Transcriptional profiling of Chlamydia trachomatis and its host in an ex vivo endocervical primary cell culture system using dual RNA sequencing

<i>Chlamydia trachomatis</i> (<i>Ct</i>) is an obligate intracellular bacterium that causes significant ocular and urogenital morbidity worldwide. Understanding host-pathogen interactions is challenging but dual RNA sequencing offers simultaneous transcriptome data for comprehensive interrogations into these interactions. While transcriptional profiling of both <i>Ct</i> and host-derived immortalized cells has been performed, this study used dual RNA sequencing to examine host-pathogen interactions in <i>ex vivo</i> human primary endocervical stromal cells infected with <i>Ct</i> strain E/Bour. At 1-hour post-infection (1hpi), 168 differentially expressed host genes (DEGs) were identified, 40% of which were non-coding RNAs, novel proteins, or pseudogenes. Pathway analysis revealed no significant enrichment at this stage, indicating a quiescent host response. At 24hpi, 212 DEGs were identified, with strong upregulation of interferon-stimulated genes and activation of the cGAS-STING and RLR pathways, despite the absence of detectable type I interferons. Pro-inflammatory and leukocyte recruitment genes were also highly expressed, suggesting an immunoreactive phenotype at this later stage. <i>Ct</i> transcriptomics identified 331 early and 903 mid-infection genes. Inclusion-membrane genes peaked at 1hpi, while hemolysin-like and polymorphic membrane protein genes were upregulated at 24hpi. Enrichment analysis identified pathways related to catalytic activity, host modulation, and bacterial survival. This study demonstrates distinct temporal dynamics in <i>Ct</i>-host interactions, including early host immune quiescence and robust mid-infection activation of innate immunity in contradistinction to previous host and <i>Ct</i> findings in immortalized cell lines. The findings emphasize the utility of <i>ex vivo</i> human primary cell culture for investigating <i>Ct</i> pathogenesis using clinically relevant <i>Ct</i> strains and provide a foundation for future exploration of uncharacterized genes and pathways critical to <i>Ct</i> infection.