Functions of Leptospira Lig Proteins in the Pathogenesis of Leptospirosis

钩端螺旋体病是一个被忽视的全球性人类健康问题，由水库传播引起 宿主的肾脏中携带致病性钩端螺旋体，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， 类（Lig）蛋白在钩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并抑制补体激活，触发纤溶酶原， 并抑制纤维蛋白形成。然而，最初的研究发现，ligA和ligB单基因敲除突变体， 有能力感染。我们的一位合作者最近的一项研究表明， 通过用TALE系统靶向其相同的启动子表达ligA和ligB两者导致了 毒力丧失，表明LigA和LigB的功能是冗余的。尽管规模庞大， 关于其结构和功能的文献越来越多，但Lig蛋白在毒力中的作用仍然很差 明白Lig蛋白的体外活性表明其作用是抵抗宿主防御 机制等我们的总体假设是，Lig蛋白的离散片段是必需的， 感染的早期阶段，以逃避宿主的杀死。对于这个项目，我们将专注于LigB，因为LigA是 在大多数致病性钩端螺旋体物种中缺失。我们将评估LigB在 细菌细胞的背景，并确定其哪些结构域负责这些功能。我们将 还开发了一种提供交叉保护免疫的LigB疫苗。为了实现这些目标，我们将 采用强大和创新的方法来跟踪感染，包括整个动物的光学成像， 高通量平行测序。这些研究对于制定有效的方法， 预防和治疗钩端螺旋体病。 具体目标1。什么时候Lig蛋白对感染至关重要？野生型和TALE配体 L.问号将被设计成表达生物发光。仓鼠将 感染生物发光菌株，并将用整个动物体内成像监测感染， 确定当Lig蛋白不产生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介导了哪些功能？ 我们将通过采用“功能获得性”方法来确定LigB介导的细菌细胞表面功能。 战略我们将把L。用ligB质粒转染双曲酶，并测试“敲入”菌株粘附的能力 宿主血浆蛋白，干扰血清杀伤，激活纤溶酶原，并减缓纤维蛋白凝块形成。我们 也将用L.问号TALE-lig敲低菌株， 携带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的哪些区域负责其毒力特性？我们将 通过突变分析确定LigB的哪些片段含有毒力所必需的位点。LigB变体 通过结构域交换和丙氨酸扫描诱变产生的DNA将从条形码化的 质粒在L.质问者表达LigB变体的钩端螺旋体将 合并并接种到仓鼠中。将通过高通量评估变体的适应性 平行测序降低体内适应性的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？可溶性重组蛋白 将产生包含LigB的不同区段的免疫原性抗体，并测试它们在免疫应答中的免疫保护潜力。 急性致死性钩端螺旋体病仓鼠模型。将用L.提问者和 L. kirschneri以评估交叉保护。将通过培养、qPCR、血清学和 免疫组织化学评估肾定植。

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.