5umi
Clostridium difficile TcdA-CROPs bound to PA50 Fab
Structural highlights
FunctionTCDA_CLODI Precursor of a cytotoxin that targets and disrupts the colonic epithelium, inducing the host inflammatory and innate immune responses and resulting in diarrhea and pseudomembranous colitis (PubMed:20844489). TcdA and TcdB constitute the main toxins that mediate the pathology of C.difficile infection, an opportunistic pathogen that colonizes the colon when the normal gut microbiome is disrupted (PubMed:19252482, PubMed:20844489). Compared to TcdB, TcdA is less virulent and less important for inducing the host inflammatory and innate immune responses (PubMed:19252482). This form constitutes the precursor of the toxin: it enters into host cells and mediates autoprocessing to release the active toxin (Glucosyltransferase TcdA) into the host cytosol (By similarity). Targets colonic epithelia by binding to some receptor, and enters host cells via clathrin-mediated endocytosis (By similarity). Binding to LDLR, as well as carbohydrates and sulfated glycosaminoglycans on host cell surface contribute to entry into cells (PubMed:1670930, PubMed:31160825, PubMed:16622409). In contrast to TcdB, Frizzled receptors FZD1, FZD2 and FZD7 do not act as host receptors in the colonic epithelium for TcdA (PubMed:27680706). Once entered into host cells, acidification in the endosome promotes the membrane insertion of the translocation region and formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane (By similarity). This activates the peptidase C80 domain and autocatalytic processing, releasing the N-terminal part (Glucosyltransferase TcdA), which constitutes the active part of the toxin, in the cytosol (PubMed:17334356, PubMed:19553670, PubMed:27571750).[UniProtKB:P18177][1] [2] [3] [4] [5] [6] [7] [8] [9] Active form of the toxin, which is released into the host cytosol following autoprocessing and inactivates small GTPases (PubMed:7775453, PubMed:24905543, PubMed:30622517, PubMed:22747490, PubMed:22267739). Acts by mediating monoglucosylation of small GTPases of the Rho family (Rac1, RhoA, RhoB, RhoC, Rap2A and Cdc42) in host cells at the conserved threonine residue located in the switch I region ('Thr-37/35'), using UDP-alpha-D-glucose as the sugar donor (PubMed:7775453, PubMed:24905543, PubMed:30622517, PubMed:22747490, PubMed:22267739). Monoglucosylation of host small GTPases completely prevents the recognition of the downstream effector, blocking the GTPases in their inactive form, leading to actin cytoskeleton disruption and cell death, resulting in the loss of colonic epithelial barrier function (PubMed:7775453). Also able to catalyze monoglucosylation of some members of the Ras family (H-Ras/HRAS, K-Ras/KRAS and N-Ras/NRAS), but with much less efficiency than with Rho proteins, suggesting that it does not act on Ras proteins in vivo (PubMed:30622517).[10] [11] [12] [13] [14] Publication Abstract from PubMedClostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces. Use of a neutralizing antibody helps identify structural features critical for binding of Clostridium difficile toxin TcdA to the host cell surface.,Kroh HK, Chandrasekaran R, Rosenthal K, Woods R, Jin X, Ohi MD, Nyborg AC, Rainey GJ, Warrener P, Spiller BW, Lacy DB J Biol Chem. 2017 Sep 1;292(35):14401-14412. doi: 10.1074/jbc.M117.781112. Epub, 2017 Jul 13. PMID:28705932[15] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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