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. Incs typically are distributed evenly around the periphery of the inclusion membrane. We have recently described a novel structure on the chlamydial inclusion membrane that is enriched with active Src-family kinases, as well as at least four inclusion membrane proteins (IncB, CT101, CT222, and CT850). Inc microdomains are localized at the point of contact of centrosomes with the inclusion membrane and may represent a complex of chlamydial and host proteins that mediates the interactions with dynein to direct migration along microtubule tracks to the MTOC. Src family tyrosine kinases appear to be significant regulators of vesicle trafficking in C. trachomatis infected cells and exhibit multiple different requirements between chlamydial species. We find that one C. trachomatis inclusion membrane protein, CT850, directly interacts with the dynein light chain DYNLT1 (Tctex1) and that this interaction promotes microtubule dependent transport. A yeast 2-hybrid system was used to screen CT850 against a HeLa cell cDNA library and identified strong interactions with DYNLT1. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 inhibits C. trachomatis trafficking to the MTOC. Collectively, the results suggest that CT850 interacts directly with DYNLT1 to promote transport to the MTOC and that this mechanism differs from that of physiological cargo or other intracellular pathogens. Nascent chlamydial inclusions migrate towards the minus end of microtubules and aggregate at the MTOC utilizing the minus-end-directed microtubule motor dynein. This interaction leads to disruption of normal centrosomal positioning leading to centrosome number defects. The association of the chlamydial inclusion membrane with centrosomes leads to failures in centrosomal partitioning and/or cytokinesis.Centrosome supernumerary defects have been implicated in chromosome instability and loss of cell cycle control in early tumors and most aggressive carcinomas. The hypothesis that bacterial infections can contribute to cancer has endured for some time, but unlike viral induced cancers, specific oncogenes and molecular mechanisms have not been clearly established. The interaction of chlamydiae with dynein and centrosomes suggests a mechanism by which chlamydial infection, through induction of abnormal centrosome numbers, may be a contributing factor in chromosome instability ultimately leading to transformation and tumor development. Secreted chlamydial proteins also control localized events such as the recruitment of actin to promote entry. A type III secreted protein, termed Tarp, is translocated and tyrosine phosphorylated while EBs are still extracellular. Tarp has been associated with the actin recruitment which is required for chlamydial internalization. Chlamydial Tarp and the inclusion membrane proteins define at least two distinct stages in chlamydial development where secreted effectors may play important roles in defining the outcome of infection. In the case of Tarp, a pre-existing effector protein is secreted across the plasma membrane from extracellular EBs, while inclusion membrane proteins require de novo synthesis and are secreted across the inclusion membrane from the RBs within. Identification of secreted effector molecules and their functions will continue to provide insights into the many adaptations chlamydiae utilize as successful pathogens. In a yeast two-hybrid screen, an interaction was found between the inclusion membrane protein CT228 and MYPT1, a subunit of myosin phosphatase. MYPT1 was recruited peripherally around the inclusion while the phosphorylated inactive form was localized to active Src-family kinase-rich microdomains. Phosphorylated myosin

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Support Year
23
Fiscal Year
2013
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$1,139,653
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Omsland, Anders; Sixt, Barbara Susanne; Horn, Matthias et al. (2014) Chlamydial metabolism revisited: interspecies metabolic variability and developmental stage-specific physiologic activities. FEMS Microbiol Rev 38:779-801
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Lutter, Erika I; Barger, Alexandra C; Nair, Vinod et al. (2013) Chlamydia trachomatis inclusion membrane protein CT228 recruits elements of the myosin phosphatase pathway to regulate release mechanisms. Cell Rep 3:1921-31
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