Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a toxin that activates the Rho GTPase.
Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.
Fiorentini
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Department of Ultrastructures, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. MD2573@mclink.it
Fabbri
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A
Flatau
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Donelli
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G
Matarrese
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P
Lemichez
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E
Falzano
L
L
Boquet
P
P
D000199:Actins / Q000096:biosynthesis; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003603:Cytotoxins / Q000494:pharmacology*; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000187:drug effects*; D006801:Humans; D008565:Membrane Proteins / Q000187:drug effects*; D009218:Myosins / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000187:drug effects; D014709:Vero Cells; D020743:rhoB GTP-Binding Protein
D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; C031815:toxB protein, Clostridium difficile; C060296:cytotoxic necrotizing factor type 1; C004717:diacetylmonoxime; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D019204:GTP-Binding Proteins; D009218:Myosins; D020743:rhoB GTP-Binding Protein; D003931:Diacetyl