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。柯克希氏菌以前专有的细胞内性质显著阻碍了推测的毒力因子的遗传特征。然而，Coxiella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发展穿梭载体、转座子突变、诱导基因表达和靶向基因缺失技术用于病原菌遗传操作。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个T4BSS效应蛋白，它们是哺乳动物细胞生产性感染所必需的。 据预测，PmrA-PmrB两组分调控系统可以调节Coxiella中IVB型的分泌。反应调节因子PmrA与靶基因上游的调控元件结合，影响基因转录。为了探讨PmrA在柯克斯体-宿主细胞相互作用中的作用，我们构建了PmrA缺失突变体，并利用新开发的基于荧光素酶的基因表达报告系统探讨了PmrA调控基因的功能。与野生型Coxiella相比，PmrA突变体表现出严重的细胞内生长缺陷。荧光素酶分析表明，编码T4BSS装置的大部分基因都受PmrA的调控。正在进行的实验包括定义剩余的PmrA调节子。 柯克斯体致病的核心是在人巨噬细胞的吞噬酶体样PV中增殖的能力。在这种通常有毒的环境中，能够生长的具体适应在很大程度上是未知的。因此，为了深入了解柯克斯体的细胞内生长策略，我们使用微阵列比较了原代人类单核细胞来源的巨噬细胞(HMDM)和ACCM中生物的转录。在感染巨噬细胞和接种ACCM后的第4天，从柯克斯体中分离到信使RNA，这一时间点与两种培养条件下的对数后期相一致。在HMDM生长过程中上调的基因包括那些编码氧化应激反应蛋白和营养转运蛋白的基因。总而言之，这些数据表明，与ACCM相比，柯克斯体PV是一个营养有限、氧化严重的环境。这份报告提供了有关柯克斯体成功寄生巨噬细胞的特定转录反应的新信息。 在人类Q热的动物模型中，已知的唯一导致柯克斯氏菌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。在我们的文库中，高传代的II期分离株不是克隆的，并且含有一小部分柯克斯氏菌亚群，仍然表达全长的I期内毒素。由此产生的混合基因型使INDELs(插入/缺失)的鉴定变得复杂，这些INDELs严格地与阶段变化有关。为了绕过这个问题，我们使用显微操作分离了九里岛、澳大利亚和加利福尼亚州高传代分离株的克隆II期群体。通过将他们的基因组DNA杂交到一个高密度微阵列中，该微阵列包含覆盖Nine Mile第一阶段分离株整个基因组的探针组，确定了第二阶段生物中可能导致内毒素生物合成缺陷的常见INDELs。 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可行模型，我们建立了SCV/LCV转换是由生长在第二代无菌培养基ACCM-2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. Coxiellas former obligate intracellular nature significantly impeded genetic characterization of putative virulence factors. However, the 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, and targeted gene deletion technologies for pathogen genetic manipulation. 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 productive infection of mammalian cells. The two-component regulatory system PmrA-PmrB is predicted to regulate type IVB secretion in Coxiella. The response regulator PmrA binds a regulatory element upstream of target genes to affect gene transcription. To explore the role of PmrA in Coxiella-host cell interactions, we constructed a PmrA deletion mutant and probed PmrA regulated gene function using a newly developed Luciferase-based gene expression reporter system. When compared to wild-type Coxiella, the PmrA mutant exhibits severe intracellular growth defects. Luciferase assays show that most genes encoding the T4BSS apparatus are regulated by PmrA. Ongoing experiments include defining the remaining PmrA regulon. Central to pathogenesis by Coxiella is the ability to proliferate in a phagolysosome-like PV of human macrophages. The specific adaptations that enable growth in this normally toxic environment are largely unknown. Therefore, to gain insight into intracellular growth strategies of Coxiella, we used microarrays to compare the transcriptomes of organisms propagated in primary human monocyte derived macrophages (HMDM) and ACCM. Messenger RNA was isolated from Coxiella at 4 days post-infection of macrophages and inoculation of ACCM, a time point that coincides with late log phase in both culture conditions. Upregulated genes during growth in HMDM include those encoding oxidative stress response proteins and nutrient transporters. Collectively, these data suggest the Coxiella PV is a nutritionally limited and oxidative and environment relative to ACCM. This report provides new information on specific transcriptional responses by Coxiella that enable successful parasitism of macrophages. The only genetic lesions known to result in attenuated Coxiella virulence in an animal model of human Q fever 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. The high passage phase II isolates in our stock collection are not clonal and contain a small subpopulation of Coxiella still expressing full-length phase I LPS. The resulting mixed genotype complicates identification of indels (insertions/deletions) strictly associated with phase variation. To circumvent this problem, we used micromanipulation to isolate clonal phase II populations of high passage Nine Mile, Australia and California isolates. By hybridizing their genomic DNA to a high-density microarray that contains probe sets encompassing the entire genome of the Nine Mile phase I isolate, common indels in phase II organisms were identified that may account for defective LPS biosynthesis. 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 second-generation axenic media, ACCM-2. 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.