HUMAN GENETIC VARIATION REGULATING SALMONELLA HOST-PATHOGEN INTERACTIONS AND DISEASE SUSCEPTIBILITY

沙门氏菌引起估计1.5亿例胃肠炎和2500万例侵袭性疾病 （肠热病和非伤寒菌血症），每年导致30万人死亡。基于近亲繁殖的结果 小鼠品系和罕见的人类突变表明沙门氏菌病的临床表现和结果 都受到宿主基因构成的影响但是，我们不了解共同的影响，自然 人类对沙门氏菌感染的遗传差异。我们的长期目标是利用高- 通过感染和自然遗传多样性的细胞表型分析来确定 易感染人类传染病。虽然全基因组关联研究（GWAS）的肠热病和 菌血症确定了调节易感性的遗传差异，这类研究受到以下因素的高度变异性的限制： 患者暴露，病原体遗传多样性，以及对已确定的 变体。我们开发了一种互补的细胞GWAS方法（Hi-HOST：高通量人体外培养）。 易感性测试; http://h2p2.oit.duke.edu）来进行人类遗传差异的高分辨率分析 影响宿主-病原体特征，同时使这些特征的实验解剖成为可能。在此前的融资中， 在此期间，我们专注于调节沙门氏菌入侵宿主细胞的人类遗传变异。我们确定了一个 VAC 14中的调节变体，其调节质膜胆固醇水平并因此限制对接 的沙门氏菌SPI-1 III型分泌系统的宿主细胞。具有VAC 14高侵袭等位基因的人类 会增加患伤寒和菌血症的风险。本申请的目的是定义和 表征改变沙门氏菌宿主-病原体全谱的人类遗传差异 并评估其对疾病的影响。 从我们独特的Hi-HOST细胞GWAS资源中构建沙门氏菌入侵，复制和宿主 在细胞因子水平上，我们已经确定了介导感染表型易感性的SNP，这些表型将经历 实验验证和机理研究。我们将确定调控SNP如何影响ARHGEF 26 和MCOLN 2的表达影响沙门氏菌的入侵和复制，以及这些基因在沙门氏菌入侵和复制过程中所起的作用。 感染小鼠。对于ARHGEF 26，一种刺激沙门氏菌诱导的膜皱褶的Rho GEF，我们的研究表明， 相关数据和实验证据表明，SNPs调节ARHGEF 26的表达，以增加侵袭 依赖于沙门氏菌效应子sopB。对于MCOLN 2，我们假设这种二价阳离子通道是一种 通过调节营养获取、金属毒性和/或免疫调节细胞内复制的新因子 细胞极化使用Arhgef 26和Mcoln 2小鼠，我们将阐明这些基因的表达改变是如何发生的。 在感染期间调节细菌负荷和免疫应答。最后，我们将测试是否SNPs识别 与人类的肠热病和菌血症有关。揭示这些机制可以 为沙门氏菌病和其他受相同遗传变异影响的疾病带来新的治疗策略。

Salmonellae cause an estimated 150 million cases of gastroenteritis and 25 million cases of invasive disease (enteric fever and non-typhoidal bacteremia), leading to 300,000 deaths per year. Findings based on inbred mouse strains and rare human mutations indicate that the clinical presentations and outcomes of salmonellosis are influenced by the host’s genetic makeup. However, we do not understand the impact of common, naturally occurring human genetic differences on Salmonella infections. Our long-term goal is to leverage high- throughput cellular phenotyping of infection and natural genetic diversity to define molecular parameters of susceptibility to human infectious diseases. While genome-wide association studies (GWAS) of enteric fever and bacteremia identified genetic differences regulating susceptibility, such studies are limited by high variability in patient exposure, pathogen genetic diversity, and a limited understanding of the pathophysiology of identified variants. We developed a complementary cellular GWAS approach (Hi-HOST: High-throughput Human in vitrO Susceptibility Testing; http://h2p2.oit.duke.edu) to perform high resolution analysis of human genetic differences that impact host-pathogen traits while enabling experimental dissection of these traits. In the previous funding period, we focused on human genetic variants that regulate Salmonella invasion of host cells. We identified a regulatory variant in VAC14 that modulates levels of plasma membrane cholesterol and thus limits the docking of the Salmonella SPI-1 type III secretion system to host cells. Humans with the high-invasion allele of VAC14 have increased risk of typhoid fever and bacteremia. The objectives of this application are to define and characterize human genetic differences that alter the full spectrum of Salmonella host-pathogen interactions and assess their impact on disease. Building from our unique resource of Hi-HOST cellular GWAS of Salmonella invasion, replication, and host cytokine levels, we have identified SNPs mediating susceptibility to infection phenotypes that will undergo experimental validation and mechanistic studies. We will determine how regulatory SNPs affecting ARHGEF26 and MCOLN2 expression impact Salmonella invasion and replication and the role these genes play during infection in mice. For ARHGEF26, a Rho GEF that stimulates Salmonella-induced membrane ruffling, our association data and experimental evidence indicate SNPs regulate ARHGEF26 expression to increase invasion dependent on the Salmonella effector sopB. For MCOLN2, we hypothesize that this divalent cation channel is a novel factor that regulates intracellular replication by modulating nutrient access, metal toxicity, and/or immune cell polarization. Using Arhgef26 and Mcoln2 mice, we will elucidate how altered expression of these genes regulates bacterial burden and immune response during infection. Finally, we will test whether SNPs identified by Hi-HOST are associated with enteric fever and bacteremia in humans. Revealing these mechanisms could lead to new therapeutic strategies for salmonellosis and other diseases impacted by the same genetic variants.