Genetics of Coxiella burnetii

伯氏柯克斯体是一种普遍存在的人畜共患细菌性病原体，也是人类急性Q热的病因，Q热是一种致残性流感样疾病。柯克斯体以前的专性细胞内特性显著阻碍了推测的毒力因子的遗传特征。然而，我们在酸性柠檬酸半胱氨酸(ACCM)中无宿主细胞(无菌)生长的开创性进展使我们能够快速开发出穿梭载体、转座子突变、诱导基因表达、靶向基因缺失和营养选择技术，用于病原体基因操作。我们还开发了一个荧光素酶基因报告系统，可以用来监测发育调节基因的表达和病原菌的应激反应。柯克斯体基因工具的全套工具现在允许使用传统的突变和互补策略来发现毒力因子。事实上，我们已经构建了30多株基因敲除菌株，包括那些编码Dot/ICM型IVB分泌系统(T4BSS)组件和分泌蛋白的基因缺失的菌株。这些研究证实，T4BSS功能对柯克斯体在巨噬细胞中的生长至关重要。突变分析还确定了几种T4BSS效应蛋白，它们是哺乳动物细胞最佳生长所必需的。 柯克斯体编码少量的转录调控因子，这些转录调控因子可能对巨噬细胞内存活和/或发育过渡至关重要。Coxiella的GacAS双组分系统(TCS)特别耐人寻味，因为其他细菌中的同源系统调控复杂的社会行为、毒力基因表达和发育转变。此外，稳定期西格玛因子rpos在应激存活中的作用尚不清楚。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、报告分析、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节级联反应。这些研究将为了解柯克斯体的毒力提供必要的见解。 在免疫活性动物模型中，唯一被证明导致柯克斯体毒力减弱的遗传损伤与脂多糖(LPS)合成缺陷有关。在体外重复传代时，具有全长内毒素的毒力I相生物转变为严重截短内毒素的无毒II期生物。鉴于内毒素在柯克斯体毒力中的关键作用，了解其相变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显示了与阶段变异相关的插入/缺失。在这些数据的基础上，正在进行等位基因交换和互补实验，以从基因上确定相变的途径。 柯克斯体经历了细胞内的两个阶段的发育周期，产生了两个不同的形态变异，可以通过超微结构和蛋白质组成来区分。小细胞变异体(SCV)不复制，含有浓缩的染色质，被认为是细胞外的生存形式。SCV分化为染色质分散的复制型大细胞变异体(LCV)。当柯克斯体进入静止生长阶段时，LCV向SCV的转变是一致的，在感染细胞培养的延长培养时间(2-4周)，出现几乎均一的SCV。作为一个有助于我们更好地理解Coxiella分化的生物学相关性的模型，我们建立了SCV/LCV转变是由生长在第三代无菌培养基ACCM-D中的微生物概括的。这一发现使柯克斯体发育生物学的研究不会遇到宿主细胞繁殖细菌的实验困难。LCV和SCV的比较转录组学和蛋白质组学现在揭示了形态分化的分子决定因素，这些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.