B-298. The Protein-Protein Interaction Map of the Chlamydia trachomatis Type Three Secretion System
The obligate intracellular pathogen Chlamydia trachomatis uses a Type III Secretion (TTS) system to deliver effector proteins into the host cytoplasm and the membrane of the pathogen-containing vacuole. A model of the chlamydial TTS apparatus has been proposed based on homologies to enteric TTS systems since there is a lack of genetic tools in Chlamydiae to directly define these components. However, because not all TTS components are conserved in C. trachomatis and several open reading frames (ORFs) of unknown function are co-transcribed with the core components, we predicted that these divergent proteins perform functions important to the assembly and function of the TTS apparatus. In this study, we generated a basic physical map of the C. trachomatis TTS system based on protein-protein interactions identified by yeast two-hybrid analysis. All ORFs associated with the three chromosomal loci encoding putative core TTS components were fused to the yeast Gal4 DNA binding domains (DBD) and activator domains (AD) and co-expressed in yeast cells. Positive chlamydial protein-protein interaction was assessed by expression of reporter genes driven by Gal4 promoters. Overall, our results suggest that predicted interactions among TTS core components are evolutionarily conserved and many ORFs of unknown function co-transcribed with TTS components interact with the secretion apparatus. Next, we expanded our search for TTS-interacting proteins to all Chlamydia-specific ORFs and putative effector proteins. This analysis revealed additional proteins that interacted with the basal ring of the TTS apparatus, with two of these proteins, CT260 and CT700, acting as central nodes of protein-protein interaction clusters. Because proteins within these clusters include known TTS effectors, we postulate that these central node proteins may be TTS secretion chaperones or escorts. Consistent with this, bioinformatic analysis indicates that of these proteins Ct260 shares structural features with known TTS chaperones. Our findings provide a framework for the biochemical analysis of TTS assembly and regulation in this clinically important pathogen.