Chlamydia, caused by the bacteria Chlamydia trachomatis, is one of the most common bacterial sexually transmitted infections of the urogenital and gastrointestinal tracts. Preliminary whole genome sequencing of C. trachomatis isolates from women and men who have sex with men (MSM) identified the gene pmpE. This gene encodes for a putative membrane protein that is a key locus of genetic variation between these two populations of isolates. Here, I performed deeper sequencing and structural analysis of natural pmpE variants to better understand how these genetically distinct strains exhibit their tissue tropism differences. PCR-based sequencing of C. trachomatis isolates was conducted against pmpE and ompA to initially type the strain, and then followed by whole genome sequencing to derive complete genomic information. Bioinformatic analysis showed that the cervical and rectal strains are sorted into two distinct clades based on pmpE. To predict possible functional differences, structures of pmpE sequence variants were computationally modeled using Alphafold v3, and analysis demonstrated that variations were confined to predicted surface-exposed loops. Based on the collective data, we hypothesize that two loops may provide functional differences in the ability of cervical versus rectal C. trachomatis strains to interact with tissue-specific host attachment proteins.

The obligate intracellular bacterium Chlamydia trachomatis is one of the leading causes of sexually transmitted bacterial infections. Despite its great prevalence and systemic impact on women's reproductive health, no vaccine has been developed. As an obligate intracellular parasite, Chlamydia trachomatis infection occurs within a host-derived vacuole, and Chlamydia have evolved numerous pathways for evading cellular immune defenses. Recent work has highlighted a mechanism through which intracellular Chlamydia interferes with a cell-intrinsic, interferon-gamma (IFN_γ) activated pathway. Our lab discovered a secreted CT virulence factor (GarD) that is important of preventing the IFN_γ -dependent recruitment of ubiquitin to CT-containing vacuoles, and we hypothesize that a gene of unknown function (ct134) immediately upstream of garD may also play a role in this immune evasion pathway. In this project, we used a new genetic system to produce a double knockout mutant for these genes, and we investigated in a cell culture model the fitness and susceptibility of mutants in cell stimulated with human IFN_γ. We expect this work to either define a broader role of this gene locus in mediating chlamydial anti-IFN_γ defenses. In addition, we generated a homologous double knockout lesion in the mouse-tropic pathogen Chlamydia muridarum, and we hypothesize that the C. muridarum homologs of ct134 and garD fulfill this phenotype will support future studies focused on investigating the in vivo role of these genes in Chlamydial infection and virulence in the upper genital tracts of infected female mice.