Summary Chlamydia trachomatis is a human pathogen associated with significant morbidity worldwide. As obligate intracellular parasites, chlamydiae must survive within eukaryotic cells for sufficient time to complete their developmental cycle. To promote host cell survival, chlamydiae express poorly understood anti-apoptotic factors. Using recently developed genetic tools, we show that three inclusion membrane proteins (Incs) out of eleven examined are required for inclusion membrane stability and avoidance of host cell death pathways. In the absence of specific Incs, premature inclusion lysis results in recognition by autophagolysosomes, activation of intrinsic apoptosis, and premature termination of the chlamydial developmental cycle. Inhibition of autophagy or knockdown of STING prevented host cell death and activation of intrinsic apoptosis. Significantly these findings emphasize the importance of Incs in the establishment of a replicative compartment that sequesters the pathogen from host surveillance systems.

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 bilobed 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 been demonstrated for only about half of them. Here, we employed recently developed genetic tools to verify the localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of epitope-tagged putative Incs identified 10 that 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 colocalization with phosphorylated Src (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 the pathogen maintains its unique intracellular niche and mediates interactions with the host. Chlamydiae are Gram-negative obligate intracellular pathogens that are of human and veterinary importance. Chlamydia trachomatis is a major cause of bacterial sexually transmitted infections and is the causative agent of trachoma, a leading cause of infectious blindness. The species includes at least 15 serovars that differ in disease state and severity, including trachoma (serovars A to C), a sexually transmitted infection (serovars D to K), and a systemic disease referred to as lymphogranuloma venereum (LGV) (serovars L1 to L3) (1). C. pneumoniae is a respiratory pathogen that is a common cause of pneumonia and bronchitis and is associated with chronic diseases, such as atherosclerosis (2). C. psittaci is a zoonotic pathogen associated with a rare but serious pneumonia (3). Other Chlamydia species, including C. muridarum, C. caviae, and C. felis, are associated with other animals, i.e., mice, guinea pigs, and cats, respectively (4-6).All chlamydiae exhibit a biphasic developmental cycle in which the bacteria alternate between an infectious, metabolically dormant elementary body (EB) and the replicative form, referred to as the reticulate body (RB) (7). Upon infection, the EB is internalized by endocytosis into a membrane-bound compartment that is rapidly modified by the pathogen to establish a replicative niche termed the inclusion (8). The inclusion avoids f...

Chlamydia trachomatis is an obligate intracellular pathogen that is the etiological agent of a variety of human diseases, including blinding trachoma and sexually transmitted infections. Chlamydiae replicate within a membrane-bound compartment, termed an inclusion, which they extensively modify by the insertion of type III secreted proteins called Inc proteins. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion of C. trachomatis inclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion in Chlamydia, we generated an incA::bla mutant. Insertional inactivation of incA resulted in the formation of nonfusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively, these results confirm in vitro membrane fusion assays identifying functional domains of IncA and expand the genetic tools available for identification of chlamydia with a method for complementation of site-specific mutants.