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. ? ? The Host-Parasite Interactions Section studies the fundamental cellular and molecular biology of intracellular pathogens. Efforts have been primarily with the genus Chlamydia and include two basic research areas: i.) the cellular interactions that promote entry and intracellular growth; primarily those controlled by secreted chlamydial effector proteins and ii.) global regulation of chlamydial gene expression by histone-like proteins. ? ? Establishment of a protected intracellular niche is a critical stage of the chlamydial developmental cycle. We have therefore focused much of our efforts over the last several years upon events occurring within these early stages of infection. Many of these events are unique among intracellular pathogens but common to the genus Chlamydia. The experimental approaches are heavily weighted toward cell biology, proteomics, and functional analysis of protein-protein interactions. An improved understanding of the distinct developmental stages and the cellular responses to them may suggest novel means of chemotherapeutic or immunologic intervention of chlamydial diseases. ? ? Chlamydiae occupy a unique vacuolar niche within the host cell. 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. 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. Another example of a chlamydial proteins controlling localized events could include 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. ? ? Chlamydial histone H1-like proteins, Hc1 and Hc2, act as global regulators of chlamydial gene expression by virtue of dramatic effects on DNA structure. The chlamydial histones are transcribed late in the developmental cycle as RBs differentiate to EBs. The mechanisms of histone release from the chlamydial chromatin at the initiation of development had been unknown. By utilizing a heterologous screening protocol in E. coli, we identified two novel means of regulation of histone activity. The affinity of histone for DNA appears to be disrupted early in infection by the synthesis of a small metabolite in the non-mevalonate pathway of isoprenoid biosynthesis. In the course of the screening procedure, we also identified a small regulatory RNA that specifically inhibits translation of Hc1.
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