The role of NQR in ROS-dependent virulence regulation in Vibrio cholerae

摘要 病原微生物的代谢必须是灵活的，因为病原细菌在微生物中的有效复制是必需的。 宿主依赖于主动适应过程。人类感染细菌病原体霍乱弧菌，需要 多种适应过程，最终导致小肠中产生强大的毒力因子。虽然 代谢状态和毒力因子表达之间的联系已在几种病原体中描述，但尚未被证实。 详细研究了霍乱弧菌。同样，膜生物能量学在细菌毒力中的作用也不太清楚 尽管大多数病原生物广泛地利用膜呼吸作为其生理学的一部分，但我们已经理解了这一点。我们 以前的结果表明，膜生物能量学的变化影响霍乱弧菌中的毒力基因表达。更 具体地说，主要霍乱弧菌呼吸酶（NQR）的丧失导致毒力基因转录的变化。在这里， 我们建议进行调查之间的联系膜呼吸和毒力基因调控的V。 胆固醇，重点是活性氧（ROS）和醌池。有充分的证据表明， 多种毒力相关转录因子通过翻译后修饰感知霍乱弧菌的氧化应激 例如硫醇开关。我们以前已经建立了NQR作为周质和细胞质ROS的来源。 此外，我们最近开发了一种方法，通过操纵表达， 同时，活细胞中细胞质ROS的水平可以使用一种新的基于荧光团的 approach.该系统使我们能够定量地将ROS水平与编码毒力的基因的表达相关联 霍乱弧菌中的因子。总的来说，我们希望获得重要的见解，这种生物本身的代谢能力， 其与致病基因表达的关系是致病的重要方面。我们对这个项目的理由是， 关于霍乱弧菌代谢及其与毒力因子产生的联系的基本信息的产生将 为新的感染治疗策略提供了潜在的机会。

ABSTRACT The metabolism of pathogenic microorganisms has to be flexible as the efficient replication of pathogenic bacteria in the host depends on an active adaptation process. Human infection by the bacterial pathogen Vibrio cholerae, requires multiple adaptation processes that ultimately lead to robust virulence factor production in the small intestine. Although a link between the metabolic status and virulence factor expression has been described in several pathogens, it has not been investigated in detail in V. cholerae. Similarly, the role of membrane bioenergetics in bacterial virulence is not well understood, although most pathogenic organisms extensively utilize membrane respiration for part of their physiology. Our previous results suggest that changes in membrane bioenergetics affect virulence gene expression in V. cholerae. More specifically, loss of the main V. cholerae respiration enzyme (NQR) leads to changes in virulence gene transcription. Here, we propose to perform an investigation into the link between membrane respiration and virulence gene regulation in V. cholerae, with an emphasis on reactive oxygen species (ROS) and the quinone pool. It has been well documented that multiple virulence-associated transcription factors sense oxidative stress in V. cholerae via post-translational modifications such as thiol-switches. We have previously established NQR as a source of both periplasmic as well as cytoplasmic ROS. Moreover, we recently developed a method to produce varying levels of ROS in V. cholerae by manipulating the expression level of NQR and in parallel, the level of cytoplasmic ROS in live cells can be monitored using a novel, fluorophore-based approach. This system enables us to quantitatively correlate ROS levels with the expression of genes encoding for virulence factors in V. cholerae. Overall, we expect to gain important insights into the metabolic capacity of this organism per se and its relation to virulence gene expression as an important aspect of pathogenesis. Our rationale for this project is that the generation of fundamental information concerning V. cholerae metabolism and its link to virulence factor production will provide potential opportunities for novel treatment strategies for infections.