Simultaneous Transcriptional Profiling of Bacteria and Their Host Cells

Abstract

We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness). Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.

Development of a vaccine for Chlamydia trachomatis: challenges and current progress

“…103 Host factors associated with scar formation and other fibrotic conditions were also recently reported as being released from epithelial cell monolayers as soon as 1 hour postinfection with C. trachomatis serovar E, and these findings have formed the basis of a hypothesis involving multiple feedback loops to explain Chlamydia-induced fibrotic scarring of fallopian tubes. 104,105 These explanations for the pathology induced by chlamydial infections add to previous work showing an involvement of heat shock protein 60 and a proposed model of molecular mimicry which has been discussed extensively by Hafner.…”

Dual RNA-seq of pathogen and host

“…The ubiquitous polyadenylation of eukaryotic mRNAs and the sequence similarity of ribosomal transcripts facilitates the experimental protocol as it enables the poly(A) enrichment or ribosomal depletion for host and pathogen in a single step. Recently a few publications have reported the application of RNA-seq to a model of bacterial infection of plant leaves (Asai et al, 2014;Socquet-Juglard et al, 2013), porcine enterocytes (Vannucci et al, 2013), or human epithelial carcinoma cells (Humphrys et al, 2013). However, these suffered from the lack of an enrichment of invaded cells, resulting in a low bacterial transcriptome coverage, and/or from a shallow sequencing depth, hampering an in-depth profiling of the host response.…”

New signaling pathways govern the host response to C. albicans infection in various niches

“…Gene expression profiling with RNA-seq is consistent with results obtained by microarrays (Liu et al 2007;Marioni et al 2008;Bradford et al 2010;Malone and Oliver 2011) but is significantly more sensitive, with a much greater dynamic range (Wang et al 2009). These traits and the probe-independent nature of RNAseq make it possible to analyze the transcriptomes of multiple species (e.g., pathogen and host) simultaneously (Tierney et al 2012;Westermann et al 2012;Humphrys et al 2013).…”