Complex in vivo RNA-seq analysis reveal reprogramming of Yersinia from virulent to persistent mode during infection [RNA-seq]. Yersinia pseudotuberculosis YPIII

We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding. Overall design: Examination of Y. pseudotuberculosis expression in two mouse cecal tissue at early and at persistent infection. Also pure bacterial cultures two at 26C and two 37C. Additionally, the bacterial content of cecal tissue during infections and before infection were analyzed with read mapping on 16SMicrobial NCBI database with non-rRNA-depleted reads.

本研究近期发现，假结核耶尔森菌（Yersinia pseudotuberculosis）可作为持续性细菌感染的研究模型。我们针对感染早期与持续感染阶段的小鼠盲肠组织活检样本中的细菌开展体内RNA测序（in vivo RNA-seq），以明确与持续性感染相关的细菌调控策略。对感染组织的混合RNA群体进行综合分析后发现，当病原体定植于盲肠时，假结核耶尔森菌在持续感染阶段会发生转录重编程，其三型分泌系统（Type Three Secretion System，T3SS）毒力基因出现显著下调。在持续感染阶段，细菌的基因表达模式在诸多方面与26℃体外培养条件下的表达模式相似，例如鞭毛基因与invA基因均出现上调。本研究结果有望为未来筛选新型抗生素的候选细菌靶标提供理论依据。持续感染阶段上调的其他基因还包括参与厌氧代谢、趋化作用以及抗氧化与抗酸性应激的相关基因，这提示不同环境信号对细菌的调控作用。我们发现，Crp/CsrA/RovA调控级联通路会影响持续感染阶段的细菌基因表达模式。此外，arcA、fnr、frdA与wrbA基因在持续性感染过程中发挥关键作用。本研究结果提出了该肠致病菌的生命周期模型：细菌从具有毒力的表型重编程为适应型表型，后者可实现持续定植并通过粪便排菌实现传播。
实验设计：分别检测2份感染早期与2份持续感染阶段的小鼠盲肠组织样本中假结核耶尔森菌的基因表达情况；同时设置2组26℃体外培养与2组37℃体外培养的纯细菌培养物作为对照。此外，采用未去除rRNA的测序reads比对NCBI 16S微生物数据库，对感染期间及感染前的盲肠组织中的细菌菌群构成进行分析。