc.gov/mmwr/preview/mmwrhtml/rr4807a2.htm]]]]

Conceptual and mathematical models have been developed by researchers to characterize the ecological interaction between vertebrate host community and distribution frequency of invasive oMGs and predict the cases of human Lyme disease. In one model, the principal natural reservoir host used in the model for the epidemic of Lyme disease in northeastern and central United States is the presence of the white-footed mice (''Peromyscus leucopus'') population, which has both high frequency distribution in all four human infectious oMGs and high transmission probabilities of oMGs A, B, I and K.<ref name="Distribution frequency of particular oMGs">PMID:16606995</ref> Ticks are least likely to parasitize inefficient reservoir hosts, thereby increasing high infection prevalence in the tick population, which enhances the risk of exposure of Lyme disease in humans. Many studies have found support for this "dilution-effect model" which proposes that maintaining high diversity of vertebrate host community may dilute the power of one host, such as the white-footed mouse by increasing the degree of specialization of ticks on inefficient hosts. This model strongly demonstrates the relationship between species diversity in the community of hosts and the risk of human exposure to Lyme disease. These ecological driving forces described in the model are useful tools in predicting the prevalence and risk of human Lyme disease.

<h3>OspC-based vaccine against Lyme disease</h3>

An OspC-based vaccine against Lyme disease is currently being developed. Because of the variability of OspC, the recombinant OspC vaccine, targeting the antigenic site of one specific OspC type is ineffective for ''B. burgdorferi'' with different OspC types. Therefore, the development of vaccine that recognizes the antigenic determinant on the variable regions of multiple OspC types is required in order to effectively activate human immune response. Based on the mapping of epitope-containing regions from oMGs: A, B, K and D, the experiment-based tetravalent chimeric vaccine is being developed to tigger anti-ABKD response. <ref>Christopher G. Earnhart, Eric L. Buckles, Richard T. Marconi. "Development of an OspC-based tetravalent, recombinant, chimeric vaccinogen that elicits bactericidal antibody against diverse Lyme disease spirochete strains, Vaccine." 25(3) 466-480 (2007). [http://dx.doi.org/10.1016/j.bbr.2011.03.031 DOI: 10.1016/j.vaccine.2006.07.052]</ref> Taking advantage of tetravalent ABKD construct, octavalent chimeric vaccine also known as OspC-A8.1, recognizing additional epitopes of oMGs: C, E, N and K, has been tested in mice. <ref name='protein'>PMID:17921702</ref>

== Antibodies to Osp A and Osp B and Lyme Disease ==

[[Image:Fab reigon.png|right|thumb|Digestion of an antibody by Papain separates the fab reigons from the antibody]]
A factor contributing to the severity of Lyme disease is its resistance to certain forms of complement-dependent immune response by the evasion of the [http://en.wikipedia.org/wiki/Alternative_complement_pathway alternative complement pathway] and the blocking of complement [http://en.wikipedia.org/wiki/Complement_component_3 C3].<ref>PMID:18080415</ref> This resistance increases the importance of the complement independent immune response when combating ''B. burgdorferi''. Certain fragment antigen binding regions ([http://en.wikipedia.org/wiki/Fragment_antigen-binding fab]) of IgG and IgM monoclonal antibodies (mAbs) are bactericidal even in the absence of complement. Binding of these fabs to their corresponding outer surface protein (OspA and OspB) of ''B. burgdoferi'' leads to the lysis of the bacteria.<ref name="connolly">PMID: 15864264</ref>

<h3>Fragment Antigen Binding (fab)</h3>
Fab consists of a [http://en.wikipedia.org/wiki/Immunoglobulin_heavy_chain heavy chain] and [http://en.wikipedia.org/wiki/Immunoglobulin_light_chain light chain] and each chain is composed of a variable and a constant region. The [http://en.wikipedia.org/wiki/Paratope paratope] is located in the N terminal of the variable region of the heavy and light chains of the fab. H6831 and CB2 are IgG mAbs that targets the C-terminal of OspB and LA-2 is an IgM mAb that targets the C-terminal of OspA.<ref>PMID:107164</ref>

<h3>OspB and H6831</h3>

<h4>Interaction between OspB and H6831</h4>
<table width='450' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1RJL' size='400' frame='true' align='center' scene='Studio:G1SecL01/1/10' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''OspB-H6831 Complex''' (<scene name='Studio:G1SecL01/1/10'>Initial Scene</scene>)<br>
<scene name='Studio:G1SecL01/1/11'>OspB</scene>''':''' <scene name='Studio:G1SecL01/1/31'>Loop 1</scene> <scene name='Studio:G1SecL01/1/32'>(w/ His 52)</scene> ·· <scene name='Studio:G1SecL01/1/33'>Loop 2</scene> ·· <scene name='Studio:G1SecL01/1/34'>Lysine 253</scene> <scene name='Studio:G1SecL01/1/35'>(CPK coloring)</scene> ·· <scene name='Studio:G1SecL01/1/36'>Loop 3</scene> <br>
<scene name='Studio:G1SecL01/1/12'>H6831</scene>''': '''
<scene name='Studio:G1SecL01/1/14'>Heavy Chain</scene> ·· <scene name='Studio:G1SecL01/1/13'>Light Chain</scene> ·· <scene name='Studio:G1SecL01/1/37'>Tyr-Trp-Glu-His</scene> <br> <scene name='Studio:G1SecL01/1/30'>OspB-H6831 Complex</scene>''': ''' <scene name='Studio:G1SecL01/1/38'>Residues 218-220</scene><br>
<scene name='Studio:G1SecL01/3/2'>OspB Unbound</scene>''':''' <scene name='Studio:G1SecL01/3/3'> Central β Sheet Strands 1-4 </scene>

</center></td></tr></table>
<scene name='Studio:G1SecL01/1/10'>Restore Original Scene</scene>

The <scene name='Studio:G1SecL01/1/30'>OspB-H6831 complex</scene> consist of two components, the outer surface protein <scene name='Studio:G1SecL01/1/11'>OspB</scene> and the <scene name='Studio:G1SecL01/1/12'>fab</scene>, which is subdivided into the <scene name='Studio:G1SecL01/1/14'>heavy chain</scene> and the <scene name='Studio:G1SecL01/1/13'>light chain</scene>. Most hydrogen bonds and electrostatic interactions that are responsible for the binding of H6831 to OspB are between the <scene name='Studio:G1SecL01/1/15'>three adjacent surface-exposed loops</scene> at the C-terminal of OspB and some <scene name='Studio:G1SecL01/1/37'>residues on the fab heavy chain</scene> that include tyrosine, tryptophan, glutamate, and histidine.<ref name=becker>PMID:15713683</ref>

The majority of hydrogen bonds and electrostatic interactions are between <scene name='Studio:G1SecL01/1/33'>Loop2</scene> (residues 250-254) and the fab heavy chain. <scene name='Studio:G1SecL01/1/34'>Lys 253</scene> in loop 2 of OspB has a necessary and major role due to its central position in the exposed loops. A mutation at its position abrogates the binding interaction and causes the resistance of the bacteria to the bactericidal effect of the fab. Lys 253 interacts with the two aromatic residues on the fab heavy chain, tyrosine and tryptophan. It also makes hydrogen bonds with the glutamate 50 in the heavy chain of the fab and forms an ionic bond. Carbonyl in <scene name='Studio:G1SecL01/1/31'>loop 1</scene> of the OspB interacts with <scene name='Studio:G1SecL01/1/32'>histidine 52</scene> in the fab heavy chain. <scene name='Studio:G1SecL01/1/36'>Loop 3</scene> of OspB interacts with fab light chain.<ref name=becker />

<h4>Structural changes to OspB in the complexed form</h4>
The binding of H6831 to OspB leads to some conformational changes in OspB compared to its <scene name='Studio:G1SecL01/3/2'> unbound form </scene>. [http://en.wikipedia.org/wiki/Crystallography Crystallography] has shown that the most significant difference is the loss of the <scene name='Studio:G1SecL01/3/3'>central β sheet strands 1-4 </scene>.<ref name=becker /> The loss of these β sheets may be due to conformational change as a result of the binding or a disorder that could have occurred during a crystallization of the complex. Both small positional shifts near the fab binding site and a few larger structural changes away from the binding site were observed. The largest shifts (7– 8 Å) correspond to the repositioning of a loop opposite the fab-binding site <scene name='Studio:G1SecL01/1/38'>at residues 218-220</scene>. In the free OspB structure, all regions that exhibit shifts are adjacent to the central sheet; in the OspB-H6831 complex they all shift toward, and slightly overlap the position of the missing sheet.

<h4>Bactericidal action</h4>
The fab binding destabilizes the [http://en.wikipedia.org/wiki/Bacterial_outer_membrane outer membrane] (OM) of B. burdorferi, with subsequent formation of [http://en.wikipedia.org/wiki/Spheroplast spheroplasts]. It has been observed that the bactericidal action, but not the binding, requires the presence of divalent cations (Mg<sup>2+</sup> and Ca<sup>2+</sup>), and
fab is unable to clear bacteria in the absence of these cations.<ref name=ding /> It is speculated that OspB-Cb2 (a fab similar to H6831) complexes could lead to the lysis of the cell by creating physical openings in the OM, allowing for rapid infusion of electrolytes and increasing the [http://en.wikipedia.org/wiki/Osmolarity osmolarity] of the [http://en.wikipedia.org/wiki/Periplasm periplasm].<ref name="escudero">PMID:9125579</ref>

<h3>OspA and LA-2</h3>

<table width='450' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='400' frame='true' align='center' scene='Studio:G1SecL01/2/1' />
</td></tr><tr><td bgcolor='#eeeeee'><center>'''OspA-LA2 Complex''' (<scene name='Studio:G1SecL01/2/1'>Initial Scene</scene>)<br>
<scene name='Studio:G1SecL01/2/1'>OspA-LA2 Complex</scene>''':''' <scene name='Studio:G1SecL01/2/2'>Three Loops</scene> '''··''' <scene name='Studio:G1SecL01/2/3'>Ser 206</scene>
</center></td></tr></table>

<h4>OspA</h4>
<scene name='Studio:G1SecL01/2/1'>Restore Original Scene</scene>

OspA is usually undetectable in the early stages of Lyme disease, and is down regulated when OspC is expressed.<ref name=kumaran>PMID:11230121</ref> OspA is 53% similar to OspB. Despite their similarity, OspB is susceptible to cleavage by exogenous [http://en.wikipedia.org/wiki/Protease proteases] both ''in vivo'' and ''in vitro'', whereas OspA is relatively resistant in both cases.<ref name=becker />

OspA is used in adhering to the tick's gut by binding with the tick receptor for OspA (TROSPA), a receptor necessary for the colonization of <i>Borrelia</i> inside the tick. High levels of both OspA and TROSPA are found prior to feeding, but are downregulated once the feeding process begins in order to initiate transmission into the host.<ref name="pal">PMID: 15537536</ref>
</p>

<h4>Interaction between OspA and LA-2</h4>
LA-2 is an IgM murine monoclonal antibody that interacts with <scene name='Studio:G1SecL01/2/2'> three exposed loops </scene> on the C-terminal of OspA. These interactions include eight direct [[hydrogen bonds]], four solvent-bridged hydrogen bonds, three ion pairs, and numerous van der Waals interactions.<ref name=ding />

<h4>Structural changes to OspA in the complexed form</h4>
Conformational changes upon the binding of OspA and LA-2 show that LA-2 recognition of OspA involves an induced fit mechanism where the conformations of loops 1-3 shift to optimize complementarity to the antigen-combining site.<ref name=ding /> The overall structure of the C-terminal of OspA is unchanged upon the binding of LA-2 with comparison to the free OspA. The maximum atomic shift is 4.7Å at the site of <scene name='Studio:G1SecL01/2/3'>Ser 206</scene>.<ref name=ding>PMID: 11183781</ref>

<h3>Medical Application</h3>

''B. burgdoferi'' is able to escape human immune response because the outer surface proteins, to which the immune system responds, are variable. The effectiveness of different Osp molecules as [http://en.wikipedia.org/wiki/Vaccine vaccines] can vary depending on their variability. OspC and OspB are highly polymorphic, with variability of OspC observed between strains collected from within a single geographical area.<ref name=kumaran>PMID:11230121</ref> Observed variations of OspB in ''B. burgdoferi'' (including its absence from the bacteria) are not accounted for by major [http://en.wikipedia.org/wiki/Dna DNA] arrangements or failure in [http://en.wikipedia.org/wiki/Transcription_(genetics) transcription] of the OspB gene. This indicates that the OspB gene may code for a variety of proteins, making OspB a poor candidate for use in vaccines.<ref>PMID:2668185</ref> OspA is the most conserved; of the three exposed loops, only loop 1 is variable while loops 2 and 3 are conserved. This makes OspA a more consistent antigen (compared to OspB and OspC) for the immune system to target and usable as a vaccine to Lyme disease.<ref name=ding />

== Osp A and Lyme Disease ==

<Structure load="1fj1" size="350" frame="true" align="right" name="OspA-secondary" caption="Outer surface protein A (OspA) ([[1fj1|1FJ1]], chain F) showing secondary structures." scene="Studio:G2SecL03/Ospa_default/5"/>
<p>
<b>Outer Surface Protein A (OspA)</b> is a major lipoprotein found on the surface of [http://en.wikipedia.org/wiki/Spirochaete spirochetes] from the genus [http://en.wikipedia.org/wiki/Borrelia <i>Borrelia</i>] and is comprised of 21 anti-parallel β-sheets and a single α-helix. OspA's expression is regulated at different points in time, from being downregulated during the tick's feeding process on its host to being upregulated in the host's cerebrospinal fluid (CSF) to induce inflammatory response, resulting in acute Lyme [http://en.wikipedia.org/wiki/Neuroborreliosis neuroborreliosis]. OspA has also been used as a vector in working towards the development of a vaccine for [http://en.wikipedia.org/wiki/Lyme_disease Lyme disease].
</p>
<p>
While feeding, OspA is downregulated in order to evade an immune response from the incoming host blood into the gut, releasing the <i>Borrelia</i> from the gut wall and migrating into the tick's salivary glands, thereby allowing it to enter the host through the bite. This is evidenced by the fact that patients with Lyme disease have been found to not possess OspA antibodies in the early stages of the disease.<ref name="connolly">PMID: 15864264</ref><ref name="rupprecht">PMID: 18097481</ref> OspA is the protein most related to acute Lyme neuroborreliosis (LNB), the neurological manifestations of Lyme disease.
</p>

<h3>OspA's Role in Invasion</h3>
<p>
Once inside the host, the <i>Borrelia</i> has a great number of mechanisms available to actively suppress the host's immune system response and neutralize its effector mechanisms, such as the expression of another outer surface protein, OspC, which prevents susceptibility to the host's [http://en.wikipedia.org/wiki/Innate_immune_system innate immunity] and [http://en.wikipedia.org/wiki/Complement_system complement systems]. Additionally, <i>Borrelia</i> is capable of suppressing many of its surface proteins to reduce its detectability, but can also utilize protective means by temporarily expressing them when needed.
</p>

<h4>Acute Lyme Neuroborreliosis (LNB)</h4>
<p>
Acute Lyme Neuroborreliosis (LNB) is part of the second stage of Lyme disease in which the spirochete invades the peripheral and central nervous systems (CNS). Symptoms of LNB include: meningoradiculitis with inflammation of the nerve roots and [http://en.wikipedia.org/wiki/Radicular_pain radiculitis] (Bannwarth’s syndrome), lymphocytic meningitis, and cranial and [http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001619/ peripheral neuritis]. In Europe, the strain predominantly found in the CSF of patients with Bannwarth's syndrome is <i>B. garinii</i>. However, in the United States, Bannwarth's syndrome is rare and the most common manifestations of Lyme neuroborreliosis is [http://en.wikipedia.org/wiki/Meningitis meningitis], caused by <i>B. burgdorferi</i>. The presence of OspA in the cerebrospinal fluid (CSF) is responsible for this complex inflammatory response in the brain that leads to the neuroborreliosis.<ref name="rupprecht">PMID: 18097481</ref>
</p>

<h4>Evasion and the Extracellular Matrix</h4>
<p>
The <i>Borrelia</i> are able to hide in the [http://en.wikipedia.org/wiki/Extracellular_matrix extracellular matrix], allowing it to survive by avoiding [http://en.wikipedia.org/wiki/Leukocytes leukocytes] circulating in the bloodstream. OspA can rapidly bind to plasminogen, which becomes [http://en.wikipedia.org/wiki/Plasmin plasmin] once activated, and degrades the extracellular matrix. By binding to plasminogen, <i>Borrelia</i> could be exploiting its function and utilizing it to invade the extracellular matrix. However, due to the fact that OspA is downregulated during feeding, and stays unexpressed, a different mechanism may be used instead. Additionally, <i>Borrelia</i> induces the local upregulation of matrix metalloproteinase-9, causing the digestion of the surrounding extracellular matrix. <i>Borrelia</i> can also bind to several proteins in the extracellular matrix, such as [http://en.wikipedia.org/wiki/Fibronectin fibronectin], [http://en.wikipedia.org/wiki/Integrins integrins] or [http://en.wikipedia.org/wiki/Decorin decorin], which can aid in the spread and survival of the spirochetes in these tissues.<ref name="rupprecht">PMID: 18097481</ref>
</p>

<h4>Migration Across the Blood-Brain Barrier</h4>
<p>
It is not fully understood how <i>Borrelia</i> get past the [http://en.wikipedia.org/wiki/Blood-brain_barrier blood-brain barrier], though some researchers suggest a paracellular route, which involves a process using transient tether-type associations, short-term dragging interactions, and stationary adhesion. There is evidence that <i>Borrelia</i> utilizes OspA in the transient tethering stage. The blood-brain barrier is composed of brain microvascular endothelial cells, astrocytes, a basement membrane, pericytes, and neurons. OspA is a major adherent molecule to brain microvascular cells by binding to the [[1aly|CD40]] receptors outside, which results in events that are typically seen when leukocytes cross the blood brain barrier.

Activation of CD40 receptors leads to the production of proinflammatory cytokines and enhanced expression of ICAM-1, E-selectin and VCAM-1, resulting in increased cell binding, and the formation of fenestrations due to increased vascular endothelial growth factor, and vascular permeability factor. OspA might be mimicking leukocytes in order to cross the blood-brain barrier. However not all strains of <i>Borrelia</i> can utilize OspA to do this, OspA only contributes about 70% to adherence, and other <i>Borrelia</i> proteins are also needed in this process. It has also been seen that OspA mediates the adhesion of <i>Borrelia</i> to murine neural and glial cell lines. <ref name="pulzova">PMID: 22355605</ref>
</p>

<h4>Role in Inflammation</h4>

[[Image:G2L03-OspA-mechanism.jpg|thumb|300px|Mechanism of the host inflammatory response to OspA]]

There are six steps involved in the host's inflammatory response to OspA: <ref name="rupprecht">PMID: 18097481</ref><br>
<ol>
<li>When the <i>Borrelia</i> enter the host’s CNS they encounter several different types of immune cells such as [http://en.wikipedia.org/wiki/Monocyte monocytes], [http://en.wikipedia.org/wiki/Macrophages macrophages], and [http://en.wikipedia.org/wiki/Dendritic_cells dendritic cells]. While in the CSF, outer surface protein A (OspA) is upregulated and it’s increased expression promotes recognition by a specific receptor on a monocyte.</li>

<li>The OspA-bound monocyte then releases proinflammatory [http://en.wikipedia.org/wiki/Cytokine cytokines] (i.e. [http://en.wikipedia.org/wiki/Interferon interferon]), as well as [http://en.wikipedia.org/wiki/Chemokine chemokines], such as [http://en.wikipedia.org/wiki/CXCL13 CXCL13]. In patients with LNB, there is an observed increase in the levels of these cytokines and chemokines in their CSF. The production of chemokines leads to the recruitment of other immune cells to the site of infection.</li>

<li>[http://en.wikipedia.org/wiki/B_lymphocyte B-lymphocytes] respond to the new concentration gradient of CXCL13 between the blood and CSF and migrate into the CSF.</li>

<li>B-lymphocytes undergo [http://en.wikipedia.org/wiki/Receptor-mediated_endocytosis receptor-mediated endocytosis], consuming the OspA antigens present in the CSF, thereby triggering its activation. The B-lymphocytes then are able to differentiate and mature into [http://en.wikipedia.org/wiki/Plasma_cells plasma cells].</li>

<li>The plasma cells create large quantities of anti-OspA antibodies specific to this strain of <i>Borrelia</i> and release them into the CSF.</li>

<li>The anti-OspA antibodies will then bind to the OspA on the spirochete’s membrane, thus killing the <i>Borrelia</i>.</li>
</ol>

<p>
This process is two-sided in the sense that the OspA aids in the pathogenesis of new symptoms (neuroborreliosis) through the chemokine’s actions, as well as initiating the signaling cascade to destroy itself.
</p>

<h3>OspA Vaccination</h3>

<table width='400' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='350' frame='true' align='center' name='complex' caption='Outer Surface Protein A (Osp A) in complex with the LA-2 Fab antibody ([[1fj1|1FJ1]]).' scene='Studio:G2SecL03/Ospafab-orig/3'/>
</td></tr><tr><td bgcolor='#eeeeee'><center><scene name='Studio:G2SecL03/Ospafab-orig/3' target="complex">Reset model</scene>,<scene name='Studio:G2SecL03/Ospafab-fab/3' target="complex">LA-2</scene> Fab antibody (Bluish regions indicate heavy chains (chains B & D of 1FJ1) and greenish regions indicate light chains (chains A & C of 1FJ1))<scene name='Studio:G2SecL03/Ospafab-ospa/3' target="complex">OspA</scene> proteins in complex with the LA-2 Fab antibody (chains E & F of 1FJ1)<scene name='Studio:G2SecL03/Ospafab-interaction/3' target="complex">Closeup</scene> of the OspA antigen : LA-2 Fab antibody interactions
</center></td></tr></table>

<p>
Risk of developing Lyme disease can be mitigated by staying clear of areas with populations of ticks, wearing proper attire to minimize easily bitten areas of the body, and using insect repellents containing [http://en.wikipedia.org/wiki/DEET DEET] (N,N-diethy-m-toluamide). However, another effective means for prevention could be possible by using an outer surface protein from <i>Borrelia</i> in the creation of a vaccine.<ref name="nigrovic">PMID: 16893489</ref>
</p>
<p>
The membrane composition of <i>Borrelia</i> is abundant in both OspA and OspB, and the two proteins share a 53% similarity in their primary sequences. Both OspA and OspB are expressed in the tick's gut and downregulated during feeding and aid in its survivability; however, OspA is overall less varied and reactive than OspB, which has greater variability.<ref name="becker">PMID: 15713683</ref> The relatively conserved sequence of OspA thus lends itself better to study and application toward the development of a vaccine for a broader range of <i>Borrelia</i> strains in the treatment of Lyme disease than that of OspB. The first vaccine used a purified recombinant form of OspA and functioned in blocking transmission of the spirochetes expressing OspA from tick to host during feeding, killing them while still attached to the tick's gut.<ref name="connolly">PMID: 15864264</ref><ref name="battisti">PMID: 18779341</ref> The vaccine, Lymerix, had shown 76% and 92% effectiveness in separate clinical trials in which patients were treated for two years following a three-dose schedule. However, the vaccination was suspended from use in 2002 when opponents claimed the [http://en.wikipedia.org/wiki/Immunoglobulin_G IgG antibodies] for OspA were associated with the onset of severe chronic arthritis, as well as other side effects affecting immunity.<ref name="connolly">PMID: 15864264</ref><ref name="plotkin">PMID: 21217175</ref> This fact, in conjunction with the desire for a more widespread vaccine treating multiple strains of <i>Borrelia</i>, has spurred research towards a new vaccine.
</p>
<p>
To address the concerns of vaccine with broader protection, creation of a chimera, mixing the OspA of different strains of <i>Borrelia</i> would be ideal. Study of the epitope of <scene name='Studio:G2SecL03/Ospafab-ospa/3' target="complex">OspA</scene> and its <scene name='Studio:G2SecL03/Ospafab-interaction/3' target="complex">interactions</scene> with the murine monoclonal antibody <scene name='Studio:G2SecL03/Ospafab-fab/3' target="complex">LA-2</scene> have proved useful in determining effectiveness of a given vaccine trial as high levels of antibodies in test sera compete against LA-2 for binding with OspA. LA-2 makes direct contact with three exposed loops of the C-terminus of OspA. The recognition of OspA by LA-2 requires an induced fit mechanism where these three loops undergo conformational changes to optimize their interaction in the complex. <ref name="ding">PMID: 11183781</ref>
</p>

<h3>Structure of OspA</h3>

<table width='400' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><Structure load='1FJ1' size='350' frame='true' align='center' name='OspA-manip' caption='Outer surface protein A (OspA) ([[1fj1|1FJ1]], chain F).' scene='Studio:G2SecL03/Ospa_3loopscartoon/3'/>
</td>
</tr>
<tr>
<td bgcolor='#eeeeee'>
<center>
<scene name='Studio:G2SecL03/Ospa_3loopscartoon/3' target="OspA-manip">Reset model</scene>,<br><scene name='Studio:G2SecL03/Ospa-3loops/4' target="OspA-manip">Three loops</scene> in C-terminus (close up)<scene name='Studio:G2SecL03/Ospa-loop1/1' target="OspA-manip">including Loop 1</scene> (residues 203-220)<scene name='Studio:G2SecL03/Ospa-loop2/1' target="OspA-manip">, Loop 2</scene> (residues 224-233)<scene name='Studio:G2SecL03/Ospa-loop3/1' target="OspA-manip">and Loop 3</scene> (residues 246-257)<scene name='Studio:G2SecL03/Ospa-3residues-nor/3' target="OspA-manip">Three residues</scene> in C-terminus (Ala208, Ala215 and Asn251 in <i>B. burgdorferi</i>; also hides R-groups). <scene name='Studio:G2SecL03/Ospa-3residues-r/2' target="OspA-manip">Display R-groups</scene> of Ala208, Ala215 and Asn251<scene name='Studio:G2SecL03/Ospa-ala208/1' target="OspA-manip">Alanine (Ala) 208</scene><scene name='Studio:G2SecL03/Ospa-ala215/1' target="OspA-manip">Alanine (Ala) 215</scene><scene name='Studio:G2SecL03/Ospa-asn251/1' target="OspA-manip">Asparagine (Asn) 251</scene><scene name='Studio:G2SecL03/Ospa-3loops3res/1' target="OspA-manip">Three loops and all three residues (with R-groups included)</scene>
</center>
</td>
</tr>
</table>

<p>
OspA is made up of 273 residues over 21 anti-parallel β-sheets and a single α-helix. It's folded conformation is divided into three main sections: a N-terminus "sandwich," a central region comprising of several β-sheets and a C-terminus "barrel" domain.<ref name="ding">PMID: 11183781</ref> The folded regions at its ends are connected by a single β-sheet layer in the middle, giving the protein the unique shape of a dumbell.<ref name="makabe">PMID: 16823038</ref>

There are <scene name='Studio:G2SecL03/Ospa-3loops/4' target="OspA-manip">three loops</scene> at the C-terminus of OspA that are important in binding with the LA-2 Fab antibody, whose interactions provide great insight into vaccine research and effectiveness. These three loops are linearly arranged and form protruding ridge at the C-terminus of OspA. Within these loops, there are <scene name='Studio:G2SecL03/Ospa-3residues-nor/3' target="OspA-manip">three residues</scene> <scene name='Studio:G2SecL03/Ospa-3residues-r/2' target="OspA-manip">(show residue R-groups)</scene> where there are distinct variations between the different strains of <i>Borrelia</i> and serve as potential targets for the creation of a broader vaccine.<ref name="ding">PMID: 11183781</ref> <scene name='Studio:G2SecL03/Ospa-3loops3res/1' target="OspA-manip">(display both the three loops and three residues together)</scene>
</p>
<p>
<scene name='Studio:G2SecL03/Ospa-loop1/1' target="OspA-manip">Loop 1</scene>, (residues 203-220), is important in showing variation amongst the different strains of <i>Borrelia</i> as well as being optimally conformed for binding without steric hindrance. <scene name='Studio:G2SecL03/Ospa-loop2/1' target="OspA-manip">Loop 2</scene> (residues 224-233) and <scene name='Studio:G2SecL03/Ospa-loop3/1' target="OspA-manip">Loop 3</scene> (residues 246-257) are more strongly conserved than Loop 1 but also help to show some variation amongst strains. The LA-2 Fab antibody readily recognizes OspA from <i>B. burgdorferi</i>, but does not recognize that from <i>B. afzelii</i> or <i>B. garinii</i>.

Between <i>B. burgdorferi</i> and <i>B. afzelii</i> genetic sequences are generally invariant, but two residues change between the species: <scene name='Studio:G2SecL03/Ospa-ala208/1' target="OspA-manip">Alanine (Ala) 208</scene> in <i>B. burgdorferi</i> is a Glutamine (Gln) in <i>B. afzelii</i>, and <scene name='Studio:G2SecL03/Ospa-asn251/1' target="OspA-manip">Asparagine (Asn) 251</scene> in <i>B. burgdorferi</i> is an Alanine (Ala) in <i>B. afzelii</i>. <i>B. garinii</i> has more variation and in addition to the previous two differences, having at least one more difference, where <scene name='Studio:G2SecL03/Ospa-ala215/1' target="OspA-manip">Alanine (Ala) 215</scene> in <i>B. burgdorferi</i> is a Lysine (Lys), and sometimes also has a deletion at <i>B. burgdorferi</i>’s Alanine 208. LA-2 and OspA of <i>B. burgdorferi</i> form a tight interface when binding, and the longer Glutamine (Gln) sidechain found in <i>B. afzelii</i> and <i>B. garinii</i> is more difficult to accommodate, causing less binding. A chimera that was weakly recognized by LA-2 was made with parts of loop 1 from <i>B. burgdorferi</i>, and loops 2 and 3 from <i>B. garinii</i>.<ref name="ding">PMID: 11183781</ref> Recently, a different kind of chimera has been made which combined the proximal region of <i>B. burgdorferi</i> and distal region of <i>B. afzelii</i>, and was able to successfully protect mice from both species.<ref name="livey">PMID: 21217174</ref>
</p>

== Osp B and Lyme Disease ==

<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="becker">PMID:15713683</ref>.

<h3>OspB Interaction with Fab of H6831</h3>
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="becker"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/7'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="becker"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="becker"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

<h4>Binding</h4>
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />
H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="becker"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="becker"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="becker"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref name="escudero">PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="becker"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain.

<h3>Potential Mechanism of Lysis</h3>

<h4>Potential Catalytic Triad</h4>

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]
The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/2'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease.

<h4>Potential Oxidative Mechanism</h4>
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement.

== VlsE and Lyme Disease ==

<table width='400' align='right' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><applet load='1l8w.pdb' size='400' frame='true' align='right' scene='Studio:G5SecL01/Main_image_vlse/1' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''Variable Major Protein (VMP)-like sequence Expressed''' ([[1l8w]]), resolution 2.3Å
[[Image:VLSE PRIMARY STRUCTURE4343.png|400px]]<br>
<nowiki>*</nowiki><small>'''This representation of VlsE illustrates the only crystal structure available on the [http://www.rcsb.org/pdb/explore/explore.do?structureId=1l8w PDB site]. There are 10<sup>30</sup> possible combinations of the VR.'''</small>
<br></center></td></tr></table>

<h3>Structural Overview</h3>

Variable Major Protein (VMP)-like sequence Expressed ('''VlsE''') is a surface lipoprotein of ''Borrelia burgdorferi''. It undergoes [http://en.wikipedia.org/wiki/Antigenic_variation antigenic variation] seemingly important in evasion of the host’s immune system. In addition, the protein is used for Lyme disease diagnosis. It is composed of four similar subunits each possessing two invariable domains and one variable domain.<ref name="A">PMID:10569796</ref> The variable domain contains six variable regions (VR<sub>1</sub>-VR<sub>6</sub>), and six invariable regions (IR<sub>1</sub>-IR<sub>6</sub>). Research suggests that the protein may exist as a dimer where each monomeric C & N termini neighbor each other forming the membrane proximal portion of the protein, and the variable regions form the membrane distal portion.<ref name="B">PMID:11923306</ref> <ref name="C">PMID:11716485</ref> The invariable regions are largely embedded in the protein and remain relatively unchanged within the host and across strains. The variable regions encompass 37% of the VlsE’s exposed surface area despite comprising only 25% of the protein.<ref name="A" /> <ref name="B" /> However, 50% of the VR surface area is exposed while IR<sub>6</sub>, a strong [http://en.wikipedia.org/wiki/Antigen antigen], exposes just 13.7% of its surface area. This leaves only <scene name='Studio:G5SecL01/Ir_6_4_residues/1'>four amino residues</scene> of the antigenic IR<sub>6</sub> unprotected: lysine-276, glutamine-279, lysine-291, and lysine-294. Thus, it is almost entirely embedded in the protein and <scene name='Studio:G5SecL01/Ir6_embedded/1'>sheilded by the variable regions </scene>.<ref name="B" />

<h3>Antigenicity</h3>

The variable regions undergo a recombination event stimulated by the host’s cytokines and absence of those cytokines results in a decreased bacterial burden.<ref name="D">PMID:11544329</ref> This leads to variation with an estimated 10<sup>30</sup> possible combinations, far exceeding the number of antibodies found in the human immune system. While the VR does exhibit antigenicity, this recombination makes it unlikely that a sufficient amount of a single VR variation will be present in large enough supply to lead to an immunodominant variable region.<ref name="E">PMID:10553085</ref> IR<sub>6</sub>, however, exhibits immunodominance while IR<sub>1</sub>-IR<sub>5</sub> are primarily nonantigenic in humans. Thus, shielding of the immunodominant IR<sub>6</sub> by VR regions not subject to antibody response allows for IR<sub>6</sub> to elicit an immune response while remaining inaccessible to antibody binding. Research suggests that the 26 amino residues of <scene name='Studio:G5SecL01/Ir6_with_epitope/1'>IR6</scene> may function as a single epitope with a central alpha helical core.<ref name="B" /> <ref name="D" /> <ref name="F">PMID:10722641</ref>

<h3>Function in Immune System Evasion</h3>

VlsE is essential to the persistence and virulence of Lyme disease and is upregulated under humoral immune pressure.<ref name="G">PMID:17714442</ref> <ref name="H">PMID:15385475</ref> While the exact mechanism for immune evasion remains unknown, several theories have been put forth. One popular theory maintains that VlsE masks other surface antigens by coating the surface of the bacteria, thereby sterically blocking the antigens from antibody binding. This is similar to other pathogens with variable regions, such as [http://en.wikipedia.org/wiki/Trypanosoma_brucei Trypanosoma brucei], the protozoa responsible for African sleeping sickness and [http://en.wikipedia.org/wiki/Neisseria_gonorrhoeae Neisseria gonorrhea], the bacterial cause of gonorrhea. However, recent studies have cast doubt on this theory. An alternate theory provides that VlsE directly stimulates B cell antibody production independent of T-cells. The robust response elicited is thought to override antibody production against other antigens.<ref name="G" />
<br>

<table width='400' align='left' cellpadding='5'><tr><td rowspan='2'> </td><td bgcolor='#eeeeee'><applet load='1l8w.pdb' size='400' frame='true' align='right' scene='Studio:G5SecL01/Chart_main/4' /></td></tr><tr><td bgcolor='#eeeeee'><center>'''VlsE''' ([[1l8w]]), resolution 2.3Å (<scene name='Studio:G5SecL01/Chart_main/4'>Original</scene>). <br>
Highlight: <scene name='Studio:G5SecL01/Chart_main/3'>IR6</scene>, <scene name='Studio:G5SecL01/Chart_vr_seagreen/1'>VR</scene>, <scene name='Studio:G5SecL01/Chart_ir1_yellow/1'>IR1</scene>, <scene name='Studio:G5SecL01/Chart_ir2_yellow/1'>IR2</scene>, <scene name='Studio:G5SecL01/Chart_ir3_yellow/1'>IR3</scene>, <scene name='Studio:G5SecL01/Chart_ir4_yellow/1'>IR4</scene>, <scene name='Studio:G5SecL01/Chart_ir5_yellow/1'>IR5</scene>. <br>
<br></center></td></tr></table>

<h3>C<sub>6</sub> Diagnostic Testing</h3>

Throughout the course of the disease, IR<sub>6</sub> produces a strong antibody response that can be identified from early to late phases. Applications in diagnostic testing have been identified as a result of this strong immune response and IR<sub>6</sub>’s relative invariability across strains.<ref name="I">PMID:10565920</ref> <ref name="F" /> A C<sub>6</sub> [http://en.wikipedia.org/wiki/ELISA ELISA] test has been developed which uses a 26 amino acid synthetic peptide, C<sub>6</sub>, containing the IR<sub>6</sub> sequence. Results show 99% specificity and 100% precision with high sensitivity. In fact, OspA vaccination does not influence C6 specificity; therefore, C<sub>6</sub> ELISA tests are valuable diagnostic tools.<ref name="I" /> The CDC currently recommends a [http://www.cdc.gov/lyme/healthcare/clinician_twotier.html two-step test] incorporating first a polyvalent, whole-cell [http://en.wikipedia.org/wiki/Sonicate sonicate] (WCS) [http://en.wikipedia.org/wiki/Immunofluorescence_assay immunofluorescent assay]. If results are positive, this is followed by IgG and IgM WCS [http://en.wikipedia.org/wiki/Western_blot Western blots] to eliminate false positives.<ref name="J">PMID:21865190</ref> Therefore, this one-step ELISA test presents an accurate and economical alternative to the current two-step model.<ref name="I" />

<br><br><br><br><br><br><br><br><br><br><br><br><br>
==References==

<references />

==Teaching at Stony Brook University==

This Proteopedia page is the product of a new introductory biology laboratory started in the spring of 2012 at Stony Brook University. Undergraduates model and print tactile 3-dimensional proteins involved in Lyme disease in order to understand and interpret contemporary structural biology research. The best student-authored summaries from spring and summer 2012 were selected for this Proteopedia page, thereby connecting students to scientists and facilitating further research experiences.<br>

Author contributions:<br>
Osp C: Irene Chen, Khine Tun<br>
Antibodies to Osp A and B: Safa Abdelhakim, Alexandros Konstantinidis, Philip J. Pipitone, Christopher Smilios<br>
Osp A: Jenny Kim Kim, Cara Lin, Andrea Mullen, Kimberly Slade<br>
Osp B: Olivia Cheng, Stephanie Maung, Ying Zhao<br>
VlsE: Frank J. Albergo, Rachel Cirineo, Tanya Turkewitz<br>
Editors, teachers: Jeff Ecklund, Joan M. Miyazaki, Christopher Morales, Carol Nicosia, Deborah A. Spikes, Raymond Suhandynata, La Zhong<br>
Technical support: Nancy A. Black, Jameson T. Crowley<br>
Collaborating research scientists, editors: Jorge L. Benach, Timothy J. LaRocca<br>
Course co-developer, writer, editor: Niamh B. O'Hara<br>
Course director, course co-developer, writer, editor: Marvin H. O'Neal III<br>
Supported by: Howard Hughes Medical Institute 52006940

==Proteopedia Page Authors==
[http://proteopedia.org/wiki/index.php/User:Safa_Abdelhakim Safa Abdelhakim],
[http://proteopedia.org/wiki/index.php/User:Frank_J._Albergo Frank J. Albergo],
[http://proteopedia.org/wiki/index.php/User:Irene_Chen Irene Chen],
[http://proteopedia.org/wiki/index.php/User:Olivia_Cheng Olivia Cheng],
[http://proteopedia.org/wiki/index.php/User:Rachel_Cirineo Rachel Cirineo],
[http://proteopedia.org/wiki/index.php/User:Jenny_Kim_Kim Jenny Kim Kim],
[http://proteopedia.org/wiki/index.php/User:Alexandros_Konstantinidis Alexandros Konstantinidis],
[http://proteopedia.org/wiki/index.php/User:Cara_Lin Cara Lin],
[http://proteopedia.org/wiki/index.php/User:Stephanie_Maung Stephanie Maung],
[http://proteopedia.org/wiki/index.php/User:Christopher_Morales Christopher Morales],
[http://proteopedia.org/wiki/index.php/User:Andrea_Mullen Andrea Mullen],
[http://proteopedia.org/wiki/index.php/User:Niamh_O'Hara Niamh B. O'Hara],
[http://proteopedia.org/wiki/index.php/User:Marvin_O'Neal Marvin H. O'Neal III],
[http://proteopedia.org/wiki/index.php/User:Philip_J._Pipitone Philip J. Pipitone],
[http://proteopedia.org/wiki/index.php/User:Kimberly_Slade Kimberly Slade],
[http://proteopedia.org/wiki/index.php/User:Christopher_Smilios Christopher Smilios],
[http://proteopedia.org/wiki/index.php/User:Raymond_Suhandynata Raymond Suhandynata],
[http://proteopedia.org/wiki/index.php/User:Khine_Tun Khine Tun],
[http://proteopedia.org/wiki/index.php/User:Tanya_Turkewitz Tanya Turkewitz],
[http://proteopedia.org/wiki/index.php/User:Ying_Zhao Ying Zhao],
[http://proteopedia.org/wiki/index.php/User:La_Zhong La Zhong].

User:Marvin O'Neal/LymeDisease

2012-08-08T14:59:42Z

Marvin O'Neal: New page: '''Title '''

'''Title '''