The Leptospiral Outer Membrane Proteome & Immunity

钩端螺旋体外膜蛋白质组

基本信息

批准号：
8633396
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 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8633396
关键词：
Adherence Affect Algorithms Amino Acids Animals Antibodies Antigens Biological Assay Borrelia burgdorferi Charge Chimeric Proteins Collaborations Environment Epitopes Escherichia coli Genes Genome Goals Growth Hamsters Health Homelessness Housing Human Immune Immune response Immune system Immunity In Vitro Individual Infection Kidney Kidney Failure Length Leptospira Leptospira interrogans Leptospirosis Lipoproteins Masks Membrane Membrane Proteins Methods Methylation Methyltransferase Military Personnel Modeling Nature Order Spirochaetales Organism Pathway interactions Peptide Vaccines Peptides Post-Translational Protein Processing Predisposition Protein Subunits Proteolysis Proteome Proteomics Recombinants Research Research Personnel Risk Role Signal Transduction Site Staging Structure Surface Surface Antigens T-Lymphocyte Testing Tissues Tungsten United States Vaccines Veterans bactericide base genetic manipulation in vitro Assay in vivo killings neglect prevent public health relevance transmission process vaccine candidate

项目摘要

The overall goal of this proposal is to define the leptospiral surface proteome and the relevance of post-translational modifications to immunity. We have identified a number of surface-exposed lipoproteins that are expressed during infection of the mammalian host. However, many leptospiral surface lipoproteins remain to be identified and those that are known appear to undergo extensive post-translational modifications that likely affect recognition by the host immune system. Lipoproteins are dominant leptospiral surface antigens. The genome of Leptospira interrogans serovar Copenhageni encodes approximately 168 lipoproteins. We have described a number of these lipoproteins, localized them to either the inner or outer membrane, and determined whether they are surface exposed. L. interrogans has genes encoding two possible lipoprotein export pathways: The LOL pathway and Type II secretion. Methods for leptospiral genetic manipulation are now available to determine the signals required to target lipoproteins to the outer membrane and leptospiral surface, as has recently been achieved for the lipoproteins of Borrelia burgdorferi by Wolfram Z¿ckert, who is an export on spirochetal surface lipoprotein export pathways and a co-investigator on this proposal. Recent proteomic studies, including those performed in collaboration with co-investigator Caroline Cameron, reveal that many leptospiral surface proteins undergo post-translational modification, particularly by methylases. We now have evidence that the major outer membrane lipoprotein, LipL32, undergoes extensive differential methylation during infection. This would explain why recombinant LipL32 produced in E. coli is ineffective as a vaccine, even though it is an abundant surface lipoprotein. Understanding the nature of surface lipoprotein methylation provides an opportunity to create effective methylated peptide vaccines that target lipoprotein surface epitopes expressed during infection. The Research Plan has the following three Specific Aims: #1. What is the leptospiral surface lipoprotein export pathway? Our hypothesis is that, as in B. burgdorferi, leptospiral lipoproteins are exported to the leptospiral surface via the LOL export pathway. We will test this hypothesis by transforming L. interrogans with genes encoding lipoprotein-GFP fusions and test their susceptibility to surface proteolysis. We will determine the length of the tether needed for targeting lipoproteins to the surface and the role of negative-charged amino acids in preventing surface localization. #2. How does in vivo LipL32 methylation alter its surface epitopes? Our hypothesis is that increased methylation during infection alters the antigenic character of LipL32. We will isolate organisms from infected tissues and further define LipL32 sites that become methylated during infection. Those sites that are predicted to be surface-exposed based on the LipL32 crystal structure will be tested for recognition by infection-derived antibodies and T-cells. #3. Which methylated peptides are most effective at inducing protective immunity? Methylated peptides that are highly recognized by infection-derived antibodies and T-cells will be examined as immunoprotective antigens in the hamster model of leptospirosis. In vitro assays examining adherence inhibition, growth inhibition, bactericidal activity, and opsonophagocytosis will be performed to determine mechanisms of protective immunity.

该提案的总体目标是定义钩端螺旋体表面蛋白质组和我们已经确定了一些翻译后修饰与免疫的相关性。哺乳动物宿主感染期间表达的表面暴露脂蛋白。然而，许多钩端螺旋体表面脂蛋白仍有待鉴定，而那些已知的似乎经历了广泛的翻译后修饰，可能会影响识别宿主免疫系统。脂蛋白是主要的钩端螺旋体表面抗原。我们已经描述了哥本哈根问号血清型编码大约 168 种脂蛋白。许多这样的脂蛋白，将它们定位于内膜或外膜，并且确定它们是否暴露在表面。问号钩体是否具有编码两种可能的基因。脂蛋白输出途径：LOL 途径和钩端螺旋体 II 型分泌方法。现在可以通过基因操作来确定将脂蛋白靶向所需的信号外膜和钩端螺旋体表面，正如最近在脂蛋白中所实现的那样伯氏疏螺旋体 (Borrelia burgdorferi) 作者：Wolfram Z¿ ckert，螺旋体表面脂蛋白的输出者出口途径和该提案的联合研究员。最近的蛋白质组学研究，包括与共同研究者合作进行的研究卡罗琳·卡梅伦 (Caroline Cameron) 揭示许多钩端螺旋体表面蛋白经历翻译后修饰，特别是通过甲基化酶，我们现在有证据表明主要的外膜。脂蛋白 LipL32 在感染过程中会发生广泛的差异甲基化。解释为什么在大肠杆菌中产生的重组 LipL32 作为疫苗无效，尽管它是了解表面脂蛋白甲基化的性质。提供了创建针对脂蛋白的有效甲基化肽疫苗的机会感染期间表达的表面表位。该研究计划有以下三个具体目标： #1.什么是钩端螺旋体表面脂蛋白输出途径？与伯氏疏螺旋体一样，钩体脂蛋白通过 LOL 输出途径。我们将通过用基因转化问号钩端螺旋体来检验这一假设。编码脂蛋白-GFP 融合体并测试它们对表面蛋白水解的敏感性。确定将脂蛋白靶向表面所需的系链长度以及带负电荷的氨基酸可防止表面定位。 #2. 体内 LipL32 甲基化如何改变其表面表位？感染过程中甲基化的增加会改变 LipL32 的抗原特性。从受感染的组织中分离生物体并进一步确定甲基化的 LipL32 位点感染期间根据 LipL32 预测会暴露于表面的部位。晶体结构将通过感染源抗体和 T 细胞的识别进行测试。 #3. 哪些甲基化肽最能有效诱导保护性免疫？被感染源性抗体和 T 细胞高度识别的甲基化肽将被在钩端螺旋体病仓鼠模型中作为免疫保护抗原进行了体外测定。检查粘附抑制、生长抑制、杀菌活性和调理吞噬作用将进行以确定保护性免疫的机制。