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性仍然是新发传染病研究中的一个知识缺口，与改善公共卫生特别相关。在蜱-哺乳动物传染周期中，通过不断适应不同的环境，蜱传疾病继续给人类健康造成负担。回归热（RF）螺旋体就是这样一种病原体，可引起严重的发病率和死亡率，但关于媒介定植和传播的机制知之甚少。当RF螺旋体进入蜱虫时，它们必须适应媒介内不同的环境以继续其生命周期，最初定植于中肠，然后是唾液腺。然而，自从RF螺旋体在1905年首次被确定为引起人类疾病以来，已经确定了两种与蜱虫相关的蛋白质。为了解决这一缺点，我们开发了一个体内系统来研究蜱-哺乳动物的完整感染循环，这是一种螺旋体。已经确定了一个包含一系列基因的位点，螺旋体在蜱定植过程中向上调节。迄今为止，这是节肢动物载体中最全面的RF螺旋体基因表达分析。预计这些基因编码外膜蛋白（Omps），鉴于Omps在宿主病原体适应和作为疫苗靶点中的重要性，这些蛋白值得进一步研究。因此，被鉴定的基因被假设为编码在蜱虫媒介的中肠和唾液腺中产生差异的Omps。目的1：确定预测的Omps在体外的表达和表面定位。针对每一种可能的重组Omp产生的兔免疫血清将用于确定螺旋体在蜱虫样条件下生长时的表面定位和增加的蛋白质产量。目的2：确定蜱的中肠和唾液腺内Omp的产生。一种方法已开发冷冻保存和切片蜱，明确区分螺旋体种群在中肠和唾液腺。

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.