Genetics of Coxiella burnetii

贝氏柯克斯体是一种普遍存在的人畜共患细菌病原体，是人类急性Q热（一种致残性流感样疾病）的病因。柯克斯体的前专性细胞内的性质显着阻碍了推定的毒力因子的遗传特征。 然而，我们在酸化柠檬酸半胱氨酸培养基（ACCM）中的宿主无细胞（无菌）生长的开创性进展使我们能够快速开发穿梭载体，转座子诱变，诱导基因表达，靶向基因缺失和营养选择技术用于病原体遗传操作。我们还开发了一个荧光素酶基因报告系统，可用于监测发育调控的基因表达和病原体胁迫反应。柯克斯体遗传工具库现在允许传统的突变和互补策略的毒力因子的发现。实际上，我们已经构建了超过30个敲除菌株，包括在编码Dot/Icm型IVB分泌系统（T4 BSS）和分泌蛋白的组分的基因中具有缺失的那些。 这些研究已经证实T4 BSS功能对于巨噬细胞中的柯克斯体生长是至关重要的。突变分析还鉴定了哺乳动物细胞中最佳生长所需的几种T4 BSS效应蛋白。 柯克斯体编码的转录调节因子，可能是至关重要的巨噬细胞内的生存和/或发展的转变的缺乏。Coxiella的GacAS双组分系统（TCS）特别有趣，因为其他细菌中的同源系统调节复杂的社会行为，毒力基因表达和发育转变。此外，固定相σ因子RpoS在应力存活中的作用是未知的。 解开GacAS和RpoS调控网络将确定重要的毒力决定因素。 利用基因敲除、报告基因测定、RNAseg和全细菌蛋白质组质谱，我们的目标是解决控制感染性、发育形式变化和应激反应的调控级联。 这些研究将提供必要的了解柯克斯体毒力。 在免疫活性动物模型中，唯一被证明导致Coxiella毒力减弱的遗传病变与脂多糖（LPS）合成缺陷有关。 在体外重复传代后，具有全长LPS的毒力I相微生物转变为具有严重截短的LPS的无毒力II相微生物。鉴于LPS在Coxiella毒力中的关键作用，了解阶段变异的分子基础是很重要的。我们收集的高传代II期菌株的全基因组测序揭示了与相变异相关的插入/缺失（插入/缺失）。基于这些数据，正在进行等位基因交换和互补实验，以从遗传学上确定相位转换的途径。 柯克斯体经历细胞内的双相发育周期，产生两种不同的形态变体，可以通过超微结构和蛋白质组成来区分。 小细胞变体（SCV）不复制，含有浓缩的染色质，被认为是细胞外存活形式。SCV分化为具有分散染色质的复制性大细胞变体（LCV）。 LCV向SCV的转变与Coxiella进入稳定生长期同时发生，在感染细胞培养物的延长孵育（2至4周）后存在几乎均一的SCV。 作为一个可行的模型，以帮助我们更好地了解柯克斯体分化的生物学相关性，我们建立了SCV/LCV转换是由第三代无菌培养基，ACCM-D中生长的生物体重演。 这一发现使柯克斯体发育生物学的研究没有遇到宿主细胞繁殖的细菌的实验困难。LCV和SCV的比较转录组学和蛋白质组学现在已经揭示了形态分化的分子决定因素，这些分子决定因素可能有助于细胞形式的独特生物学特征。

Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. Coxiella's former obligate intracellular nature significantly impeded genetic characterization of putative virulence factors. However, our seminal advance of host cell-free (axenic) growth of Coxiella in acidified citrate cysteine medium (ACCM) enabled us to quickly develop shuttle vector, transposon mutagenesis, inducible gene expression, targeted gene deletion, and nutritional selection technologies for pathogen genetic manipulation. We also developed a Luciferase gene reporter system that can be used to monitor developmentally-regulated gene expression and pathogen stress responses. The repertoire of Coxiella genetic tools now allows traditional mutation and complementation strategies for virulence factor discovery. Indeed, we have constructed over 30 knockout strains including those with deletions in genes encoding components of the Dot/Icm type IVB secretion system (T4BSS) and secreted proteins. These studies have confirmed that T4BSS function is critical for Coxiella growth in macrophages. Mutational analysis has also identified several T4BSS effector proteins that are required for optimal growth in mammalian cells. Coxiella encodes a paucity of transcriptional regulators that are likely critical for intramacrophage survival and/or developmental transitions. The GacAS two-component systems (TCS) of Coxiella are especially intriguing as homologous systems in other bacteria regulate complex social behaviors, virulence gene expression, and developmental transitions. Moreover, the role the stationary phase sigma factor RpoS is stress survival is unknown. Unraveling GacAS and RpoS regulatory networks will identify important virulence determinants. Using gene knockouts, reporter assays, RNAseg, and whole bacterial proteome mass spectrometry, we aim to resolve regulatory cascades governing infectivity, developmental form changes, and stress responses. These studies will provide needed insight into Coxiella virulence. The only genetic lesions proven to result in attenuated Coxiella virulence in an immunocompetent animal model are associated with defective lipopolysaccharide (LPS) synthesis. Virulent phase I organisms with full-length LPS transition to avirulent phase II organisms with severely truncated LPS upon repeated in vitro passage. Given the critical role of LPS in Coxiella virulence, it is important to understand the molecular basis of phase variation. Whole genome sequencing of high passage phase II strains in our collection has revealed indels (insertions/deletions) associated with phase variation. Based on these data, allelic exchange and complementation experiments are being conducted to genetically define pathways of phase conversion. Coxiella undergoes an intracellular biphasic developmental cycle that generates two distinct morphological variants that can be distinguished by ultrastructure and protein composition. Small cell variants (SCV) do not replicate, contain condensed chromatin, and are considered extracellular survival forms. SCV differentiate into replicative large cell variants (LCV) with dispersed chromatin. Transition of LCV back to SCV occurs coincident with entry of Coxiella into stationary growth phase with nearly homogeneous SCV present upon extended incubation (2 to 4 weeks) of infected cell cultures. As an amenable model to help us better understand the biological relevance of Coxiella differentiation, we established that SCV/LCV transitions are recapitulated by organisms growing in the third-generation axenic media, ACCM-D. This discovery enables studies of Coxiella developmental biology without experimental difficulties encountered with host cell-propagated bacteria. Comparative transcriptomics and proteomics of LCV and SCV have now revealed molecular determinants of morphological differentiation that likely contribute to the unique biological characteristics of cell forms.