Investigating the Contribution of the Coxiella Cell Wall to Intracellular Pathogenesis

项目摘要 贝氏柯克斯体是Q热的高度传染性病原体，可表现为急性或非急性感染性。 诱发慢性疾病。这种天然专性细胞内细菌感染肺泡巨噬细胞， 在类似于成熟吞噬溶酶体的隔室中。C.伯内特群岛 它能够进入这些细胞，而不激活可检测的先天免疫防御或将细胞极化为 促炎表型与这种致病模式有关的毒力因子是其独特的抗- 炎性脂多糖（LPS）和由其IVb型分泌系统（T4 SS）分泌的效应物。到 鉴定与C. LPS或T4 SS无关的未鉴定的毒力因子。burnetii，一种基因筛选， TnSeq比较了Coxiella转座子突变体在Vero细胞中与在原代巨噬细胞中的复制。 筛选确定了一系列与细胞壁功能相关的基因突变体，这些基因仅为复制所需 在原代巨噬细胞中。这些数据表明，细菌细胞壁成分可能属于 一类未被重视的柯克斯体毒力因子这个项目的长期目标是描述 所鉴定的细胞壁组分促进细胞内发病的分子机制 关于Coxiella中心假设是C.贝氏菌细胞壁特别适于抵抗或逃避 先天防御，存在于其首选的细胞宿主巨噬细胞中。本申请的目的 是为了确定1）分子功能和2）单个细胞壁组分的毒力贡献， 是柯克斯体在原代巨噬细胞内存活和增殖所必需的。细胞壁组分的作用 在柯克斯体发病机制中的作用将通过追求以下具体目标来定义：1）确定功能， 候选细胞壁相关毒力基因的选择，以及2）确定这些细胞壁相关毒力基因的作用 突变体C. burnetii宿主相互作用。具体目标1将集中在这些分子的贡献 候选毒力基因的生存和抗性，特别是在巨噬细胞内遇到的压力。 我们将使用多种实验方法，包括互补分析，PEP的生化分析， 潮聚糖和脂质，发育和形态学分析，以及体外功能测定，以确定 这些细胞壁组分对柯克斯体存活的贡献。具体目标2将调查 巨噬细胞感染这些有趣的细胞壁突变体。通过使用RNAseq，酶活性 分析，和分泌测定，我们将建立一个整体的看法，主机响应这些突变体。我们预计 拟议的一系列实验将为一个强大的、长期的研究计划奠定基础， 将细胞壁的完整性与贝氏柯克斯体独特的环境稳定性和无与伦比的 在一个杀微生物的小生境中生存。

Project Summary Coxiella burnetii is the highly infectious causative agent of Q fever, which can manifest as an acute or debil- itating chronic disease. This naturally obligate intracellular bacterium infects alveolar macrophages, replicating within a compartment that resembles a mature phagolysosome. A distinguishing characteristic of C. burnetii is its ability to enter these cells without activating detectable innate immune defenses or polarizing the cell to a proinflammatory phenotype. The virulence factors credited with this pathogenic paradigm are its unique anti- inflammatory lipopolysaccharide (LPS), and the effectors secreted by its Type IVb Secretion system (T4SS). To identify unappreciated virulence factors unrelated to LPS or T4SS of C. burnetii, a genetic screen employing TnSeq compared the replication of Coxiella transposon mutants in Vero cells to that in primary macrophages. The screen identified a series of mutants in genes related to cell wall function were required for replication only in the context of the primary macrophages. These data suggested that bacterial cell wall components may belong to an unappreciated class of Coxiella virulence factors. The long-term goal of this project is to describe the molecular mechanism(s) by which the identified cell wall components contribute to the intracellular pathogenesis of Coxiella. The central hypothesis is that the C. burnetii cell wall is specifically adapted to resist or evade the innate defenses that are present in its preferred cellular host, macrophages. The objectives of this application are to determine the 1) molecular functions and 2) virulence contributions of individual cell wall components that are required for Coxiella’s survival and proliferation within primary macrophages. The role(s) of cell wall compo- nents in Coxiella pathogenesis will be defined by pursuing the following specific aims: 1) determine the func- tions of the candidate cell wall-related virulence genes, and 2) determine the effect of these cell wall mutants on C. burnetii-host interactions. Specific aim 1 will focus on the molecular contribution of these candidate virulence genes to survival and resistance to stresses specifically encountered within macrophages. We will use multiple experimental approaches including complementation assays, biochemical analyses of pep- tidoglycan and lipids, developmental and morphological analyses, and in vitro functional assays to define the contribution of these cell wall components to Coxiella survival. Specific aim 2 will investigate the response of macrophages to infection with these interesting cell wall mutants. By employing RNAseq, enzymatic activity assays, and secretion assays, we will build a holistic view of the host response to these mutants. We anticipate that the proposed series of experiments will lay the foundation for a strong, long-lived research program that links cell wall integrity to Coxiella burnetii’s distinguishing environmental stability and incomparable ability to survive in a microbicidal niche.