Virulence Proteins of Pathogenic Leptospira Species

摘要 钩端螺旋体病被疾病控制中心认为是最普遍的 世界上最大的人畜共患病。慢性肾小管感染的宿主通常会传播 致病性钩端螺旋体通过尿液排出给人类。湖北省钩端螺旋体感染情况分析 人类经常导致暴发性肝功能障碍、肾功能衰竭和严重的肺部疾病。 出血综合征，病死率为10%。钩端螺旋体病已成为主要的 风险与贫困和老鼠接触密切相关的城市贫民窟的公共卫生负担。L. 问号是从城市褐鼠中分离出来的最常见的生物体，也是 城市贫民窟中人类钩端螺旋体病的主要原因。 这项建议的总体目标是鉴定钩端螺旋体毒力蛋白和 了解它们在发病机制中的作用(S)。鉴于钩端螺旋体病原体都持续存在于 环境和定植寄主组织作为其生命周期的必要部分，它不是 令人惊讶的是，它们的基因组中至少有152种不同的信号转导 蛋白质。高通量大规模平行转座子测序结果鉴定 几个新的毒力基因包括LIC12327，一个推定的腺苷酸环化酶。此外，我们还拥有 发现LIC11484，一种独特的带有环核苷酸的钩端螺旋体转录调控因子 结合结构域和螺旋-转弯-螺旋DNA结合结构域是毒力所必需的。这些数据 提出了cAMP是问号钩端螺旋体毒力中枢信使的假说。 我们提出了以下两个目标：1)钩端螺旋体需要哪些基因 致命性？我们将筛选体内适合性缺陷的突变体转座子突变体文库 通过高通量大规模并行转座子测序。我们将确认毒性缺陷 对突变株和互补株进行定量聚合酶链式反应和半数致死量研究。这类基因的鉴定 将有助于更好地了解钩端螺旋体的毒力。2)是环核苷酸 参与钩端螺旋体毒力调节？我们将确定LIC12327是否为 使用对cAMP或cAMP有选择性反应的大肠杆菌报告菌株的腺苷环化酶 和cGMP。我们将测量问号钩端螺旋体中的cAMP和/或cGMP，并检查如何循环 核苷酸水平对体内类似条件的反应发生变化，如生理渗透压和 温度范围从环境到37°C。我们将测量LIC11484对循环的亲和力 核苷酸。RNAseq将对野生型问号乳杆菌、LIC12327突变体和 LIC11484突变体，用于识别cAMP和/或LIC11484可能调控的基因。

ABSTRACT Leptospirosis is considered by the Centers for Disease Control to be the most widespread zoonosis in the world. Reservoir hosts with chronic renal tubular infection typically transmit pathogenic Leptospira species to humans through urinary shedding. Leptospiral infection in humans frequently results in fulminant liver dysfunction, kidney failure, and severe pulmonary hemorrhage syndrome with a mortality rate of >10%. Leptospirosis has emerged as a major public health burden in urban slums where risk is strongly linked to poverty and rat exposure. L. interrogans is the most common organism isolated from the urban brown rat and is also the predominant cause of human leptospirosis in urban slums. The overall goal of this proposal is to identify leptospiral virulence proteins and understand their role(s) in pathogenesis. Given that leptospiral pathogens both persist in the ambient environment and colonize host tissues as essential parts of their life cycle, it is not surprising that their genomes are richly endowed with at least 152 different signal transduction proteins. High throughput massively parallel transposon sequencing resulted in identification of several novel virulence genes including LIC12327, a putative adenylate cyclase. Further, we have discovered that LIC11484, a unique leptospiral transcriptional regulator with a cyclic nucleotide binding domain and helix-turn-helix DNA binding domain, is required for virulence. These data have led to the hypothesis that cAMP is a central virulence messenger of L. interrogans. We propose the following two Aims: 1) What genes are required for leptospiral virulence? We will screen a transposon mutant library for mutants with in vivo fitness defects by high throughput massively parallel transposon sequencing. We will confirm virulence defects of mutants and complemented strains by qPCR and LD50 studies. Identification of such genes will facilitate a greater understanding of leptospiral virulence. 2) Are cyclic nucleotides involved in leptospiral virulence regulation? We will determine whether LIC12327 is an adenylate cyclase using E. coli reporter strains that respond selectively to either cAMP or cAMP and cGMP. We will measure cAMP and/or cGMP in L. interrogans and examine how cyclic nucleotide levels change in response to in vivo-like conditions such as physiologic osmolarity and temperatures ranging from ambient to 37°C. We will measure the affinity of LIC11484 for cyclic nucleotides. RNAseq will be performed with wildtype L. interrogans, LIC12327 mutants, and LIC11484 mutants to identify genes potentially regulated by cAMP and/or LIC11484.