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

据估计，沙门氏菌导致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。基于近亲交配的研究结果 小鼠菌株和罕见的人类突变表明沙门氏菌病的临床表现和结果 受宿主基因构成的影响。然而，我们并不了解共同的影响，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 通过感染的细胞表型和自然遗传多样性来确定分子参数 对人类传染病的易感性。虽然肠热病和肠道热的全基因组关联研究(GWAs) 菌血症确定了调节易感性的遗传差异，这些研究受到高度变异性的限制 患者暴露，病原体遗传多样性，以及对已确定的病理生理学有限的了解 变种。我们开发了一种互补的细胞GWAS方法(Hi-host：高通量人类体外培养 敏感度测试；http://h2p2.oit.duke.edu)对人类遗传差异进行高分辨率分析 这影响了宿主-病原体的特征，同时使这些特征能够进行实验解剖。在之前的资助中 在此期间，我们专注于调节沙门氏菌入侵宿主细胞的人类基因变体。我们确定了一个 VAC14中调节质膜胆固醇水平从而限制对接的调节性变异体 沙门氏菌SPI-1 III型分泌系统对宿主细胞的影响。携带VAC14高侵袭性等位基因的人类 会增加伤寒和菌血症的风险。本应用程序的目标是定义和 描述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。 从我们独特的高宿主细胞资源构建沙门氏菌入侵、复制和宿主 细胞因子水平，我们已经确定了SNPs介导对感染表型的易感性 实验验证和机理研究。我们将确定监管SNPs如何影响ARHGEF26 和MCOLN2的表达影响沙门氏菌的入侵和复制，以及这些基因在 小鼠的感染。对于ARHGEF26，一种刺激沙门氏菌诱导的膜褶皱的Rho环境基金，我们的 相关数据和实验证据表明，SNPs调节ARHGEF26的表达以增加侵袭性 依赖于沙门氏菌效应物SopB。对于MCOLN2，我们假设这个二价阳离子通道是一个 通过调节营养获取、金属毒性和/或免疫来调节细胞内复制的新因子 细胞极化。使用Arhgef26和Mcoln2小鼠，我们将阐明这些基因的表达是如何改变的 在感染过程中调节细菌负荷和免疫反应。最后，我们将测试是否识别出SNPs Hi-host与人类的肠热病和菌血症有关。揭示这些机制可能会 为沙门氏菌病和受相同基因变异影响的其他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.