Cellular Biology Of Host/parasite Interactions

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Abstract

Chlamydia trachomatis is the etiological agent of several significant diseases of humans including trachoma, the leading cause of infectious blindness worldwide. It is also the most common cause of sexually transmitted disease in the USA. Other species of medical importance include C. pneumoniae, a causative agent of upper respiratory tract infections and possibly associated with atherosclerosis, and C. psittaci, which is primarily a pathogen of animals but occasionally is transmitted to humans. Chlamydiae are obligate intracellular bacteria that must be taken up by host cells to progress through their intracellular growth cycle and cause infection. Chlamydia trachomatis rapidly induces its entry into host cells. Initial attachment is mediated by electrostatic interactions to heparan sulfate moieties on the host cell, followed by irreversible binding to an unknown secondary receptor. This secondary binding leads to the recruitment of actin to the site of attachment, formation of a pedestal-like structure underneath the chlamydiae, and finally internalization of the bacteria. How chlamydiae induce this process is unknown. We have identified a high-molecular weight protein that is rapidly tyrosine-phosphorylated upon attachment to the host cell. Immunoelectron microscopy revealed a tyrosine-phosphorylated protein localized to the cytoplasmic face of the plasma membrane at the site of attachment of surface-associated chlamydiae. Analysis of this protein isolated by immunoprecipitation with the antiphosphotyrosine antibody 4G10 identified it as the chlamydial protein CT456, a hypothetical protein of unknown function. The chlamydial protein (Tarp) appears to be translocated into the host cell via type III secretion as it is exported in a Yersinia heterologous secretion assay. C. trachomatis Tarp possesses up to six direct repeats of approximately 50 amino acids each. The majority of the tyrosine residues are found within this repeat region. We have ectopically expressed distinct domains of Tarp in HeLa 229 cells and demonstrate that tyrosine phosphorylation occurs primarily within the repeat region while recruitment of actin is mediated by the C-terminal domain of the protein. A comparison of other sequenced chlamydial genomes revealed that each contains an ortholog of Tarp although C. muridarum, C. caviae, and C. pneumoniae Tarp lack the large repeat region. Immunofluorescence and immunoblotting using an anti-phosphotyrosine antibody show no evidence of phosphotyrosine at the site of entry of C. muridarum, C. caviae, and C. pneumoniae although each species similarly recruits actin. Ectopic expression of full-length C. trachomatis and C. caviae Tarp confirmed that both recruit actin but only C. trachomatis Tarp is tyrosine phosphorylated. The data indicate that the C-terminal domain of Tarp is essential for actin recruitment and that tyrosine phosphorylation may not be an absolute requirement for actin recruitment. The results further suggest the potential for additional, unknown signal transduction pathways associated specifically with C. trachomatis. Chlamydiae, like many Gram-negative pathogenic bacteria, subvert the responses of the eukaryotic host by the secretion of specific effector proteins directly into the cytosol of the host cell using a specific transport system known as type III secretion to inject bacterial proteins into the host cell. Chlamydiae possess the components of a complete type III secretion system although the identify and function of the secreted effector proteins and even when they are secreted are unknown. Over the past year, we have shown that chlamydiae secrete at least two distinct classes of effector proteins throughout the developmental cycle. One protein is pre-existing in elementary bodies and is secreted into the host cell where it is recognized by the host and serves to trigger the recruitment of actin to promote internalization. Other secreted effector proteins are not synthesized until the chlamydiae are intracellular. Several of these latter proteins are known to be inserted into and modify the vacuolar (inclusion) membrane which encompasses the replicating chlamydiae. Understanding the initial events in chlamydial differentiation including the transition in properties of the endocytic vesicle to one which intersects an exocytic pathway remain significant challenges in defining the pathogenic mechanisms of chlamydiae. Based on a demonstrated role of T3S-specific chaperones in secretion of anti-host proteins by Gram-negative pathogens, we investigated putative Chlamydia T3S chaperones in an effort to gain mechanistic insight into the Chlamydia T3SS and to identify Chlamydia-specific secreted products. Expression of two such chlamydial proteins in Yersinia resulted in their type III-dependent secretion, and localization studies in C. trachomatis-infected cells indicated that both were secreted by Chlamydia. Additional studies have focused upon the early signals to intiate chlamydial development from the metabolically dormant elementary bodies to the a replicative form. A key step in this process is the dissociation of the chlamydial chromatin from a condensed state maintained by the presence of histone-like proteins. Elementary bodies are characterized by a condensed chromatin, which is maintained by a histone H1-like protein, Hc1. Differentiation of elementary bodies to reticulate bodies is accompanied by dispersal of the chromatin as chlamydiae become transcriptionally active although the mechanisms of Hc1 release from DNA have remained unknown. Dissociation of the nucleoid requires chlamydial transcription and translation with negligible loss of Hc1. A genetic screen was therefore designed to identify chlamydial genes rescuing E. coli from the lethal effects of Hc1 overexpression. CT804, a gene homologous to ispE, an intermediate enzyme of the non-mevalonate methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, was shown to produce a small metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis that releases histone from chromatin. In addition, we have identified a small regulatory RNA (sRNA) that negatively regulates Hc1 synthesis. Co-expression of the sRNA with Hc1 in E. coli inhibited Hc1 translation but did not affect hctA mRNA transcription or stability. IhtA (inhibitor of hctA translation) is present in purified RBs only while Hc1 is present only in EBs. IhtA, but not Hc1, is present in carbenicillin induced aberrant RBs and is down regulated while Hc1 is up regulated upon carbenicillin removal and RB to EB differentiation. The downregulation of gene expression at high levels of Hc1 and alteration of expression patterns at sub-condensing levels requires that chlamydiae strictly control Hc1 activity. Thus, in addition to transcriptional control and inhibition of DNA binding by a small metabolite, chlamydiae also employ an addtional checkpoint restricting Hc1 translation. We propose that IhtA is part of a global regulatory circuit that controls differentiation at the end of the chlamydial life cycle.