Bioinformatic analysis of host cell gene expression and chromatin accessibility in response to Chlamydia trachomatis infection

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Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a wide range of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection (STI) worldwide and is the leading cause of trachoma (infectious blindness) in disadvantaged populations. If left untreated, infections can lead to more complex disease outcomes including infertility, ectopic pregnancy, epididymitis, prostatitis, and pelvic inflammatory disease. Due to widespread rates of infection and disease around the world and the associated economic costs, chlamydial infections remain a serious public health concern. All chlamydial species are defined by their unique intracellular developmental cycle. However, this has been a significant barrier restricting traditional molecular microbial investigation, such as transformation. As a result, we still do not have a comprehensive understanding of chlamydial gene function, particularly secreted effector proteins that modulate many host cell interactions. In the absence of a reliable and efficient transformation system, next generation sequencing (NGS) approaches enable the recovery of genome-wide expression patterns from a chlamydial or host point of view to aid in uncovering these functions and interactions. To help with further characterisation and identification of these host-chlamydial interactions, this work applied three novel NGS approaches using in vitro models of infection with C. trachomatis. Chapter 3 examines chromatin accessibility dynamics across the developmental cycle (1, 12, 24 and 48 hours) to identify epigenomic changes to host cells; Chapter 4 utilises single cell RNA-sequencing (scRNA-seq) from host cells to examine early developmental time points (3, 6 and 12 hours); and Chapter 5 simultaneously examines host and chlamydial expression (dual RNA-seq) from two time points (1 and 24 hours), with an experimental design aimed to examine different depletion techniques and to optimise the ratio of EBs per cell for infection models. Examination of the host cell epigenome identified both conserved and distinct temporal changes genome-wide. Differentially accessible chromatin regions were associated with immune responses, re-direction of host cell nutrients, intracellular signalling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. Temporally enriched transcription factors identified a novel family of Krüppel-like-factors (KLFs) which are ubiquitously expressed in reproductive tissues and associated with a variety of uterine pathologies. Analyses from scRNA-seq highlight infection-specific host cell biology, including two distinct clusters separating 3 hour cells from 6 and 12 hours. Pseudotime analysis identified a possible infection-specific cellular trajectory for Chlamydia-infected cells, and differential expression identified temporally expressed genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis and cellular stress. Dual RNA-seq analysis showed that combining depletion methods (polyA and rRNA) increases the capture rate of chlamydial transcripts, but negatively impacts host-cell expression. Different MOIs (0.1, 1 and 10) highlighted that an MOI of 10 captures significantly more transcripts and is more beneficial for capturing chlamydial transcripts. Overall, this work highlights the complex nature of chlamydial infections, uncovering novel biological functions and regulatory activities. These results and analyses also provide further considerations and improvements for future in vitro experiments, but also enable the application of these genome-scale techniques to the investigation of complex disease models in vivo and in human tissues ex vivo.
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