Difference in F-actin depolymerization induced by toxin B from the Clostridium difficile strain VPI 10463 and toxin B from the variant Clostridium difficile serotype F strain 1470.
Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the causative agent of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis (PMC). TcdB from the C. difficile strain VPI10463 mono-glucosylates (thereby inactivates) the small GTPases Rho, Rac, and Cdc42, while Toxin B from the variant  C. difficile strain serotype F 1470 (TcdBF) specifically mono-glucosylates Rac but not Rho(A/B/C). TcdBF is related to lethal toxin from C. sordellii (TcsL) that glucosylates Rac1 but not Rho(A/B/C). In this study, the effects of Rho-inactivating toxins on the concentrations of cellular F-actin were investigated using the rhodamine-phalloidin-based F-actin ELISA. TcdB induces F-actin depolymerization comparable to the  RhoA-inactivating exoenzyme C3 from C. limosum (C3-lim). In contrast, the  Rac-glucosylating toxins TcdBF and TcsL did not cause F-actin depolymerization. These observations led to the conclusion that F-actin depolymerization depends on the toxin's capability of glucosylating RhoA. Furthermore, the integrity of focal adhesions (FAs) was analyzed using paxillin and p21-activated kinase (PAK) as FA marker proteins. Paxillin dephosphorylation was observed upon treatment of cells with TcdB, TcdBF, or C3-lim. In conclusion, the Rho-inactivating toxins induce loss of cell shape by either F-actin depolymerization (upon RhoA inactivation) or the disassembly of FAs (upon  Rac1 inactivation).
Institute of Toxicology Hannover Medical School, Hannover D-30625, Germany. martinmay@gmx.de
Martin
M
May
Tianbang
T
Wang
Micro
M
Müller
Harald
H
Genth
D000199:Actins / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D015415:Biomarkers / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D045744:Cell Line, Tumor; D048430:Cell Shape / Q000187:drug effects; D016360:Clostridioides difficile / Q000472:pathogenicity / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006031:Glycosylation; D006367:HeLa Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D051419:Paxillin / Q000378:metabolism; D058105:Polymerization / Q000187:drug effects*; D012703:Serotyping; D013045:Species Specificity; D053778:Thiazolidines / Q000494:pharmacology; D054462:p21-Activated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015415:Biomarkers; D019086:Bridged Bicyclo Compounds, Heterocyclic; D051419:Paxillin; D053778:Thiazolidines; C031815:toxB protein, Clostridium difficile; D054462:p21-Activated Kinases; D020742:rhoA GTP-Binding Protein; C037068:latrunculin B