Understanding the chlamydial developmental cycle at the single cell level

“Understanding the chlamydial developmental cycle at the single cell level”. Project Summary/Abstract The bacteria in the chlamydiales order are obligate intracellular parasites of eukaryotic cells. They are reliant on a developmental cycle consisting of at least three 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 the distinct cell forms are currently poorly understood. Our preliminary and recently published data indicates the cycle should be broken down into four stages; EB germination, RB amplification/maturation, IB production, and EB formation. Understanding these forms and their contributions to the developmental cycle and pathogenesis has been obfuscated by the mixed cell environment of the chlamydial inclusion. Our data show that the chlamydial replication niche contains mixed phenotypic cells as early as after the first few rounds of replication. Therefore, two aims focusing on understanding the regulation of gene expression in these populations. In Aim 1, we will develop a CRISPRi knockdown screen to assess the role of essential genes on cell type regulation. For Aim 2, the spatial gene expression pattern of cell type specific genes and the DNA elements that generate this expression pattern will be determined at the single cell level. Completion of these aims will lead to a more complete understanding of the developmental cycle and allow for careful dissection of the effects of mutations, chemical agents, ectopic expression of regulatory proteins, and therapeutic treatments on the different aspects of the developmental cycle.