Actin Re-Organization Induced by Chlamydia trachomatis Serovar D - Evidence for a Critical Role of the Effector Protein CT166 Targeting Rac

Abstract

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166's participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.

Chlamydial Intracellular Survival Strategies

“…Therefore, the entry process is complex, requiring a concerted activation of growth factor receptors, cytoplasmic tyrosine kinases, small GTPases, and TARP to synergistically remodel the actin cytoskeleton and promote bacterial uptake. The actin rearrangements that occur during entry are transient and may be terminated by secreted chlamydial effectors such as CT166, which glucosylates Rac1 (Thalmann et al 2010), or CT694, which interacts and colocalizes with AHNAK, an actin-binding protein (Hower et al 2009). Additional host factors that contribute to uptake into nonphagocytic cells include clathrin (Boleti et al 1999;Hybiske and Stephens 2007a) and cholesterol-rich microdomains (Norkin et al 2001;Jutras et al 2003;Stuart et al 2003;Gabel et al 2004).…”

Rho GTPases as pathogen targets: Focus on curable sexually transmitted infections

“…It is possible that these kinases might function redundantly in the internalization step. 29,37 The last stage in the cascade is probably regulated by another bacterial protein, CT166, which inactivates Rac1 (but not Rho A) via glucosylation, 38 thus completing the activation/ inactivation cycle of Rac1. In summation, this is a complex and tightly regulated process in which diverse bacterial and host proteins play essential roles in the attachment and entry of Chlamydia, in which Rac1 plays an important function throughout the process (Fig.…”

Mutational Analysis of the Chlamydia muridarum Plasticity Zone

“…While high-multiplicity infections were important in associating a toxin-like activity with chlamydial EB, these doses may not be relevant in natural chlamydial disease. Roles for the chlamydial cytotoxins in host cell attachment and entry, vesicle trafficking, and modification of host cell GTPases have been reported (35)(36)(37). The C. muridarum cytotoxins have also been proposed to mediate evasion of the inhibitory effects of IFN-␥ responses in murine cells (12).…”