Abstract
Chlamydia are Gram-negative obligate intracellular bacterial pathogens responsible for a variety of disease in humans and animals worldwide. Chlamydia trachomatis causes trachoma in disadvantaged populations, and is the most common bacterial sexually transmitted infection in humans, causing reproductive tract disease. Antibiotic therapy successfully treats diagnosed chlamydial infections, however asymptomatic infections are common. High-throughput transcriptomic approaches have explored chlamydial gene expression and infected host cell gene expression. However, these were performed on large cell populations, averaging gene expression profiles across all cells sampled and potentially obscuring biologically relevant subsets of cells. We generated a pilot dataset, applying single cell RNA-Seq (scRNA-Seq) to C. trachomatis infected and mock-infected epithelial cells to assess the utility, pitfalls and challenges of single cell approaches applied to chlamydial biology, and to potentially identify early host cell biomarkers of chlamydial infection. Two hundred sixty-four time-matched C. trachomatis-infected and mock-infected HEp-2 cells were collected and subjected to scRNA-Seq. After quality control, 200 cells were retained for analysis. Two distinct clusters distinguished 3-h cells from 6- and 12-h. Pseudotime analysis identified a possible infection-specific cellular trajectory for Chlamydia-infected cells, while differential expression analyses found temporal expression of metallothioneins and genes involved with cell cycle regulation, innate immune responses, cytoskeletal components, lipid biosynthesis and cellular stress. We find that changes to the host cell transcriptome at early times of C. trachomatis infection are readily discernible by scRNA-Seq, supporting the utility of single cell approaches to identify host cell biomarkers of chlamydial infection, and to further deconvolute the complex host response to infection.
Highlights
Chlamydia are Gram-negative obligate intracellular bacterial pathogens that cause disease in humans and a wide variety of animals
We identify cell cycle stage as a likely confounding effect (Barron and Li, 2016) that was removed from our subsequent analyses, it may be relevant to the infection and growth strategies of Chlamydia
We further examined differentially expressed (DE) genes firstly by comparing infected and mock-infected cells at 6 and 12 h, respectively, and secondly by comparing the 3 h infected cells against cluster 2, as the 3 h mockinfected cells were removed after initial quality control steps
Summary
Chlamydia are Gram-negative obligate intracellular bacterial pathogens that cause disease in humans and a wide variety of animals. Chlamydia trachomatis typically infects cells within the ocular and genital mucosa, causing the most prevalent bacterial sexually transmitted infections (STI; Reyburn, 2016), inducing acute and chronic reproductive tract diseases that impact all socioeconomic groups, and trachoma in disadvantaged populations (Burton and Mabey, 2009). Disease outcomes arise from complex inflammatory cascades and immune-mediated host processes that can lead to tissue damage and fibrotic scarring in the upper genital tract or the conjunctiva (Taylor et al, 2014; Menon et al, 2015). Antibiotic therapy with azithromycin or doxycycline successfully treats diagnosed infections, asymptomatic infections are common (Hafner et al, 2014; Ali et al, 2015). Asymptomatic infection rates are estimated to exceed diagnosed infection rates by at least 4.3-fold (Ali et al, 2015)
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