Investigating the Contribution of the Coxiella Cell Wall to Intracellular Pathogenesis

项目摘要 伯氏柯克斯体是Q热的高传染性病原体，可表现为急性或慢性- 像是慢性病。这种自然专属的细胞内细菌感染肺泡巨噬细胞，复制 在一个类似于成熟的吞噬小体的隔间里。伯氏拟青霉的一个显著特征是 它能够进入这些细胞，而不会激活可检测到的先天性免疫防御系统，也不会使细胞两极分化为 致炎表型。这种致病模式的毒力因素是它独特的抗 炎性脂多糖(LPS)及其IVb型分泌系统(T4SS)分泌的效应物。至 鉴定与伯氏卡氏杆菌的内毒素或T4SS无关的未被欣赏的毒力因子，这是一种利用 TnSeq比较了柯克斯体转座子突变体在Vero细胞和原代巨噬细胞中的复制。 筛选发现，与细胞壁功能相关的基因中的一系列突变体只需进行复制 在初级巨噬细胞的背景下。这些数据表明细菌细胞壁成分可能属于 一类不受赏识的柯克斯氏菌毒力因子。该项目的长期目标是描述 细胞壁组分参与细胞内致病的分子机制(S) 柯克西拉病毒。中心假说是伯氏梭菌细胞壁被特别地适应来抵抗或逃避 存在于其首选的细胞宿主巨噬细胞中的先天防御系统。本应用程序的目标是 是为了确定1)分子功能和2)单个细胞壁成分的毒力贡献 是柯克斯体在原代巨噬细胞内生存和增殖所必需的。细胞壁复合体的角色(S)- 在柯克斯氏菌致病机制中，NETS将通过追求以下具体目标来定义：1)确定功能-- 候选的细胞壁相关毒力基因的片段，以及2)决定这些细胞壁的作用 伯氏拟青霉与寄主相互作用的突变体。具体目标1将集中在这些分子的贡献上 生存和抵抗巨噬细胞所特有的压力的候选毒力基因。 我们将采用多种实验方法，包括互补分析、生化分析等。 脂多糖和脂类，发育和形态分析，以及体外功能分析，以确定 这些细胞壁成分对柯克斯体存活的贡献。《特定目标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，以及分泌物分析，我们将建立宿主对这些突变体反应的整体视图。我们期待着 拟议的一系列实验将为一个强大、持久的研究计划奠定基础 将细胞壁的完整性与伯氏柯克斯体独特的环境稳定性和无与伦比的 在杀灭微生物的利基环境中生存。

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.