Identifying host genetic determinants of Coxiella burnetii pathogenesis

项目概要/摘要： 贝氏柯克斯体是一种革兰氏阴性、专性胞内嗜巨噬细胞细菌， 人畜共患病Q热的病原体。人类疾病的急性期特征是 主要是流感样症状，大约3-5%的感染者发展为慢性 感染，其特征是心内膜炎。宿主遗传背景与 已经在人类和动物中证实了Coxiella感染的发展;然而， 与感染易感性相关的基因在很大程度上仍然未知。一个强大的工具来研究的影响 致病性感染的宿主遗传学是模式生物黑腹果蝇。果蝇 遗传学参考组（DGRP）是来自自然种群的蝇系的完全测序组， 作为一个活的文库来测试多态性如何影响复杂的宿主反应。因此，为了解决 关于宿主遗传学如何影响柯克斯体致病机制的知识缺口，我们建议鉴定宿主 利用黑腹果蝇研究与柯克斯体感染易感性有关的遗传变异。 我们的初步数据表明，果蝇表现出死亡率和宿主免疫反应 在用C.脂多糖缺失的贝氏菌 合成基因，但保留其他毒力因子。II期菌株具有类似的巨噬细胞内复制 动力学与BSL 3阶段I C相同。burnetii并导致蜡螟幼虫死亡。同样地，果蝇也屈服于 Coxiella II期克隆4，因此可用作Coxiella感染的新宿主模型。而且我们 途径无偏筛选方法将显着改善正在进行的研究柯克斯体在 哺乳动物系统我们假设DGRP系将揭示免疫应答中的遗传变异- 与Coxiella感染易感性相关的基因。在具体目标1中，我们将使用DGRP线， 进行全基因组关联研究（GWAS），并筛选与 对柯克斯体感染的易感性。在具体目标2中，我们将使用基因重组苍蝇品系， 敲低或过表达具有鉴定的变体的基因以进行验证和进一步功能分析的测定 评估该基因对柯克斯体发病机制的影响。此外，由于大多数已知的人类 疾病基因在果蝇中有可识别的同源物，我们将利用这些遗传 相似性，以询问在我们的筛选中鉴定出的同源基因在人类Coxiella感染期间如何发挥作用 细胞 通过这项工作，我们将确定宿主对柯克斯体易感性的遗传决定因素 感染果蝇和哺乳动物宿主。我们的发现将通过识别宿主产生广泛的影响， 当功能障碍时，这些因素赋予对柯克斯体感染的易感性。这些结果将对 更好地预测国内反刍动物宿主中的病原体传播和人类疾病进展。

Project Summary/Abstract: Coxiella burnetii is a Gram-negative, obligate intracellular, macrophage-tropic bacterium and the causative agent of the zoonotic disease Q fever. Acute phase of the disease in humans is characterized primarily by influenza-like symptoms, and approximately 3-5% of the infected individuals develop chronic infection, which is characterized by endocarditis. Association between host genetic background and the development of Coxiella infection has been demonstrated both in humans and animals; however, specific genes associated with susceptibility to infection remain largely unknown. A powerful tool to study the impact of host genetics on pathogenic infection is the model organism Drosophila melanogaster. The Drosophila Genetics Reference Panel (DGRP) is a fully sequenced panel of fly lines derived from a natural population that is used to as a living library to test how polymorphisms affect complex host responses. Thus, to address the knowledge gap regarding how host genetics affects Coxiella pathogenesis, we propose to identify host genetic variants implicated in susceptibility to Coxiella infection using Drosophila melanogaster. Our preliminary data demonstrate that Drosophila exhibit mortality and host immune responses following infection with the BSL2 phase II clone 4 strain of C. burnetii that has a deletion in lipopolysaccharide synthesis genes but retains other virulence factors. The phase II strain has similar intra-macrophage replication kinetics as BSL3 phase I C. burnetii and causes lethality in wax moth larvae. Similarly, Drosophila succumb to Coxiella phase II clone 4 and thus can be used as a novel host model for Coxiella infection. Moreover, our pathway-unbiased screening approach will significantly improve upon ongoing studies of Coxiella in mammalian systems. We hypothesize that the DGRP lines will reveal genetic variants in immune response- related genes associated with susceptibility to Coxiella infection. In Specific Aim 1 we will use DGRP lines to perform a genome-wide association study (GWAS) and screen for genetic variants associated with susceptibility to Coxiella infection. In Specific Aim 2 we will use genetically recombinant fly lines and in vitro assays to knockdown or over-express genes with identified variants for validation and further functional evaluation of the gene's impact on Coxiella pathogenesis. Additionally, since a majority of known human disease genes have recognizable homologues in Drosophila, we will take advantage of these genetic similarities to ask how homologous genes identified in our screen function during Coxiella infection in human cells. Through the proposed work we will identify host genetic determinants of susceptibility to Coxiella infection in Drosophila and mammalian hosts. Our findings will have broad-ranging impacts by identifying host factors that when dysfunctional confer susceptibility to Coxiella infection. These results will be important for better prediction of pathogen spread in domestic ruminant reservoirs and disease progression in humans.