The Leptospiral Outer Membrane Proteome & Immunity

这项建议的总体目标是定义钩端螺旋体表面蛋白质组和 翻译后修饰与免疫力的相关性。我们已经确认了一个号码 在哺乳动物宿主感染期间表达的暴露于表面的脂蛋白。 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改，这可能会影响 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 文森氏血清型编码约168种脂蛋白。我们已经描述了 许多这样的脂蛋白，将它们定位在内膜或外膜上，以及 已确定它们是否暴露在表面。问号钩端螺旋体有编码两种可能的基因 脂蛋白输出途径：LOL途径和II型分泌。钩端螺旋体的治疗方法 基因操作现在可以用来确定靶向脂蛋白所需的信号 外膜和钩端螺旋体表面，就像最近对脂蛋白所达到的那样 Wolfram Z？ckert的伯氏疏螺旋体，他是螺旋体表面脂蛋白的出口商 出口途径和一名关于这项提议的联合调查员。 最近的蛋白质组学研究，包括与合作研究员合作进行的研究 Caroline Cameron，揭示许多钩端螺旋体表面蛋白经历翻译后 修饰，尤其是通过甲基酶的修饰。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。这将会 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。理解表面脂蛋白甲基化的本质 提供了创造有效的针对脂蛋白的甲基化多肽疫苗的机会 在感染过程中表达的表面表位。 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 就像在伯氏杆菌中一样，钩端螺旋体的脂蛋白通过 LOL输出途径。我们将通过用基因转化问号杆菌来检验这一假设 编码脂蛋白-绿色荧光蛋白融合，并测试其对表面蛋白分解的敏感性。我们会 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方面。 #2.体内LipL32甲基化如何改变其表面表位？我们的假设 感染过程中甲基化增加改变了LipL32的抗原性。我们会 从受感染的组织中分离生物，并进一步定义甲基化的LipL32位点 在感染期间。根据LipL32预测将暴露在表面的那些部位 将测试晶体结构，以识别感染来源的抗体和T细胞。 #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是 在钩端螺旋体病的仓鼠模型中被检测为免疫保护性抗原。体外试验 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。

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.