Virulence Proteins of Pathogenic Leptospira Species

致病性钩端螺旋体的毒力蛋白

基本信息

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
项目类别：
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

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

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项目摘要

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.

抽象的疾病控制中心认为钩端螺旋病是最广泛的世界上的动物病。水库患有慢性肾块茎感染的宿主通常会传播致病性钩端螺旋体通过尿液脱落给人类。钩端螺旋体感染人类经常导致暴发性肝功能障碍，肾衰竭和严重的肺部死亡率> 10％的出血综合征。钩端螺旋体病已成为主要公共卫生在城市贫民窟中燃烧，那里的风险与贫困和老鼠暴露密切相关。 L. 探究是从城市棕色老鼠中孤立的最常见的生物，也是城市贫民窟中人类钩端螺旋体病的主要原因。该提案的总体目标是确定钩端螺旋体病毒蛋白和了解它们在发病机理中的作用。鉴于钩端螺旋体病原体都持续存在环境环境并将宿主组织定为生命周期的重要部分，不是令人惊讶的是，它们的基因组至少具有152个不同的信号转导。蛋白质。高吞吐量大量平行的转座子测序导致鉴定几种新型病毒基因，包括LIC12327，一种推定的腺苷酸环化酶。此外，我们还有发现LIC11484是一种具有环状核苷酸的独特的钩螺旋转录调节剂结合结构域和螺旋 - 旋转螺旋DNA结合结构域是病毒所必需的。这些数据已经提出了一个假设，即营地是L. discentogans的中央病毒使者。我们提出以下两个目标：1）钩端螺旋体需要哪些基因毒力？我们将筛选一个具有体内适应性缺陷突变体的转座子突变库通过高吞吐量大规模平行的转座子测序。我们将确认病毒缺陷通过QPCR和LD50研究完成的突变体和完成的菌株。鉴定这种基因将促进对钩端螺旋体病毒的更多了解。 2）是环状核苷酸参与钩端螺旋体病毒调节？我们将确定LIC12327是否是使用大肠杆菌报告菌株有选择性地响应CAMP或CAMP的腺苷酸环化酶和CGMP。我们将测量L. ereadogans中的营地和/或CGMP，并检查如何循环核苷酸水平在响应体内样条件（例如生理渗透压和）的响应中发生变化温度从环境到37°C不等。我们将衡量LIC11484对循环的亲和力核苷酸。 RNASEQ将使用Wildtype L. erentogans，LIC12327突变体和 LIC11484突变体识别可能受CAMP和/或LIC11484调节的基因。