Borrelia burgdorferi Interactions with Mammalian Host Cells

描述(申请人提供)：莱姆病，由螺旋体伯氏疏螺旋体细菌引起，是美国最流行的节肢动物传播感染，由于报告病例数量的显著增加，已被归类为重新出现的媒介传播感染。莱姆病是一个重要的公共卫生问题，特别是在流行州，在这些州，莱姆病的发病率很高。伯氏杆菌在哺乳动物中引起感染的能力取决于它快速改变基因表达的能力，以响应它通过扁虱叮咬传播时遇到的高度不同的环境信号。几个全基因组的转录分析，比较了在模拟扁虱载体或哺乳动物宿主的条件下培养的伯氏杆菌，发现在哺乳动物宿主特有的条件下，线性质粒54(IP54)上的几个质粒编码基因上调。对其中几个差异表达的ORF进行的基于序列的二级结构分析表明，BBA34在N-末端有一个精氨酸-甘氨酸-谷氨酸(RGD)基序，该基序可能是表面暴露的。此外，基于序列相似性，BBA34是寡肽渗透酶A(OppA5)蛋白的同源蛋白，但不像其他染色体编码的OPA同源蛋白那样与七肽结合，在传播到哺乳动物宿主时在伯氏杆菌中上调表达。 这项建议的目的是确定BBA34上的RGD基序是否会促进与哺乳动物细胞的相互作用，可能是通过与整合素的直接相互作用。第一个特定目的是确定重组BBA34蛋白与哺乳动物细胞纯化或表达的各种整合素的相互作用。我们将使用全长、N端和C端的重组BBA34蛋白来确定RGD基序在与各种整合素结合中的作用。第二个特定目的是确定伯氏杆菌bba34突变株在莱姆病小鼠模型中的结合、传染性和组织传播特性。第三个特异性目的是鉴定伯氏杆菌bba34-/bbk32-和bba34-/p66-双突变体在莱姆病小鼠模型中的结合和感染性。我们将确定多种硼决定因素的丢失对宿主细胞黏附的意义。这些研究将有助于确定宿主-病原体之间的相互作用，这些相互作用对伯氏杆菌在哺乳动物宿主上的定殖至关重要。

DESCRIPTION (provided by applicant): Lyme disease, caused by the spirochetal bacterium Borrelia burgdorferi, is the most prevalent arthropod-borne infection in the United States and has been classified as a re-emerging vector-borne infection due to a significant increase in the number of reported cases. Lyme disease is an important public health issue, particularly in endemic states where it contributes to significant rates of morbidity. The ability of B. burgdorferi to cause infection in mammals is dependent on its ability to alter gene expression rapidly in response to highly disparate environmental signals that it encounters upon transmission through a tick bite. Several genome-wide transcriptional analyses, comparing B. burgdorferi cultivated under conditions that mimic the tick vector or the mammalian host, revealed that several plasmid encoded genes on linear plasmid 54 (Ip54) are up-regulated under mammalian host-specific conditions. Sequence based secondary structure analysis of several of these differentially expressed ORFs revealed that BBA34 has an arginine-glycine-glutamic acid (RGD) motif at the N-terminal region and that this motif is probably surface-exposed. Moreover, based on sequence similarity, BBA34 is an orthologue of oligopeptide permease A (OppA5) protein but does not bind heptapeptides like other chromosomally encoded OppA orthologues and is up-regulated in B. burgdorferi upon transmission to mammalian host. The objective of this proposal is to determine if the RGD motif on BBA34 will facilitate interactions with mammalian cells presumably through direct interactions with integrins. The first specific aim is to determine the interactions of recombinant BBA34 proteins with various integrins either purified or expressed on mammalian cells. We will determine the role of RGD motif in binding to various integrins using full-length, N-terminal and C-terminal recombinant BBA34 proteins. The second specific aim is to determine the binding, infectivity and tissue dissemination characteristics of a bba34- mutant of B. burgdorferi in the murine model of Lyme disease. The third specific aim is to characterize the binding and infectivity of bba34-/bbk32- and bba34-/p66- double mutants of B. burgdorferi in the murine model of Lyme disease. We will determine the significance of the loss of multiple borrelial determinants on host-cell adherence. These studies will help to determine the host-pathogen interactions that are critical for the colonization of the mammalian host by B. burgdorferi.