The Leptospiral Outer Membrane Proteome & Immunity

描述（由申请人提供）： 这个建议的总体目标是定义钩端螺旋体表面蛋白质组和免疫的翻译后修饰的相关性。我们已经确定了一些表面暴露的脂蛋白，表达在感染的哺乳动物宿主。然而，许多钩端螺旋体表面脂蛋白仍有待鉴定，并且已知的那些脂蛋白似乎经历了广泛的翻译后修饰，这可能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。我们已经描述了一些这样的脂蛋白，将它们定位于内膜或外膜，并确定它们是否暴露于表面。L.问号线虫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用于确定将脂蛋白靶向外膜和钩端螺旋体表面所需的信号，正如Wolfram Zuckert最近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出口，也是该提案的共同研究者。 最近的蛋白质组学研究，包括与共同研究者Caroline卡梅隆合作进行的研究，揭示了许多钩端螺旋体表面蛋白质经历翻译后修饰，特别是 甲基化酶。我们现在有证据表明，主要的外膜脂蛋白，LipL 32，在感染过程中经历了广泛的差异甲基化。这可以解释为什么重组LipL 32在大肠杆菌中产生。大肠杆菌作为疫苗是无效的，即使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提供了一个机会，以创造有效的甲基化肽疫苗，在感染过程中表达的脂蛋白表面表位的目标。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，就像在B。在伯氏螺旋体中，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。我们将通过变换L来检验这个假设。用编码脂蛋白-GFP融合的基因转染问号线虫，并测试它们对表面蛋白水解的易感性。康贝特人将以 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LipL 32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LipL 32的抗原特性。我们将从受感染的组织中分离出有机体 并进一步定义感染期间甲基化的LipL 32位点。将测试基于LipL 32晶体结构预测为表面暴露的那些位点是否被感染源性抗体和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？在钩端螺旋体病仓鼠模型中，将检查被感染源性抗体和T细胞高度识别的甲基化肽作为免疫保护性抗原。将进行体外试验，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。

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.