Relapsing fever spirochete protein production within the vector

描述(由申请人提供)：了解微生物在不同自然环境中的适应仍然是新发传染病研究中的一个知识空白，对改善公共健康特别相关。硬虱传播的疾病通过在硬虱-哺乳动物感染周期中不断适应变化的环境，继续给人类健康带来负担。复发性热(RF)螺旋体就是这样一种病原体，可导致严重的发病率和死亡率，但有关病媒定植和传播的机制却知之甚少。当RF螺旋体进入扁虱体内时，它们必须适应媒介内不同的环境才能继续它们的生命周期，最初在中肠和唾液腺定植，然而自从1905年RF螺旋体首次被发现导致人类疾病以来，已经鉴定出两种与扁虱相关的蛋白。为了解决这一缺陷，我们建立了一套体内系统来研究RF螺旋体中的疏螺旋体-哺乳动物感染的完整循环。已经确定了一个基因座，其中包含一系列基因，螺旋体在壁虱定植过程中上调这些基因。到目前为止，这是节肢动物载体中最全面的RF螺旋体基因表达分析。这些基因被预测为编码外膜蛋白(OMP)，鉴于OMPS在宿主内病原体适应和作为疫苗靶标方面的重要性，这些蛋白值得进一步研究。因此，已识别的基因被假设为OMP的编码，这些OMP是在扁虱载体的中肠和唾液腺中差异产生的。以下目的将验证这一假设：目的1：确定预测的OMPS在体外的表达和表面定位。针对每个可能的重组OMP产生的兔免疫血清将用于确定螺旋体在类扁虱条件下生长时的表面定位和增加的蛋白质产量。目的2：测定硬蜱中肠和唾液腺的OMP产量。已经开发出一种方法来冷冻保存和切片扁虱，清楚地区分中肠和唾液腺中的螺旋体种群。 扁虱切片将用针对给定OMP产生的兔血清进行探测，以确定中肠和唾液腺中蛋白质的产生。完成这一项目将与NIAID更好地了解和预防疾病的使命不谋而合。该项目将对RF螺旋体在中肠和唾液腺中产生的表面蛋白进行最全面的鉴定，并指导研究扁虱定植和传播过程中蛋白质功能和OMPS需求的机制研究。这一结果还将指导针对RF螺旋体的疫苗开发，针对唾液腺中产生的细菌OMP，从而在细菌进入宿主时中和它们。

DESCRIPTION (provided by applicant): Understanding microbial adaptation within different natural environments remains a gap in knowledge in emerging infectious disease research and is particularly relevant toward improving public health. Tick-borne diseases continue to burden human health by continuously adapting in varying environments during the tick- mammalian infectious cycle. Relapsing fever (RF) spirochetes are one such pathogen, causing severe morbidity and mortality, yet little is known regarding the mechanisms involved with vector colonization and trans- mission. As RF spirochetes enter the tick they must adapt to disparate environments within the vector to continue their life-cycle, initially colonizing the midgut and thn the salivary glands, yet since RF spirochetes were first identified to cause human disease in 1905, two tick-associated proteins have been identified. To address this shortcoming, an in vivo system has been developed to study the complete tick- mammalian infectious cycle of Borrelia turicatae, a species of RF spirochete. A locus has been identified containing a series of genes that the spirochetes up-regulate during tick colonization. To date, this is the most comprehensive gene expression analysis of RF spirochetes within the arthropod vector. The genes are predicted to encode for outer membrane proteins (Omps), and given the importance of Omps in pathogen adaptation within the host and as vaccine targets, the proteins warrant further investigation. Thus, the identified genes are hypothesized to encode for Omps that are differentially produced within the midgut and salivary glands of the tick vector. The following aims will test the hypothesis: Aim 1: Determine the expression and surface localization of the predicted Omps in vitro. Rabbit immune serum generated against each putative recombinant Omp will be used to determine the surface localization and increased protein production when spirochetes are grown in tick-like conditions. Aim 2: Determine Omp production within the midgut and salivary glands of the tick. A method has been developed to cryopreserve and section ticks, clearly distinguishing spirochete populations within the midgut and salivary glands. Tick sections will be probed with rabbit serum generated against a given Omp to deter- mine protein production in the midgut and salivary glands. Completing this project will coincide with NIAID's mission to better understand and prevent disease. This project will result in the most comprehensive identification of surface proteins RF spirochete produce with- in the midgut and salivary glands, and guide mechanistic studies investigating protein function and the requirement of Omps during tick colonization and transmission. The results will also direct vaccine development against RF spirochetes, targeting bacterial Omps produced within the salivary glands, consequently neutralizing the bacteria as they enter the host.