Virulence Proteins of Pathogenic Leptospira Species

致病性钩端螺旋体的毒力蛋白

• 批准号: 9387341
• 负责人: DAVID A HAAKE
• 金额: $ 19.49万
• 依托单位: BRENTWOOD BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9387341
• 关键词: Adenylate Cyclase; Affinity; Antigens; Attenuated; Binding; Blood; Cells; Centers for Disease Control and Prevention (U.S.); Chronic; Collaborations; Complement; Cyclic AMP; Cyclic GMP; Cyclic Nucleotides; DNA Binding Domain; Data; Defect; Dose; Environment; Escherichia coli; Failure; Genes; Genetic; Genetic Transcription; Genome; Goals; Hamsters; Health; Helix-Turn-Helix Motifs; Hemorrhage; Human; Immunoglobulins; Infection; Iron; Kidney; Kidney Failure; Kinetics; Leptospira; Leptospira interrogans; Leptospirosis; Lethal Dose 50; Libraries; Life Cycle Stages; Link; Liver; Liver Dysfunction; Lung; Measures; Membrane; Methods; Organism; Osmolar Concentration; Pathogenesis; Pathogenicity; Physiological; Pilot Projects; Pneumonia; Post-Transcriptional Regulation; Poverty; Proteins; Public Health; Rattus; Regulation; Regulator Genes; Reporter; Reporting; Research; Risk; Role; Signal Transduction; Slum; Stimulus; Syndrome; System; TYRP1 gene; Temperature; Tissues; Tropism; Tubular formation; Virulence; attenuation; base; fitness; in vivo; interest; mortality; mutant; neglect; novel; pathogen; protein B; response; transcriptome sequencing; urinary

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.

摘要 钩端螺旋体病被疾病控制中心认为是最广泛的 最大的动物传染病慢性肾小管感染的储血宿主通常传播 致病性钩端螺旋体通过尿液排出到人类。钩端螺旋体感染 人类经常导致暴发性肝功能障碍、肾衰竭和严重的肺损伤。 出血综合征，死亡率> 10%。钩端螺旋体病已成为一种主要的 城市贫民窟的公共卫生负担，那里的风险与贫困和老鼠接触密切相关。L. 问号线虫是从城市褐鼠中分离出的最常见的生物体， 城市贫民窟人类钩端螺旋体病的主要原因。 该提案的总体目标是鉴定钩端螺旋体毒力蛋白， 了解它们在发病机制中的作用。考虑到钩端螺旋体病原体都存在于 环境和殖民宿主组织作为其生命周期的重要组成部分，它不是 令人惊讶的是，它们的基因组富含至少152种不同的信号转导， proteins.高通量大规模平行转座子测序导致鉴定了 几个新的毒力基因，包括LIC 12327，一个假定的腺苷酸环化酶。您因前述 LIC 11484是一种独特的钩端螺旋体转录调节因子， 结合结构域和螺旋-转角-螺旋DNA结合结构域是毒力所必需的。这些数据 cAMP是L.质问者 我们提出以下两个目的：1）钩端螺旋体需要什么样的基因 毒力？我们将筛选转座子突变体库中的突变体在体内的健身缺陷 通过高通量大规模平行转座子测序。我们将确认毒力缺陷 通过qPCR和LD 50研究对突变体和补充菌株进行分析。鉴定这些基因 将有助于更好地了解钩端螺旋体的毒力。2)是环状核苷酸 参与钩端螺旋体毒力调节我们将确定LIC 12327是否为 腺苷酸环化酶，选择性应答cAMP或cAMP的大肠杆菌报告菌株 cGMP。我们将测量L中的cAMP和/或cGMP。疑问句和检查如何循环 核苷酸水平响应于体内样条件如生理渗透压和 温度范围从环境温度到37°C。我们将测量LIC 11484对环状寡核苷酸的亲和力。 个核苷酸将使用野生型L进行RNAseq。问号，LIC 12327突变体，和 LIC 11484突变体，以鉴定可能受cAMP和/或LIC 11484调节的基因。