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

<div><p><i>Francisella tularensis</i> is a zoonotic intracellular pathogen that is capable of causing potentially fatal human infections. Like all successful bacterial pathogens, <i>F. tularensis</i> 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 <i>F. tularensis</i> 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 <i>F. tularensis</i> 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 <i>F. tularensis</i> 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.</p> </div

Comparison of the genes up- and down-regulated at each time point.

<p>The Venn diagrams depict the number of genes with significant changes in expression at both the 4 and 8-hour post-infection time points, with the number in the middle representing genes up- or down-regulated at both time points. A) Up-regulated genes. B) Down-regulated genes.</p

Differentially expressed genes plotted across the <i>F. tularensis</i> genome.

<p>All genes that had at least a 4-fold change in expression at either 4 hours (red) or 8 hours (blue) were plotted according to their gene ID number across the genome. The two copies of the FPI are highlighted in the up-regulated portion of the figure, and the ribosomal proteins and ATP synthase subunits are highlighted in the down-regulated portion of the figure.</p

Heat map of virulence factor genes up- and down-regulated at each time point.

<p>Change in expression was determined for previously identified <i>F</i>. <i>tularensis</i> virulence factor genes at both post-infection time points, and then clustered to identify genes that are coordinately regulated. The cluster analysis segregated the genes into three groups. Cluster 1, in which the genes are up-regulated at both post-infection time points, is comprised entirely of genes in the FPI.</p