How Listeria senses and responds to different host environments

项目总结/摘要 单核细胞增生李斯特菌是兼性胞内食源性病原体， 适用于宿主-病原体相互作用基础研究的模型。这一建议是根据一项 遗传筛选揭示了细菌氧化还原敏感对毒力基因上调的关键作用 体内表达。我们发现L.单核细胞增生谷胱甘肽合成酶在感染过程中上调 并确定谷胱甘肽是主要毒力转录因子PrfA的变构激活剂。 基于这些发现，经过多个小组数十年的调查，我们成功地 通过简单地将还原剂添加到生长在培养基中的细菌中， 化学成分确定的合成培养基。这一发现导致了另一种基因筛选，以确定细菌突变体 对高浓度的生长抑制特性更敏感或更耐受的细胞， 还原剂对筛选出的抗性突变体进行分析，发现L. 单核细胞增多症具有基于黄素的细胞外电子传递链（FLEET）， 超过100，000个电子/细菌/秒到存在于两种细胞外脂蛋白PplA上的黄素部分 和FrdA，其可以通过激活FrdA将电子转移到三价铁和富马酸盐，FrdA编码 延胡索酸还原酶我们表明，FLEET可以介导厌氧生长使用三价铁或 富马酸盐作为电子受体。对还原剂更敏感的突变体聚集在perR中， 氧化还原敏感转录因子，控制对氧化应激的转录反应。我们 假设氧化应激是由FLEET产生的，我们发现FLEET在体内产生超氧化物， 氧气的存在。在包括病原体在内的100多个厚壁菌属物种中发现了FLEET直向同源物 和微生物群的成员，我们认为它代表了一个多功能的电子传递中心， 在不同的革兰氏阳性菌中，这为厌氧生长的细菌提供了选择性优势， 肠子我们还提出，FLEET活动有其他后果有氧，可能有 对感染、毒力和免疫力的细胞生物学产生深远影响。

Project Summary/Abstract Listeria monocytogenes is facultative intracellular food-borne pathogen that provides an extremely amenable model for basic studies on host-pathogen interactions. This proposal is based on the results of a genetic screen that revealed a critical role of bacterial redox sensing for the upregulation of virulence gene expression in vivo. We found that L. monocytogenes glutathione synthase was upregulated during infection and determined that glutathione is the allosteric activator of the major virulence transcription factor PrfA. Based on these findings, and after decades of investigation by multiple groups, we successfully recapitulated intracellular virulence gene expression by simply adding reducing agents to bacteria grown in a chemically defined synthetic media. This finding led to another genetic screen to identify bacterial mutants that were either more sensitive or resistant to the growth inhibitory property of high concentrations of reducing agents. Analysis of the resistant mutants that arose from this screen led to the discovery that L. monocytogenes possess a Flavin-based Extracellular Electron Transport chain (FLEET) that can transport over 100,000 electrons/bacterium/second to flavin moieties present on two extracellular lipoproteins, PplA and FrdA, which can transfer electrons to ferric iron and fumarate via activation of FrdA, which encodes a fumarate reductase. We show that FLEET can mediated anaerobic growth using either ferric iron or fumarate as electron acceptors. Mutants that were more sensitive to reducing agents clustered in perR, a redox-sensing transcription factor that controls the transcriptional response to oxidative stress. We hypothesize that the oxidative stress is being generated by FLEET that we show is producing superoxide in the presence of oxygen. FLEET orthologues were found in 100s of Firmicute species including pathogens and members of the microbiota and we propose that it represents a versatile electron transfer hub present in diverse Gram-positive bacteria, that provides a selective advantage to bacteria growing anaerobically in the intestine. We also propose that FLEET activity has other consequences aerobically that may have profound effects on the cell biology of infection, virulence, and immunity.