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）中无宿主细胞（无菌）生长的开创性进展使我们能够快速开发一个完整的遗传学工具箱。最近，我们开发了一种成分确定的培养基，支持柯克斯体的稳健生长，称为ACCM-D，含有氨基酸作为唯一的碳源和能源。 柯克斯体缺乏生物合成中的最后步骤，因此Arg、Lys、Pro和Tyr营养缺陷型。嗜肺军团菌argGH、lysA和proAB以及大肠杆菌tyrB的异源表达分别挽救了Arg、Lys、Pro和Tyr缺失培养基中的生长，从而为柯克斯体转化体的营养选择提供了四种方法。这允许遗传转化体的强的、非基于抗生素的选择，考虑到基于抗生素抗性的选择标记对于该选择剂是有限的，这是一个重要的进步。 总的来说，我们的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发现。事实上，我们已经在毒性和无毒力的柯克斯体中构建了敲除菌株，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。此外，使用Cre-lox，我们已经创建了一个32.4 kb的dot/icm突变体的毒性九英里第一阶段的菌株，缺乏完整的dot/icm基因座所需的合成的T4 BSS。该突变体在合成培养基中生长旺盛，但不能在细胞内生长。 突变分析还确定了与毒力相关的LPS阶段变异的遗传机制。 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PhoBR双组分系统（TCS）特别有趣，因为其他细菌中的同源系统调节毒力基因表达。此外，稳定相σ因子RpoS在压力存活中的作用是未知的。解开PhoBR和RpoS调控网络将确定重要的毒力决定因素。使用基因敲除、报告基因测定、RNAseg和全细菌蛋白质组质谱，我们解析了RpoS和PhoBR的调控级联。其他TCS的特性也在进行中。 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。我们使用等位基因交换和互补来从遗传上定义LPS相转化的途径。 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，这些决定因素可能有助于细胞形式独特的生物学特征。 与分化相关的基因现在正在被灭活，突变体的表型。 我们发现，柯克斯体的遗传多样性与疾病的发病机制。在豚鼠感染模型中评价了柯克斯体属6个主要遗传群中的13个菌株的致病性。根据温度和脾肿大，菌株分为高、中等或低/无毒力。发病机制的两个极端是高毒力NMI蜱分离株（组1）和无毒力Dugway啮齿动物分离株（组VI）。 有趣的是，感染杜格威的动物仍然发生血清转化，这表明存在生产性感染。为了深入了解致病型特异性毒力，我们产生了用于毒力测试的突变体。

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 a a complete genetics tool box. Most recently, we developed a defined medium that supports robust growth of Coxiella called ACCM-D that contains amino acids as sole carbon and energy sources. Coxiella is auxotrophic for Arg, Lys, Pro and Tyr by lacking the final steps in biosynthesis. Heterologous expression of Legionella pneumophila argGH, lysA and proAB and E.coli tyrB rescues growth in Arg, Lys, Pro and Tyr dropout media, respectively, thus providing four methods for nutritional selection of Coxiella transformants. This allows strong, non-antibiotic-based selection of genetic transformants, an important advance considering selectable markers based on antibiotic resistance are limited for this select agent. Collectively, our repertoire of Coxiella genetic tools now allows traditional mutation and complementation strategies for virulence factor discovery. Indeed, we have constructed knockout strains in both virulent and avirulent Coxiella, 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. Moreover, using Cre-lox, we have created a 32.4 kb dot/icm mutant of the virulent Nine Mile phase I strain that lacks the entire dot/icm locus required for synthesis of the T4BSS. The mutant displays vigorous growth in synthetic medium but cannot grow intracellularly. Mutational analysis has also identified genetic mechanisms of LPS phase variation associated with virulence. Coxiella encodes a paucity of transcriptional regulators that are likely critical for intramacrophage survival and/or developmental transitions. The PhoBR two-component system (TCS) of Coxiella is especially intriguing as homologous systems in other bacteria regulate virulence gene expression. Moreover, the role the stationary phase sigma factor RpoS in stress survival is unknown. Unraveling PhoBR and RpoS regulatory networks will identify important virulence determinants. Using gene knockouts, reporter assays, RNAseg, and whole bacterial proteome mass spectrometry, we resolved regulatory cascades of RpoS and PhoBR. Characterization of other TCS's is also being conducted. The only genetic lesions proven to result in attenuated Coxiella virulence in an immunocompetent animal model are associated with defective 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. We used allelic exchange and complementation to genetically define pathways of LPS 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 Coxiella entry 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 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. Genes associated with differentiation are now being inactivated and mutants phenotyped. We found that Coxiella genetic diversity correlates with disease pathogenesis. Thirteen strains within the six major genetic groups of Coxiella were evaluated for pathogenicity in a guinea pig model of infection. Based on temperature and splenomegaly, strains grouped as high, intermediate, or low/no virulence. Two extremes in pathogenesis are the highly virulent NMI tick isolate (Group 1) and the avirulent Dugway rodent isolate (group VI). Interestingly, animals infected with Dugway still seroconvert, indicating productive infection. To gain insight into pathotype-specific virulence, we generated mutants for virulence testing.