Members of the genus Chlamydia are bacterial obligate intracellular parasites of eukaryotic cells. They constitute an important group of pathogenic bacteria that are responsible for multiple medically significant conditions. The species Chlamydia trachomatis is comprised of at least fifteen serologically defined groups or serovars that are associated with human diseases. Trachoma, the world's leading cause of infectious blindness, is caused by serovars A, B, Ba, and C. Chlamydial sexually transmitted disease (STD) is the most common reportable disease in the United States. Serovars D though K are most commonly associated with STDs. The more serious sequelae of these diseases, blindness from trachoma and pelvic inflammatory disease from chlamydial STD, are immunopathological responses to chronic or repeated infections. While trachoma and sexually transmitted infections are primarily localized to the mucosal epithelium, a more systemic infection, lymphogranuloma venereum (LGV), caused by C. trachomatis serovars L1, L2, and L3, is also a sexually transmitted infection that causes inflammation of the inguinal lymph nodes. C. pneumoniae, is a common cause of community acquired pneumonia and is currently of interest due to possible associations with a variety of chronic diseases. C. psittaci is a zoonotic disease that infects many different types of poultry and livestock thus is of economic importance to agricultural industries and is occasionally transmitted to humans. Chlamydiae undergo their entire intracellular developmental cycle within a parasitophorous vacuole, termed an inclusion, that is unique among intracellular parasites. Chlamydiae are endocytosed into a tightly membrane-bound vesicle which grows throughout the developmental cycle to accommodate an increasing number of intracellular bacteria. The chlamydial inclusion, unlike vacuoles containing other intracellular pathogens, is not interactive with endocytic vesicular trafficking pathways but is instead fusogenic with an incompletely understood exocytic pathway which delivers sphingomyelin and cholesterol from the Golgi apparatus to the plasma membrane. Although all species of Chlamydia intersect this pathway, no other intracellular parasites have yet been found to similarly interact with this host vesicular trafficking pathway. Sequestration of chlamydiae within a vesicle that intersects an exocytic pathway is hypothesized to provide a unique, protected intracellular niche in which the chlamydiae replicate. Entry into this pathway is an active process on the part of the chlamydiae as both de novo transcription and translation are required. Virtually all of these interactions are specific and localized to the inclusion. This specificity strongly suggests modification of the exposed inclusion membrane. Examples of cis-acting modifications to the nascent inclusion membrane include: evasion of lysosomal fusion, interactions with microtubules to deliver the nascent inclusion to the peri-Golgi region and microtubule organizing center, initiation of fusion with exocytic vesicular traffic from the Golgi apparatus, and recruitment of, but not fusion with, recycling endosomes containing transferrin and its receptor. Many of these interactions are temporally associated with the exposure of inclusion membrane proteins to the host cell cytoplasm by a chlamydial type III secretion system. C. trachomatis expresses up to fifty predicted inclusion membrane proteins characterized by a long, bilobed hydrophobic domain of approximately 40 amino acids in length. Incs are exposed on the cytosolic face of the inclusion membrane and thus are likely candidates for factors controlling interactions with the host cell. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. Further mechanistic studies aimed at elucidating effector function will further our understanding of how this pathogen maintains its unique intracellular niche and mediates interactions with the host. Chlamydial infection requires the formation of a membrane-bound vacuole, termed the inclusion, that undergoes extensive interactions with select host organelles. The importance of the Inc protein CT229 in the formation and maintenance of the chlamydial inclusion was recently highlighted by studies demonstrating that its absence during infection results in reduced bacterial replication, premature inclusion lysis, and host cell death. Previous reports have indicated that CT229 binds Rab GTPases; however, the physiological implications of this interaction are unknown. Here, we show that CT229 regulates host multivesicular trafficking by recruiting multiple Rab GTPases and their cognate effectors to the inclusion. We demonstrate that CT229 specifically modulates clathrin-coated vesicle trafficking and regulates the trafficking of transferrin and the mannose-6-phosphate receptor, both of which are crucial for proper chlamydial development. This study highlights CT229 as a master regulator of multiple host vesicular trafficking pathways essential for chlamydial infection. Chlamydia trachomatis is an obligate intracellular bacterium that replicates within a vacuole termed an inclusion. At the end of their intracellular developmental cycle, chlamydiae are released either by lysis of the host cell or extrusion of the intact inclusion. The inclusion membrane is extensively modified by the insertion of type III secreted inclusion membrane proteins, Incs, which contribute to inclusion membrane structure and facilitate host-pathogen interactions. An interaction was identified between the inclusion membrane protein, MrcA, and the Ca2+ channel inositol-1,4,5-trisphosphate receptor, type 3 (ITPR3). ITPR3 was recruited and localized to active Src-family-kinase rich microdomains on the inclusion membrane as was the Ca2+ sensor, STIM1. Disruption of MrcA by directed mutagenesis resulted in loss of ITPR3 recruitment and simultaneous reduction of chlamydial release by extrusion. Complementation of MrcA restored ITPR3 recruitment and extrusion. Inhibition of extrusion was also observed following siRNA depletion of host ITPR3 or STIM1. Chlamydial extrusion was also inhibited by the calcium chelator BAPTA-AM. Each of these treatments resulted in a concomitant reduction in phosphorylation of the myosin regulatory light chain (MLC2) and a loss of myosin motor activity at the end of the developmental cycle which is consistent with the reduced extrusion formation. These studies suggest that Ca2+ signaling pathways play an important role in regulation of release mechanisms by C. trachomatis.
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