SummaryThe obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N '-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.

SummaryThe obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole and employs a type III secretion mechanism to translocate host-interactive proteins. These proteins most likely contribute to pathogenesis through modulation of host cell mechanisms crucial for the establishment and maintenance of a permissive intracellular environment. Using a surrogate Yersinia type III secretion system (T3SS), we have identified the conserved gene product CT847 as a chlamydial T3SS substrate. Yeast two-hybrid studies using CT847 as bait to screen a HeLa cell cDNA library identified an interaction with mammalian Grap2 cyclin D-interacting protein (GCIP). Immunoblot analyses of C. trachomatis-infected HeLa cells showed that GCIP levels begin to decrease (as compared with mockinfected HeLa cells) between 8 h and 12 h post infection. GCIP was virtually undetectable in 24 h time point material. This decrease was inhibited by proteasome inhibitors lactacystin and MG-132, and the T3SS inhibitor Compound 1. CT847 was detectible in purified reticulate body but not elementary body lysates, and reverse transcription polymerase chain reaction (RT-PCR) expression analyses indicate a mid-cycle expression pattern. Both of these findings are consistent with CT847 contributing to the observed effect on GCIP. Given the established roles of GCIP, we believe that we have discovered a novel C. trachomatis antihost protein whose activity is relevant to chlamydial pathogenesis.

Chlamydia spp. are among the many pathogenic Gram-negative bacteria that employ a type III secretion system (T3SS) to overcome host defenses and exploit available resources. Significant progress has been made in elucidating contributions of T3S to the pathogenesis of these medically important, obligate intracellular parasites, yet important questions remain. Chief among these is how secreted effector proteins traverse eukaryotic membranes to gain access to the host cytosol. Due to a complex developmental cycle, it is possible that chlamydiae utilize a different complement of proteins to accomplish translocation at different stages of development. We investigated this possibility by extending the characterization of C. trachomatis CopB and CopB2. CopB is detected early during infection but is depleted and not detected again until about 20 h postinfection. In contrast, CopB2 was detectible throughout development. CopB is associated with the inclusion membrane. Biochemical and ectopic expression analyses were consistent with peripheral association of CopB2 with inclusion membranes. This interaction correlated with development and required both chlamydial de novo protein synthesis and T3SS activity. Collectively, our data indicate that it is unlikely that CopB serves as the sole chlamydial translocation pore and that CopB2 is capable of association with the inclusion membrane.