Tlr1: Functional and Genetic Variation in Urinary Tract Infection

描述(申请人提供)：尿路感染(UTI)是发达国家最常见的细菌感染，致尿性大肠杆菌(UPEC)占这些感染的80%以上。发生在2-7%的女性中的频繁复发(RUTI)的最强烈的危险因素之一是RuTI的家族史，这表明UTI易感性的遗传因素。这一结论进一步得到了UTI小鼠模型的支持，该模型表明小鼠对UTI的易感性存在高水平的菌株对菌株的变异。 对UTI易感品系和耐药品系中保守的单核苷酸多态(SNPs)进行的全基因组电子分析发现，在天然免疫受体Toll样受体1(TLR1)的蛋白编码部分存在几个非同义SNP，以及一个SNP被预测破坏了该基因的一个剪接点。TLR1蛋白是一种模式识别受体，它与TLR2复合，检测细菌脂蛋白，并激活免疫系统，以响应这种刺激。初步结果表明，与耐药株C57BL/6相比，耐药株C3H/HEN的巨噬细胞对TLR1特异性激动剂Pam3cys的反应性显著降低。此外，敏感株的Tlr1基因表达较耐药株降低。这项研究的目的是研究TLR1在尿路感染中的作用，并确定Tlr1基因的多态如何影响对尿路感染的易感性。这将通过检测C56B1/6Tlr1-/-小鼠和其他具有不同Tlr1基因多态的菌株的尿路感染敏感性来实现。在这些研究之后，将使用基于组织培养的表达和功能分析来检测遗传变异对TLR1在VITR中的表达、翻译、蛋白运输和信号转导的影响。在体内，Tlr1基因变异对不同小鼠基因组背景下尿路感染易感性的影响将通过检测其他Tlr1基因序列改变的小鼠品系或通过靶向育种引入特定等位基因来评估。基因组背景和TLR1功能对骨髓源性免疫细胞在影响尿路感染敏感性中的作用将通过建立小鼠骨髓嵌合体并评估其对尿路感染的敏感性来阐明。最后，将通过对250个有频繁RUTI的个体的Tlr1基因座进行测序，并将这些序列与人类基因组序列的通用数据库和不容易患RUTI的对照人群进行比较，来检验Tlr1变异与人类人群中UTI易感性之间是否存在相关性。这一分析将为使用患者的Tlr1序列来确定他们的尿路感染倾向提供证据。了解TLR1及其多态性在尿路感染易感性中的作用，将有助于更好地了解尿路感染的发病机制，以及针对RUTI患者的靶向治疗和预测结果。

DESCRIPTION (provided by applicant): Urinary tract infections (UTI) are the most common bacterial infection in the developed world and uropathogenic E. coli (UPEC) account for greater than 80% or these infections. One of the strongest risk factors for the development of frequent recurrent (rUTI), which occurs in 2-7% of women, is a family history of rUTI, suggesting a genetic component to UTI susceptibility. This conclusion is further supported by mouse models of UTI, which demonstrate high levels of strain to strain variation in mouse susceptibility to UTI. An in silico whole genome analysis of single nucleotide polymorphisms (SNPs) conserved in UTI susceptible mouse strains and excluded from UTI resistant mouse strains identified several non-synonymous SNPs in the protein coding portion of the innate immune receptor toll-like receptor 1 (TLR1), as well as a SNP predicted to disrupt a splice site in this gene. The TLR1 protein is a pattern recognition receptor that complexes with TLR2 to detect bacterial lipoproteins and activate the immune system in response to this stimulus. Preliminary results suggest that macrophages from the UTI susceptible mouse strain C3H/HeN are dramatically less responsive to the TLR1 specific agonist Pam3cys than macrophages from the UTI resistant mouse strain C57Bl/6. In addition, gene expression of Tlr1 is reduced in the susceptible strain relative to the resistant strain. The goal of this study is to examine the role of TLR1 in UTI and o determine how polymorphisms in the Tlr1 gene influence susceptibility to UTI. This will be accomplished by examining the UTI susceptibility in C56Bl/6 Tlr1 -/- mice and other strains with various Tlr1 polymorphisms. These studies will be followed by an examination of the effects of genetic variation on the expression, translation, protein trafficking and signaling of TLR1 in vitr, using tissue culture based expression and functional analysis. In vivo, the effect of Tlr1 genetic variation on UTI susceptibility in different mouse genomic backgrounds will be assessed by examining other mouse strains with altered Tlr1 gene sequences or by introducing specific alleles via targeted breeding. Genomic background effects and the role of TLR1 function on bone marrow derived immune cells in influencing UTI susceptibility will be elucidated by generating mouse bone marrow chimeras and assessing their sensitivity to UTI. Finally, the existence of a correlation between Tlr1 variation and UTI susceptibility in the human population will be examined by sequencing the Tlr1 locus of 250 individuals with frequent rUTI and comparing these sequences to general databases of human genome sequences and to a control population not prone to rUTI. This analysis will provide evidence for the use of Tlr1 sequences from patients to determine their propensity to UTI. Understanding the role of TLR1 and its polymorphisms in UTI susceptibility will allow for a better understanding of the pathogenesis of UTI as well as targeted therapies and predictive outcomes for individuals afflicted with rUTI.