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.
Highlights
Chlamydia trachomatis is a gram negative, obligate intracellular bacterium
Actin Re-organization Induced by CT166-containing C. trachomatis Serovar D Resembles That Induced by C. difficile Toxin B
To corroborate the hypothesis that CT166 induces actin re-organization HeLa cells were infected with C. trachomatis D and analyzed for actin re-organization
Summary
Chlamydia trachomatis is a gram negative, obligate intracellular bacterium. It causes infections of the eyes, the urogenital tract, or the lungs of newborns. Infections with the serovars D–K range from acute to chronic inflammatory diseases of the urogenital tract with sequelae such as infertility or reactive arthritis. The serovars L1–L3 cause Lymphogranuloma venereum, a more severe sexually transmitted urogenital infection that affects the inguinal lymph nodes. Chlamydiae share a unique developmental cycle: A metabolically inactive, infectious form called the elementary body (EB) enters the host-cell. In a host-derived inclusion it differentiates into its metabolically active form called the reticulate body that multiplies by binary fission. 20 h post infection (p.i.), the reticulate bodies start to differentiate back into a new generation of infectious EBs
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