Cellular Biology Of Host/parasite Interactions

National Institute Of Allergy And Infectious Diseases

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Abstract

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 and limited opportunities for genetic knockout. 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. Host proteins critical for regulating intracellular calcium homeostasis have been shown to interact with the inclusion membrane. These include ITPR3 and STIM1 which localize to the inclusion membrane via an uncharacterized interaction. During development, host proteins critical for regulating intracellular calcium (Ca2+) homeostasis interact with the inclusion membrane. The inclusion membrane protein, MrcA, interacts with the inositol-trisphosphate receptor (IP3R), an ER cationic channel that conducts Ca2+. Stromal interaction molecule 1 (STIM1), an ER transmembrane protein important for regulating store-operated Ca2+ entry (SOCE), localizes to the inclusion membrane via an uncharacterized interaction. We therefore examined Ca2+ mobilization in C. trachomatis infected cells. Utilizing a variety of Ca2+ indicators to assess changes in cytosolic Ca2+ concentration, we demonstrate that C. trachomatis impairs host cell SOCE. Ca2+ regulates many cellular signaling pathways. We find that the SOCE-dependent NFAT/calcineurin signaling pathway is impaired in C. trachomatis infected HeLa cells and likely has major implications on host cell physiology as it relates to C. trachomatis pathogenesis possibly by downregulation of chemokine or cytokine production. Obligate intracellular bacteria have evolved multiple means to promote their intracellular survival and replication within the otherwise harsh environment of the eukaryotic cell. Nutrient acquisition and avoidance of cellular defense mechanisms are critical to an intracellular lifestyle. Mitochondria are critical cellular organelles that perform a wide variety of functions including energy production and immune regulation. To perform these diverse functions, mitochondria contain approximately 1,500 proteins, the majority of which are translated in the cytoplasm and translocated to the mitochondria using distinct mitochondria targeting sequences (MTS). It has been shown that bacterial proteins can also contain MTS and localize to the mitochondria. For the obligate intracellular human pathogen, Chlamydia trachomatis, interaction with host cell organelles provides essential nutrients, promotes intracellular replication and aids in maintaining inclusion stability. However, the extent and mechanisms through which Chlamydia interact directly with mitochondria remain unclear. In this study, we investigated the presence of MTS in the C. trachomatis genome and discovered 30 genes with around 70% or greater probability of mitochondrial localization. Five are translocated to the mitochondria upon ectopic expression in HeLa cells. Mass spectrometry of isolated mitochondria from infected cells revealed that two of these proteins localize to the mitochondria during infection. Comparison of mitochondria from infected and uninfected cells suggests that chlamydial infection affects mitochondrial protein composition. Around 125 host proteins were significantly decreased or absent in infected cells. Among these are pro-apoptotic factors and those related to mitochondrial fission/fusion dynamics. Conversely, 82 host proteins were increased in or specific to infected cells, many of which act as anti-apoptotic factors and upregulators of cellular metabolism. These data support the notion that C. trachomatis specifically targets host mitochondria to manipulate cell fate decisions and metabolic function to support pathogen survival and replication.