Actin re-organization induced by Chlamydia trachomatis serovar D--evidence for a critical role of the effector protein CT166 targeting Rac.
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.
Thalmann
Jessica
J
Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany.
Janik
Katrin
K
May
Martin
M
Sommer
Kirsten
K
Ebeling
Jenny
J
Hofmann
Fred
F
Genth
Harald
H
Klos
Andreas
A
D000199:Actins / Q000737:chemistry; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000378:metabolism*; D002692:Chlamydia trachomatis / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D016366:Open Reading Frames; D010641:Phenotype; D011994:Recombinant Proteins / Q000737:chemistry; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000096:biosynthesis; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis
D000199:Actins; D001426:Bacterial Proteins; C107953:RAC1 protein, human; D011994:Recombinant Proteins; C066737:RHOA protein, human; C554842:CT166 protein, Chlamydia trachomatis; D005964:Glucosyltransferases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D005947:Glucose