Surface Lipoproteins and Serum Resistance in Borrelia burgdorferi

描述(申请人提供)：莱姆病是由螺旋体引起的，这些螺旋体被分成三个不同的基因种：伯氏疏螺旋体、阿氏疏螺旋体和加里尼氏疏螺旋体。有趣的是，在感染期间，这三个基因种似乎都存在于哺乳动物宿主的不同生态位中。例如，伯氏杆菌和阿氏杆菌通常与涉及心脏、关节和皮肤的疾病有关，而加里尼氏杆菌通常会导致中枢神经系统异常。最近发现伯氏杆菌和阿氏杆菌都能与宿主血清补体抑制因子H结合在细胞表面，这可能增强了它们对血清的抵抗力。相比之下，大多数加里尼氏杆菌分离株不结合H因子，可被哺乳动物血清杀死。这一观察结果表明，加里尼双胞菌生物持续感染中枢神经系统，因为它是一个有限的细胞毒性补体的环境。最近，我们等人在体外发现了几种能与因子H结合的伯氏杆菌外表面脂蛋白(OSP)。最相关的H因子结合蛋白包括与OSPE相关的脂蛋白家族以及名为CSPA的脆性表面脂蛋白。与它们结合因子H的能力一致，所有这些表面脂蛋白都被证明可以增强血清抵抗力。我们和其他人之前已经报道过，在血清敏感的加里尼氏杆菌株中表达伯氏杆菌297 OSPE蛋白会增加对血清介导的杀伤的抵抗力。我们还表明，在伯氏杆菌强毒株中突变CSPA基因会导致突变，该突变对血清介导的杀伤非常敏感。此外，我们最近还表明，伯氏杆菌在吸食哺乳动物血液的过程中，需要CSPA的表达才能在硬蜱的中肠中存活。这些联合观察结果与我们的假设一致，即CSPA和OSPE表面脂蛋白是伯氏杆菌血清耐药性的组成部分。为了直接检验这一潜在的假设，并进一步检验这些蛋白质在伯氏杆菌从扁虱传播到哺乳动物中所起的机制作用，我们提出了以下两个特定的目标。在具体目标1中，我们将使用我们最近产生的几个新菌株来研究CSPA在毒力和疾病发病机制中的作用，使用莱姆病的小鼠模型。在具体目标2中，我们将检测一株在伯氏杆菌野生型和我们的CSPA突变株中都缺乏OSPE相关脂蛋白表达的菌株，以更好地评估它们在血清抵抗和莱姆病发病机制中的作用(S)。最后，在修订的提案中，我们包括了更多基于机制的研究，以确定因子H与CSPA和OSPE结合是否真的促进了C3b在该生物体表面降解为ICC3b，这是该领域目前的教条。自从我们上次提交以来，我们已经产生了有趣的数据，表明H因子结合蛋白可能毕竟不会促进伯氏杆菌表面iC3b的生成，我们将在我们修订的目标2中更详细地研究这一观察结果。 与公共卫生相关：联合研究可能导致确定新的机制，使莱姆病细菌在感染后逃避最初的宿主免疫监测系统。从拟议的研究中获得的信息可以用来产生一种新的疫苗或莱姆病的疾病调节疗法。鉴于莱姆病是美国最流行的扁虱传播感染，从拟议的研究中获得的独特见解可能会导致莱姆病的新疫苗战略，这将极大地有利于美国和国外的整体公众健康。

DESCRIPTION (provided by applicant): Lyme disease is caused by spirochetes that have been grouped into three different genospecies, Borrelia burgdorferi, B. afzelii, and B. garinii. Interestingly, all three genospecies seem to populate different niches in the mammalian host during infection. For example, B. burgdorferi and B. afzelii are commonly associated with disease involving the heart, joints, and skin, while B. garinii often causes central nervous system abnormalities. It was recently shown that both B. burgdorferi and B. afzelii bind host serum complement inhibitor factor H on their cell surface, which possibly enhances their resistance to serum. By contrast, most B. garinii isolates do not bind factor H and are killed by mammalian serum. This observation has led to the suggestion that B. garinii organisms persistently infect the central nervous system because it is an environment limited in cytotoxic complement. Recently, several B. burgdorferi outer surface lipoproteins (Osp) were identified by us and others that can bind factor H in vitro. The factor H binding proteins that are most relevant include a family of lipoproteins related to OspE as well as a borrelial surface lipoprotein designated CspA. Consistent with their ability to bind factor H, all of these surface lipoproteins have been shown to enhance serum resistance. We and others have previously reported that expressing the B. burgdorferi strain 297 OspE protein in a serum sensitive B. garinii strain results in increased resistance to serum-mediated killing. We also have shown that mutating the cspA gene in a virulent strain of B. burgdorferi results in a mutant that is exquisitely sensitive to serum-mediated killing. Furthermore, we also have recently shown that CspA expression is required for B. burgdorferi to survive in the midgut of ticks as they engorge on mammalian blood. The combined observations are consistent with our hypothesis that CspA and the OspE surface lipoproteins are integral to serum resistance in B. burgdorferi. To directly examine this underlying hypothesis and further examine the mechanistic role played by these proteins in transmission of B. burgdorferi from the tick to mammal, we propose the following two Specific Aims. In Specific Aim 1 we will use several novel strains we recently generated to examine the role of CspA in virulence and disease pathogenesis using the mouse model of Lyme disease. In Specific Aim 2 we will examine a strain that lacks expression of the OspE-related lipoproteins in both wild type B. burgdorferi and in our CspA mutant strain to better assess their role(s) in serum resistance and Lyme disease pathogenesis during the borrelial enzootic cycle. Finally, in the revised proposal we have included more mechanism-based studies to determine if factor H binding to CspA and OspE actually enhances degradation of C3b to iC3b on the surface of this organism, which is the current dogma in the field. Since our last submission, we have generated interesting data indicating that factor H binding proteins may not enhance generation of iC3b on the surface of B. burgdorferi after all, and we will examine this observation in more detail in our revised Aim 2. PUBLIC HEALTH RELEVANCE: The combined studies could result in the identification of new mechanisms that allow Lyme disease bacteria to evade the initial host immune surveillance system after infection. Information derived from the proposed studies could be used to generate a new vaccine or disease modulating therapeutic for Lyme disease. Given that Lyme disease is the most prevalent tick-borne infection in the United States, unique insight from the proposed studies that could lead to a new vaccine strategy for Lyme disease could greatly benefit overall public health in the United States and abroad.