Tlr1: Functional and Genetic Variation in Urinary Tract Infection

描述（由申请人提供）：尿路感染（UTI）是发达国家最常见的细菌感染，尿路致病性大肠杆菌。大肠埃希菌（UPEC）占80%以上。发生在2-7%女性中的频繁复发性（鲁蒂）的最强风险因素之一是鲁蒂家族史，表明UTI易感性的遗传成分。这一结论得到了UTI小鼠模型的进一步支持，该模型证明了小鼠对UTI的易感性存在高水平的品系间变异。 对UTI易感小鼠品系中保守的单核苷酸多态性（SNP）进行计算机全基因组分析，并从UTI抗性小鼠品系中排除，在先天免疫受体toll样受体1（TLR 1）的蛋白质编码部分鉴定出几种非同义SNP，以及预测破坏该基因中剪接位点的SNP。TLR 1蛋白是一种模式识别受体，与TLR 2复合以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，与来自UTI抗性小鼠品系C57 B1/6的巨噬细胞相比，来自UTI易感小鼠品系C3 H/HeN的巨噬细胞对TLR 1特异性激动剂Pam 3cys的响应显著更低。此外，相对于抗性品系，在敏感品系中Tlr 1的基因表达降低。本研究的目的是研究TLR 1在UTI中的作用，并确定TLR 1基因多态性如何影响UTI的易感性。这将通过检查C56 Bl/6 Tlr 1-/-小鼠和具有各种Tlr 1多态性的其他品系中的UTI易感性来实现。这些研究之后将检查遗传变异对TLR 1在体外的表达、翻译、蛋白运输和信号传导的影响，使用基于组织培养的表达和功能分析。在体内，将通过检查具有改变的Tlr 1基因序列的其他小鼠品系或通过靶向育种引入特定等位基因来评估Tlr 1遗传变异对不同小鼠基因组背景中UTI易感性的影响。通过产生小鼠骨髓嵌合体并评估其对UTI的敏感性，将阐明基因组背景效应和TLR 1功能对骨髓源性免疫细胞在影响UTI易感性中的作用。最后，将通过对250名频繁发生鲁蒂的个体的Tlr 1基因座进行测序，并将这些序列与人类基因组序列的一般数据库和不倾向于鲁蒂的对照人群进行比较，来检查Tlr 1变异与人群中UTI易感性之间的相关性。该分析将为使用患者的Tlr 1序列来确定其UTI倾向提供证据。了解TLR 1及其多态性在UTI易感性中的作用将有助于更好地了解UTI的发病机制以及针对鲁蒂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.