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

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.土拉弗朗西斯菌（Francisella tularensis）是一种人畜共患胞内病原体，可引发具有潜在致死性的人类感染。如同所有成功的细菌性病原体一般，土拉弗朗西斯菌在宿主细胞感染期间，以及遭遇宿主细胞内的各类微环境时，均可快速响应环境变化。这种可对感染相关挑战作出恰当响应的能力，依赖于基因表达模式发生快速且全局性的改变。本研究借助新型病原体转录本富集策略与全转录组测序（RNA-Seq），对鼠巨噬细胞感染过程中土拉弗朗西斯菌LVS菌株的基因表达快速全局性变化展开了精细化表征。我们针对感染进程中的两个关键阶段——吞噬体逃逸与胞质增殖——对所有细菌基因开展了差异表达分析。通过将感染这两个阶段的土拉弗朗西斯菌转录组与体外培养的细菌转录组进行比对，我们成功鉴定出感染过程中存在差异表达的基因簇。该分析揭示了多个已知及推定转录调控因子与毒力因子的时序动态表达特征，为解析其在感染过程中的功能提供了重要线索。此外，我们还鉴定出若干在感染过程中显著上调，但此前未被归类为毒力因子的土拉弗朗西斯菌基因。这类功能未知的基因有望成为极具应用前景的治疗靶点或疫苗候选靶点。