Functions of Leptospira Lig Proteins in the Pathogenesis of Leptospirosis

钩端螺旋体病是由水库传播引起的一个被忽视的全球人类健康问题 在肾脏中含有致病钩端螺旋体的宿主，并通过 他们的尿液。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(S)。 类(Lig)蛋白在钩端螺旋体致病和免疫机制中的作用LIGA和LIGB 蛋白质表现出与宿主配体的高亲和力结合，抑制补体激活，触发纤溶酶原， 并抑制纤维蛋白的形成。然而，初步研究发现，LIGA和LIGB单基因敲除突变体 都有能力被感染。我们的一位合作者最近的一项研究表明，击倒 通过用TALL系统靶向其相同的启动子来表达LIGA和LigB，导致 毒力丧失，说明LigA和LigB的功能是多余的。尽管有很大的 关于它们的结构和功能的文献越来越多，但关于Lig蛋白在毒力中的作用仍然很少 明白了。Lig蛋白的体外活性表明它们的作用是抵抗宿主防御。 机械装置。我们的总体假设是，Lig蛋白的离散片段在 感染的早期步骤，以躲避宿主的杀戮。对于这个项目，我们将重点关注LigB，因为LIGA是 从大多数致病钩端螺旋体物种中缺失。我们将评估LigB在 细菌细胞的环境，并确定它的哪些区域负责这些功能。我们会 同时开发一种能提供交叉保护免疫的LigB疫苗。为了实现这些目标，我们将 使用强大的创新方法来跟踪感染，包括整个动物的光学成像和 高通量并行测序。这些研究对于发展有效的方法至关重要。 预防和治疗钩端螺旋体病。 具体目的1.Lig蛋白何时对感染起关键作用？野性的和童话般的 敲除的问号钩端螺旋体菌株将被改造成表达生物发光。仓鼠将会是 感染的生物发光菌株，感染将监测整个动物的活体成像 当没有产生Lig蛋白时，确定何时何地停止感染。 特定目的2.细菌细胞表面表达的LigB有哪些功能？ 我们将通过使用“功能增益”来确定由LigB介导的细菌细胞表面功能 策略。我们将用一个igB质粒转化双弯乳杆菌，并测试其黏附能力。 承载血浆蛋白，干扰血清杀伤，激活纤溶酶原，减缓纤维蛋白凝块的形成。我们 也将对问号钩端螺旋体的童话唇基因敲除株和 以确定LigB在这些致病功能中的作用。 具体目的3.LigB的哪些区域负责它们的毒力特性？我们会 通过突变分析确定LigB的哪些片段包含毒力所必需的位点。LigB变体 通过结构域交换和丙氨酸扫描突变产生的将从条形码表达 问号钩端螺旋体基因敲除株中的质粒。表达LigB变异体的钩端螺旋体 被集中起来并接种到仓鼠体内。变体的适合性将通过高吞吐量进行评估 并行测序。降低体内适合度的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？可溶性重组蛋白 将产生包含不同片段的LigB，并在 急性致死性钩端螺旋体病仓鼠模型。接种疫苗的仓鼠将受到问号志贺氏菌和 L.kirschneri来评估交叉保护。肾脏将通过培养、定量聚合酶链式反应、血清学和 免疫组织化学方法评估肾脏定植情况。

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.