The Leptospiral Outer Membrane Proteome & Immunity

钩端螺旋体外膜蛋白质组

基本信息

批准号：
8811327
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/8811327
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 Methylation 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 transmission process vaccine candidate

项目摘要

DESCRIPTION (provided by applicant): 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 Zuckert, 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.

描述（由申请人提供）：该提案的总体目标是定义钩端螺旋体表面蛋白质组以及翻译后修饰与免疫的相关性。我们已经鉴定出许多在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，已知的那些似乎经历了广泛的翻译后修饰，可能影响宿主免疫系统的识别。脂蛋白是主要的钩端螺旋体表面抗原。问号钩端螺旋体哥本哈根血清型的基因组编码大约 168 种脂蛋白。我们已经描述了许多这样的脂蛋白，将它们定位于内膜或外膜，并确定它们是否暴露在表面。问号钩端螺旋体具有编码两种可能的脂蛋白输出途径的基因：LOL途径和II型分泌途径。现在可以使用钩体基因操作方法来确定将脂蛋白靶向外膜和钩体表面所需的信号，正如最近由 Wolfram Zuckert 对伯氏疏螺旋体的脂蛋白所实现的那样，Wolfram Zuckert 是螺旋体表面脂蛋白输出途径的输出者，该提案的联合研究员。最近的蛋白质组学研究，包括与共同研究员 Caroline Cameron 合作进行的研究，揭示了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 在感染过程中经历了广泛的差异甲基化。这可以解释为什么在大肠杆菌中产生的重组 LipL32 作为疫苗无效，尽管它是一种丰富的表面脂蛋白。了解表面脂蛋白甲基化的性质为创建针对感染期间表达的脂蛋白表面表位的有效甲基化肽疫苗提供了机会。该研究计划有以下三个具体目标：#1。钩端螺旋体表面脂蛋白输出途径是什么？我们的假设是，与伯氏疏螺旋体一样，钩体脂蛋白通过 LOL 输出途径输出到钩体表面。我们将通过用编码脂蛋白-GFP融合的基因转化问号钩体来检验这一假设，并测试它们对表面蛋白水解的敏感性。我们将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在防止表面定位中的作用。 #2.体内 LipL32 甲基化如何改变其表面表位？我们的假设是感染过程中甲基化的增加改变了 LipL32 的抗原特征。我们将从受感染的组织中分离出生物体并进一步确定在感染过程中甲基化的 LipL32 位点。根据 LipL32 晶体结构预计会暴露在表面的那些位点将被测试以检测感染衍生抗体和 T 细胞的识别情况。＃3。哪些甲基化肽在诱导保护性免疫方面最有效？感染源性抗体和 T 细胞高度识别的甲基化肽将作为钩端螺旋体病仓鼠模型中的免疫保护性抗原进行检查。将进行检查粘附抑制、生长抑制、杀菌活性和调理吞噬作用的体外测定，以确定保护性免疫机制。