Mary M. Weber, Laura D. Bauler, Jennifer Lam, Ted Hackstadt

Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a membrane bound vacuole termed the inclusion. Early in the infection cycle, the pathogen extensively modifies the inclusion membrane through incorporation of numerous type III secreted effector proteins, called inclusion membrane proteins (Incs). These proteins are characterized by a bi-lobed hydrophobic domain of 40 amino acids. The presence of this domain has been used to predict up to 59 putative Incs for C. trachomatis, however, localization to the inclusion membrane with specific antibodies has only been demonstrated for about half of those. Here we employed recently developed genetic tools to verify localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of epitope-tagged putative Incs identified 10 which were previously unverified as inclusion membrane localized and thus authentic Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by co-localization with p-Src. Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. Using Yersinia as a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they may not be true Incs. Collectively our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will advance our understanding of how this pathogen maintains its unique intracellular niche and mediates interactions with the host.