Use of a Capture-Based Pathogen Transcript Enrichment Strategy for RNA-Seq Analysis of the Francisella Tularensis LVS Transcriptome during Infection of Murine Macrophages

Abstract

Francisella tularensis is a zoonotic intracellular pathogen that is capable of causing potentially fatal human infections. Like all successful bacterial pathogens, F. tularensis rapidly responds to changes in its environment during infection of host cells, and upon encountering different microenvironments within those cells. This ability to appropriately respond to the challenges of infection requires rapid and global shifts in gene expression patterns. In this study, we use a novel pathogen transcript enrichment strategy and whole transcriptome sequencing (RNA-Seq) to perform a detailed characterization of the rapid and global shifts in F. tularensis LVS gene expression during infection of murine macrophages. We performed differential gene expression analysis on all bacterial genes at two key stages of infection: phagosomal escape, and cytosolic replication. By comparing the F. tularensis transcriptome at these two stages of infection to that of the bacteria grown in culture, we were able to identify sets of genes that are differentially expressed over the course of infection. This analysis revealed the temporally dynamic expression of a number of known and putative transcriptional regulators and virulence factors, providing insight into their role during infection. In addition, we identified several F. tularensis genes that are significantly up-regulated during infection but had not been previously identified as virulence factors. These unknown genes may make attractive therapeutic or vaccine targets.

Transcriptomic Analysis of Yersinia enterocolitica Biovar 1B Infecting Murine Macrophages Reveals New Mechanisms of Extracellular and Intracellular Survival

“…Using RNA-Seq to analyze bacterial gene expression profiles through the course of an infection has proved to be a highly informative method for better understanding the pathways and mechanisms that bacteria use to colonize different host environments (22,(73)(74)(75). It has significant advantages over most other transcriptional profiling techniques because it provides an unbiased way to analyze the temporally and spatially dynamic expression of every bacterial gene, in wild-type bacteria, as they inhabit diverse host environments.…”

“…However, analysis of the bacterial transcriptome during infection is all but impossible without an enrichment step that increases the proportion of bacterial transcripts in the host-dominated sample. In the present study, we utilized our previously described capture-based enrichment strategy (22,23) to analyze the complete transcriptome of Y. enterocolitica biovar 1B in different media and host environments, including a transcriptomic comparison of bacteria attached to the surface of macrophages to those that had been internalized by the macrophages. This analysis revealed a suite of genes whose expression levels change over the course of an infection, providing insight into the various mechanisms the bacteria use to infect macrophages and remain viable within them after being internalized.…”

“…Enrichment of bacterial transcripts from the host-dominated background was performed as previously described with some minor modifications (22,23). Biotinylated Y. enterocolitica probes were generated from genomic DNA, including the plasmid pYV, using the BioPrime DNA labeling system according to the manufacturer's directions (Life Technologies).…”

“…However, it is exceedingly difficult to sequence the bacterial transcriptome in the context of an infection as the vast majority of transcripts belong to the host (21). To overcome this obstacle, we developed a novel bacterial transcript enrichment strategy that increases the number of bacterial reads, allowing an accurate and unbiased analysis of the entire transcriptome (22,23). In the present study, we use this technique to analyze the complete transcriptome of Y. enterocolitica biovar 1B through the course of a murine macrophage infection.…”

A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing

“…This is because it is extremely difficult to obtain sufficient pathogen transcripts from an infected host sample, particularly early in the infection when the pathogen is least abundant, but during which time the pathogen may be actively adapting to the host environment, or evading host immune response. Typically host transcripts outnumber pathogen transcripts by well over 100 fold [6–8], meaning that using a standard RNA-seq library prep to sequence the pathogen transcripts in a mixed sample can be an expensive and computationally wasteful proposition [9, 10]. …”