Glucosyltransferase toxins from clostridia : molecular interactions with cells

Abstract: An enzymatic activity was recently described for the group of Large Clostridial Cytotoxins (LCTs). This activity is glucosyltransfer and consists in the covalent addition of glucose, from UDP-glucose (UDP-Glc) to threonine 35 of small GTPases of the Rho subfamily. To address the pathophysiology of the diseases induced by LCTs, the cytotoxicity on mammalian cells was used as model. A mutant cell line resistant to C. difficile toxin B (TcdB-10463) was generated earlier in order to understand events associated with the intoxication induced by LCTs. The TcdB-10463-induced modification of Rho was shown to be dependent on the dose of added UDP-Glc. The cellular UDP-Glc level was quantified and its level found to be lower in the mutant than in the wild type. TcdB-10463-exposed mutant cells microinjected with the nucleotide recovered the sensitivity demonstrating the low level of UDP-GIc to be responsible for the resistant phenotype. The mutant cell was used to screen for other LCTs' mechanisms of action. It was found to be resistant to TcsL from C. sordellii. This toxin was shown to transfer glucose from UDP-Glc to 21-25 kDa proteins in cell lysates. The modified substrates were identified as the GTPases Ras, Rae and Rap by the use of recombinant purified proteins in an in vitro glucosylation assay. Intoxication of cells with TcsL was shown to inhibit the Ras-dependent activation of MAP kinases, indicating a functional inactivation of Ras in vivo. The cytopathic effect (CPE) induced by a variant toxin B from C difficile (TcdB-1470) was studied. Homology studies indicated that this toxin possesses high similarity with TcdB-10463 in the domain responsible for receptor binding. However, the similarity is significantly lower in the domain that carries the enzymatic activity. TcdB-1470 induces a CPE that resembles more that induced by TcsL than that induced by TcdB-10463. TcdB-1470 was demonstrated to bind the same specific receptor as TcdB-10463, but it glucosylated the same proteins as TcsL, i.e., Ral, Rap and Rac. In addition the small GTP-binding protein R-Ras was identified as a target for TcdB-1470 and TcsL. Transfection of cells with constitutively active R-Ras protected against intoxication by these toxins, indicating that R-Ras is a relevant substrate in vivo. Other substrates did not confer the same degree of protection and R-Ras transfection did not protect against toxins not affecting this GTPase. R-Ras glucosylation induced complete rounding of the cell body and detachment from the substrate, contrasting the arborizing CPE induced by Rho modifying toxins like TcdB-10463 or TcdA. The genesis of these two different types of CPE induced by LCTs was characterized. The arborizing CPE was shown to be dependent on the presence of stress fibers and on the fact that integrins are not inactivated by Rho modifying toxins. The rounded CPE was shown to be dependent on integrin inactivation due to R-Ras modification accompanied by Cdc42 activation resulting in the formation of filopodia. TcdA is known as the C. difficile enterotoxin because it reproduces intestinal pathological events whereas TcdB is referred to as the cytotoxin for being 1000 times more efficient than TcdA in producing a CPE. The reason for the 1000-fold difference in cytotoxicity was explored. TcdB was found to be 100 times more efficient as a glucosyltransferase enzyme than TcdA. In addition, it bound 10 times more efficiently to cell surface receptors. Altogether these data have contributed to understand some aspects of the cytotoxic mechanism exerted by LCTs. The knowledge on bacterial virulence factors is expected to improve current clinical therapies against pathogenic bacteria.