The Leptospiral Outer Membrane Proteome & Immunity

描述(由申请人提供)： 这项建议的总体目标是定义钩端螺旋体表面蛋白质组和翻译后修饰与免疫的相关性。我们已经确定了一些暴露在表面的脂蛋白，它们在哺乳动物宿主感染期间表达。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，可能会影响宿主免疫系统的识别。脂蛋白是钩端螺旋体的主要表面抗原。问号钩端螺旋体不同血清型的基因组编码约168个脂蛋白。我们已经描述了一些这样的脂蛋白，将它们定位在内膜或外膜上，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。钩端螺旋体基因操作的方法现在可以确定将脂蛋白靶向外膜和钩端螺旋体表面所需的信号，就像Wolfram Zuckert最近对伯氏疏螺旋体脂蛋白所做的那样，他是螺旋体表面脂蛋白输出途径的出口商，也是这一提议的联合研究员。最近的蛋白质组学研究，包括与合作研究员卡罗琳·卡梅隆合作进行的研究，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。这将解释为什么在大肠杆菌中生产的重组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本质为创造有效的甲基化多肽疫苗提供了机会，该疫苗针对感染期间表达的脂蛋白表面表位。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？我们的假设是，与伯氏杆菌一样，钩端螺旋体脂蛋白通过LOL输出途径输出到钩端螺旋体表面。我们将通过将编码脂蛋白-GFP融合的基因转化为问号钩端螺旋体来检验这一假设，并测试它们对表面蛋白降解的敏感性。我们将确定 将脂蛋白定位到表面所需的系绳长度以及带负电荷的氨基酸在防止表面局部化中的作用。#2.体内LipL32甲基化如何改变其表面表位？我们的假设是，感染过程中甲基化增加会改变LipL32的抗原性。我们将从受感染的组织中分离出生物 并进一步定义在感染过程中发生甲基化的LipL32位点。根据LipL32晶体结构预测将暴露在表面的那些部位将接受感染衍生抗体和T细胞的识别测试。#3.哪些甲基化多肽在诱导保护性免疫方面最有效？感染衍生抗体和T细胞高度识别的甲基化多肽将在钩端螺旋体病的仓鼠模型中作为免疫保护性抗原进行检测。体外检测黏附抑制、生长抑制、杀菌活性和吞噬细胞功能，以确定保护性免疫的机制。

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.