Functions of Leptospira Lig Proteins in the Pathogenesis of Leptospirosis

钩端螺旋体Lig蛋白在钩端螺旋体病发病机制中的功能

基本信息

批准号：
10265369
负责人：
DAVID A HAAKE
金额：
--
依托单位：
VA GREATER LOS ANGELES HEALTHCARE SYSTEM
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-01-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10265369
关键词：
Acute Affinity Alanine Animals Antigens Bar Codes Binding Bioluminescence Cell surface Cells Coagulation Process Complement Activation Contracts Data Development Disease Engineering Environment Epitopes Evaluation Exhibits Fibrin Genes Goals Hamsters Health Host Defense Mechanism Human Immunity Immunization Immunize Immunoglobulins Immunohistochemistry In Vitro Infection Institutes Kidney Knock-in Leptospira Leptospira interrogans Leptospirosis Life Ligands Literature Mediating Membrane Proteins Modeling Monitor Mutagenesis Mutation Analysis Osmolar Concentration Pathogenesis Pathogenicity Plasma Proteins Plasmids Plasminogen Play Property Protein Family Proteins Rattus Recombinant Proteins Recombinants Renal Tissue Risk Role Scanning Serology Serum Site Structure Surface System Temperature Testing Therapeutic Agents Tissues United States Urine Vaccines Variant Veterans Virulence base design experimental study fitness gain of function host colonization in vitro activity in vivo in vivo imaging innovation knock-down knockout gene mutant neglect optical imaging pathogen prevent promoter transmission process urban area vector

项目摘要

Leptospirosis is a neglected global human health problem caused by transmission from reservoir hosts that harbor pathogenic Leptospira species in their kidneys and shed them into the environment via their urine. Our goal is to elucidate the role(s) of the surface-exposed leptospiral immunoglobulin- like (Lig) proteins in mechanisms of leptospiral pathogenesis and immunity. The LigA and LigB proteins exhibit high affinity binding to host ligands and inhibit complement activation, trigger plasminogen, and inhibit fibrin formation. However, initial studies found that ligA and ligB single gene knockout mutants were competent for infection. A recent study by one of our collaborators showed that knocking down expression of both ligA and ligB by targeting their identical promoters with the TALE system resulted in the loss of virulence, indicating that the functions of LigA and LigB are redundant. Despite the large and growing literature on their structure and function, the roles of the Lig proteins in virulence remain poorly understood. The in vitro activities of the Lig proteins suggest that their role is to resist host defense mechanisms. Our overall hypothesis is that discrete segments of the Lig proteins are required at an early step of infection to evade killing by the host. For this project, we will focus on LigB since ligA is missing from a majority of pathogenic Leptospira species. We will evaluate the functions of LigB in the context of the bacterial cell and determine which of its domains are responsible for these functions. We will also develop a LigB vaccine that provides cross-protective immunity. To accomplish these goals, we will employ powerful and innovative approaches to track infection including whole animal optical imaging and high-throughput parallel sequencing. These studies are vital for development of effective approaches for protection from and treatment of leptospirosis. Specific Aim 1. When are the Lig proteins critical for infection? The wild-type and TALE-lig knockdown strains of L. interrogans will be engineered to express bioluminescence. Hamsters will be infected with the bioluminescent strains, and infection will be monitored with whole animal in vivo imaging to determine when and where infection is halted when the Lig proteins are not generated. Specific Aim 2. What functions are mediated by LigB expressed on the bacterial cell surface? We will determine the bacterial cell surface functions mediated by the LigB by employing a “gain-of-function” strategy. We will transform L. biflexa with a ligB plasmid and test the ability of the “knock-in” strain to adhere to host plasma proteins, interfere with serum killing, activate plasminogen, and slow fibrin clot formation. We will also perform these experiments with the L. interrogans TALE-lig knockdown strain and with the knockdown strain harboring ligB plasmid to determine the contribution of LigB to these pathogenic functions. Specific Aim 3. What regions of LigB are responsible for their virulence properties? We will determine by mutation analysis which segments of LigB contain sites necessary for virulence. LigB variants generated by domain-swapping and alanine-scanning mutagenesis will be expressed from barcoded plasmids in the TALE-lig knockdown strain of L. interrogans. Leptospires expressing the LigB variants will be pooled and inoculated into hamsters. The fitness of the variants will be assessed by high throughput parallel sequencing. LigB variants that reduce fitness in vivo will undergo functional evaluation in vitro. Specifc Aim #4. Does LigB generate cross-protective immunity? Soluble recombinant proteins comprising different segments of LigB will be generated and tested for their immunoprotective potential in the hamster model of acute lethal leptospirosis. Immunized hamsters will be challenged with L. interrogans and L. kirschneri to assess cross-protection. Kidneys will be evaluated by culture, qPCR, serology, and immunohistochemistry to assess renal colonization.

钩端螺旋体病是由储存库传播引起的一个被忽视的全球人类健康问题宿主的肾脏中含有致病性钩端螺旋体物种，并通过以下途径将其排入环境中：我们的目标是阐明表面暴露的钩端螺旋体免疫球蛋白的作用。 LigA 和 LigB 是钩端螺旋体发病机制和免疫机制中的类似 (Lig) 蛋白。蛋白质表现出与宿主配体的高亲和力结合并抑制补体激活，触发纤溶酶原，然而，初步研究发现ligA和ligB单基因敲除突变体。我们的一位合作者最近的一项研究表明，击倒。通过使用 TALE 系统靶向 ligA 和 ligB 的相同启动子来表达 ligA 和 ligB，结果毒力丧失，表明 LigA 和 LigB 的功能是冗余的。关于 Lig 蛋白结构和功能的文献越来越多，但其在毒力中的作用仍然很差 Lig 蛋白的体外活性表明它们的作用是抵抗宿主防御。我们的总体假设是，Lig 蛋白的离散片段在某个时间点是必需的。感染的早期步骤，以避免被宿主杀死，对于这个项目，我们将重点关注 LigB，因为 ligA 是。大多数致病性钩端螺旋体物种中都缺失了 LigB。我们将了解细菌细胞的背景并确定其哪些结构域负责这些功能。还开发了一种提供交叉保护性免疫的 LigB 疫苗。为了实现这些目标，我们将。采用强大且创新的方法来追踪感染，包括整个动物光学成像和这些研究对于开发有效的方法至关重要。预防和治疗钩端螺旋体病。具体目标 1. Lig 蛋白何时对感染至关重要？敲除问号钩端螺旋体菌株将被改造为表达生物发光的仓鼠。感染生物发光菌株，并通过整个动物体内成像监测感染情况确定当不产生 Lig 蛋白时感染停止的时间和地点。具体目标 2. 细菌细胞表面表达的 LigB 介导哪些功能？我们将通过“功能获得”来确定 LigB 介导的细菌细胞表面功能我们将用 ligB 质粒转化 L. biflexa，并测试“敲入”菌株的粘附能力。宿主血浆蛋白，干扰血清杀灭，激活纤溶酶原，并减缓纤维蛋白凝块的形成。还将使用问号 L. TALE-lig 敲除菌株和含有 ligB 质粒的敲低菌株以确定 LigB 对这些致病功能的贡献。具体目标 3. LigB 的哪些区域对其毒力特性负责？通过突变分析确定 LigB 的哪些片段包含 LigB 变异所需的位点。通过域交换和丙氨酸扫描诱变生成的将由条形码表达表达 LigB 变体的问号钩端螺旋体 TALE-lig 敲低菌株中的质粒将。将通过高通量评估变体的适应性。降低体内适应性的 LigB 变体将在体外进行功能评估。具体目标#4。LigB 是否产生交叉保护性免疫？将生成包含 LigB 不同片段的组合物，并测试它们在急性致死性钩端螺旋体病的仓鼠模型将受到问号钩端螺旋体的攻击。 L. kirschneri 将通过培养、qPCR、血清学和评估交叉保护来评估肾脏。免疫组织化学评估肾脏定植。