Cell form determination during the chlamydial intracellular infectious cycle

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Project Summary/Abstract: The bacteria in the Chlamydiales order are intracellular parasites of eukaryotic cells. They are reliant on a developmental cycle consisting of phenotypically distinct cell forms termed the reticulate body (RB), the intermediate body (IB) and the elementary body (EB). The EB is infectious but does not replicate. The RB replicates in the host cell but is non-infectious, while the IB is an intermediate form that transitions to the EB form. Completion of this developmental cycle is central to chlamydial pathogenesis. Within this order, the genus Chlamydia contains the causative agents of a number of important pathogens of humans. C. psittaci causes zoonotic infections resulting in pneumonia, while C. pneumoniae is a human pathogen that causes respiratory disease and is linked to atherosclerosis. Biovars of C. trachomatis are the causative agents of trachoma, the leading cause of preventable blindness worldwide, as well as sexually transmitted infections with the potential to cause pelvic inflammatory disease and infertility. Irrespective of the resulting disease, all chlamydial species share the same obligate intracellular life cycle and developmental cell forms. The mechanisms that regulate the development of these distinct cell forms are currently poorly understood. The EB cell has limited transcription and translation after formation in the inclusion and never initiates DNA replication. However, upon entering a new host cell through pathogen directed endocytosis the EB initiates a germination program ultimately resulting in replicating RB cells. EB germination is an important knowledge gap, our preliminary data suggest a role for 1) DNA replication, 2) the DNA binding protein Euo in the previous infection and, 3) the DNA binding protein DdbA in the germination process. Therefore, in Aim 1 we will Determine the mechanisms involved in EB germination/RB reformation. In Aim 2 we will Characterize the IB form and its role in regenerating the infectious EB. Ectopic expression of Euo reversibly arrests the developmental cycle at an early IB stage. Therefore we will leverage this arrest/recovery observation to determine the mechanism regulating gene expression during the RB to IB/EB transition. Additionally, our preliminary data showed that structural components of the T3SS were expressed exclusively in the IB cell form. Therefore, we will determine in which cell forms the T3SS is expressed and how cell form restriction is regulated. Aim 3 we will Determine the role of HctA, HctB and Euo in EB maturation and gain of infectivity. HctA and HctB are DNA binding proteins that play a role in the structure of the chlamydial nucleoid. HctA is transiently expressed early during IB to EB transition (1-2 hours after the committed step) while HctB is expressed at the very end of the EB formation process (~8-10 hours after the committed step). Additionally we have shown that euo expression is required for infectious EBs. Therefore, we hypothesize that HctA, HctB and Euo play important and distinct roles in regulating gene expression during EB formation.