Role of Shigella two component regulation systems in intracellular adaptation

描述(申请人提供)：福氏志贺氏菌是一种兼性的细胞内病原体，是人类志贺氏菌病(细菌性痢疾)的病原体。旨在了解这种细胞内病原体对真核宿主细胞适应性的基础研究将有助于设计更有效的治疗方法，并将产生适用于其他细胞内病原体的信息。志贺氏菌在人体内与多种类型的细胞相互作用，包括结肠上皮细胞和肠道巨噬细胞。细菌适应这些环境的机制尚不完全清楚，但需要通过改变细菌基因表达来感知和响应当前环境的能力。因此，当志贺氏菌在细胞内时，就会诱导一组独特的编码蛋白质的基因表达，这些蛋白质有助于细菌在真核细胞内生存和/或繁殖。由于志贺氏菌的毒力在很大程度上取决于逃避巨噬细胞杀伤和在结肠上皮细胞内繁殖的能力，因此该研究计划的长期目标是确定志贺氏菌在这些环境中生存和/或繁殖的生理过程，以及在这些环境中调节志贺氏菌基因表达的因素。这个项目的目标是确定6个福氏志贺氏菌双组分调控系统(TCRS)对志贺氏菌适应真核细胞的贡献，并将通过完成三个特定目标来实现。第一项工作(具体目标1)将构建6个缺乏特定TCRS的突变志贺氏菌株(Baes/Baer、Bara/UvrY、EvgS/EVGA、NtrB/NTRC、CREC/CREB和YfhK/YfhA)，以便研究这些系统对志贺氏菌毒力的贡献。具体目的2是通过评估细胞单层上斑块的形成，分析每个TCRS缺失突变对志贺氏菌在上皮细胞培养中存活和生长能力的影响。如果Tcrs突变株没有形成斑块或形成比亲本志贺氏菌株形成的斑块更小的斑块，这将表明TCRS激活了一种基因(S)的表达，该基因增强了志贺氏菌的生存、繁殖或扩散到邻近上皮细胞的能力。具体目的3是通过分析6个Tcrs突变体在巨噬细胞中的存活和诱导巨噬细胞凋亡的能力，来评估这6个Tcrs系统是否激活了有助于志贺氏菌在巨噬细胞内存活的基因(S)的表达。 志贺氏菌是人类志贺氏菌病(细菌性痢疾)的病原体，每年在工业化国家导致150万病例，在不发达国家导致1.65亿病例，并导致100多万人死亡。志贺氏菌病是前往欠发达国家旅行的美国公民腹泻的常见原因，也是美国军事行动的关切，这既是因为志贺氏菌是一种潜在的生物武器制剂，也是因为志贺氏菌病影响志贺氏菌流行国家的部队健康。这项建议中描述的研究将有助于设计更有效的治疗方法，并将产生适用于其他类似病原体的信息。

DESCRIPTION (provided by applicant): Shigella flexneri, a facultative intracellular pathogen, is a causative agent of shigellosis (bacterial dysentery) in humans. Basic research aimed at understanding the adaptation of this intracellular pathogen to the eukaryotic host cell will be useful for designing more effective therapies and will yield information applicable to other intracellular pathogens. Shigella interacts with several human cell types while within the human host including colonic epithelial cells and intestinal macrophage cells. The mechanisms by which the bacterium adapts to these environments are not completely understood, but require the ability to sense and respond accordingly to the current environment by altering bacterial gene expression. Thus, expression of a unique set of genes encoding proteins that help the bacterium survive and/or multiply within the eukaryotic cell is induced when Shigella is intracellular. Since Shigella virulence depends largely on the ability to evade the macrophage killing and multiply within the colonic epithelial cells, the long-term goal of the research program is to identify the physiological processes that Shigella employs to survive and/or multiply within these environments, as well as the elements that regulate Shigella gene expression in these environments. The goal of this particular project is to determine the contribution of six S. flexneri two component regulatory systems (TCRS) to Shigella adaptation to the eukaryotic cells and will be accomplished by the completion of three specific aims. The first undertaking (specific aim 1) will be to construct six mutant Shigella strains that lack particular TCRS (BaeS/BaeR, BarA/UvrY, EvgS/EvgA, NtrB/NtrC, CreC/CreB, and YfhK/YfhA) so that the contribution of these systems to Shigella virulence can be examined. Specific aim 2 is to analyze the effects of the deletion mutations in each TCRS on the ability of Shigella to survive and grow in epithelial cell culture by assessing plaque formation on cell monolayers. If a TCRS mutant does not form plaques or forms plaques that are smaller than those formed by the parental Shigella strain, this will suggest that the TCRS activates expression of a gene(s) that enhances the ability of Shigella to survive, multiply, or spread to adjacent epithelial cells. Specific aim 3 is to assess whether the six TCRS systems activate expression of a gene(s) that contribute to survival of Shigella within macrophage cells by analyzing each TCRS mutant for the ability to survive in macrophages and induce macrophage apoptosis. Shigella species are the causative agents of shigellosis (bacterial dysentery) in humans that results in 1.5 million cases in industrialized countries, 165 million cases in underdeveloped countries, and over one million deaths annually. Shigellosis is a frequent cause of diarrhea in US citizens traveling to underdeveloped countries and is of concern to US military operations, both because Shigella is a potential biowarfare agent and because shigellosis impacts troop health in countries where Shigella is endemic. The research described in this proposal will be useful for designing more effective therapies and will yield information applicable to other similar pathogens.