Rac1 inactivation by lethal toxin fromClostridium sordelliimodifies focal adhesions upstream of actin depolymerization

Abstract

SummaryInactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT-82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT-82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1 V12. Rac1 inactivation by LT-82 modifies interactions between proteins from AJ and FA complexes as shown by pull-down assays. We showed that in Triton X-100-insoluble membrane proteins from these complexes, namely E-cadherin, b-catenin, p120-catenin and talin, are decreased upon LT-82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we proposed that Rac inactivation by LT-82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization. Introductionc mi_1392 217..232

Rho/Ras-GTPase-dependent and -independent activity of clostridial glucosylating toxins

“…Indeed, the level of talin, a protein that belongs to the protein complex forming FA, decreases in detergent-insoluble membranes of TcsL-82-intoxicated cells compared to that present in control cells. Several AJ proteins, including E-cadherin, b-catenin and p120 catenin, as well as PI4,5P 2 , are also decreased in detergent-insoluble membrane structures of TcsL-82-intoxicated cells compared to control resting cells (Geny et al, 2010). These changes in lipid raft protein content are probably related to modifications in polyphosphoinositides of these membrane structures induced by TcsL-82.…”

“…A Rac pathway involves modifications of PAK-LIM-kinase and cofilin phosphorylation, thereby inhibiting actin depolymerization (Arber et al, 1998;Chen & Macara, 2006;Yang et al, 1998). Since Tcs-82 does not change the expression or phosphorylation of cofilin, this pathway is probably not involved in the toxin process (Geny et al, 2010). However, we have found that TcsL-82 induces a rapid dephosphorylation of paxillin and thus decreases the number of focal adhesions (FAs).…”

“…Paxillin dephosphorylation does not appear to result from inactivation of the Rac major tyrosine kinase, Src or FAK (FA kinase), but rather from an earlier event. Rac inactivation might activate some phosphatase and thereby dephosphorylate proteins from FA complexes such as paxillin (Geny et al, 2010). Several studies have reported the involvement of Rac1 in the control of AJ regulation, probably through reorganization of actin cytoskeleton (Braga et al, 1997(Braga et al, , 1999Fukata & Kaibuchi, 2001;Takaishi et al, 1997).…”

“…Upon cell intoxication with TcsL-82, and thus Rac1 inactivation, interactions between b-catenin, paxillin and talin are markedly decreased, indicating that FA and AJ complexes and their interactions are loosened. Moreover, protein interactions inside FA complexes also appear to be loosened, as the level of talin, which co-immunoprecipitates with paxillin, is decreased noticeably after cell intoxication (Geny et al, 2010). These findings are in agreement with the critical role of Rac1 in FA complex assembly (Guo et al, 2006) and the recent work of the group of Birukov, which demonstrates such an interaction by showing that FA and AJ proteins can be coimmunoprecipitated and that interactions between FA and AJ complexes via paxillin and b-catenin association involve Rac1 as a regulator (Birukova et al, 2007(Birukova et al, , 2008.…”

TcsL Is an Essential Virulence Factor inClostridium sordelliiATCC 9714

“…Collectively, these toxins are monoglucosyltransferases that act by glucosylating and disrupting the functional properties of small Rho GTPases in the host cell cytosol. These enzymes control the cell cycle, apoptosis, transcription, and the structural consequences of actin polymerization, such as cell morphology, migration, and polarity (21,27,29). Injection of purified TcsL into mice reproduces key features of the toxic shock syndrome observed in C. sordelliiinfected humans, with lung vascular endothelial cells being particularly susceptible to the toxin (22).…”