The Leptospiral Outer Membrane Proteome & Immunity

该提案的总体目标是定义钩端螺旋体表面蛋白质组， 翻译后修饰与免疫的相关性。我们已经确认了一个号码 在哺乳动物宿主感染过程中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， 似乎经历了广泛的翻译后修饰，这可能会影响识别的 宿主免疫系统 脂蛋白是钩端螺旋体的主要表面抗原。钩端螺旋体的基因组 哥本哈根型问号线虫编码约168种脂蛋白。我们已经描述 这些脂蛋白的数量，定位于内膜或外膜， 确定它们是否暴露在表面。L.问号有两种可能的基因编码 脂蛋白输出途径：LOL途径和II型分泌。钩端螺旋体检测方法 基因操作现在可用于确定靶向脂蛋白所需的信号， 外膜和钩端螺旋体表面，如最近对脂蛋白所实现的 疏螺旋体burgdorferi由Wolfram Z <$ckert，谁是螺旋体表面脂蛋白的出口 出口途径和一名共同调查员。 最近的蛋白质组学研究，包括与合作研究者合作进行的研究 Caroline卡梅隆等揭示了许多钩端螺旋体表面蛋白经过翻译后 修饰，特别是通过甲基化。我们现在有证据表明 脂蛋白LipL 32在感染期间经历广泛的差异甲基化。这将 解释了为什么重组LipL 32在E.大肠杆菌作为疫苗是无效的，尽管它是 丰富的表面脂蛋白。了解表面脂蛋白甲基化的性质 提供了一个机会，创造有效的甲基化肽疫苗， 在感染期间表达的表面表位。 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 就像在B中一样。螺旋体脂蛋白通过螺旋体膜输出到螺旋体表面。 LOL输出路径。我们将通过变换L来检验这个假设。带有基因的问号 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。我们将 确定将脂蛋白靶向表面所需的系链的长度以及 带负电荷的氨基酸防止表面定位。 #2.体内LipL 32甲基化如何改变其表面表位？我们的假设 在感染过程中甲基化的增加改变了LipL 32的抗原特性。我们将 从感染组织中分离生物体并进一步确定甲基化LipL 32位点 在感染期间。根据LipL 32预测为表面暴露的部位 晶体结构将被感染衍生的抗体和T细胞识别。 #3.哪种甲基化肽在诱导保护性免疫方面最有效？ 被感染源性抗体和T细胞高度识别的甲基化肽将被 在钩端螺旋体病的仓鼠模型中作为免疫保护性抗原检查。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 以确定保护性免疫的机制。

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.