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迅速开发出一个完整的遗传学工具箱。最近，我们开发了一种支持柯克斯氏菌强劲生长的特定培养基名为ACCM-D，它包含氨基酸作为唯一的碳和能源。柯克斯氏菌对Arg、Lys、Pro和Tyr具有营养缺陷性，因为它们缺乏生物合成的终末酶。嗜肺军团菌Arggh、Lysa和proAB和E.ColityrB的异源表达分别挽救了Arg、Lys、Pro和Tyr ACCM-D辍学培养基中的生长，为Coxiella转化子的营养选择提供了四种方法。考虑到基于抗生素耐药性的可选择标记对于这种选择剂来说是有限的，强的、非基于抗生素的遗传转化子的选择是一个重要的进步。 到目前为止测序的所有伯氏杆菌分离株都含有一个自主复制的质粒(QpH1、QpDG、Qprs、QpDV)或具有整合到染色体上的Qprs样质粒序列。这些序列上存在的基因的作用尚不清楚，但有些基因编码Dot/ICM型IVB分泌底物。对QPH1上编码基因的作用进行了研究。利用含有伯氏隐翅虫QpH1复制起点和新的营养选择标记(酪氨酸)的新型穿梭载体(pBR322-TyrB-QpH1ori)，我们成功地治愈了伯氏隐翅虫九里相II株的QpH1。QpH1菌株在无菌条件下生长正常，但在Vero细胞中有明显的生长缺陷，表明该质粒对伯氏梭菌的毒力是关键的。我们开发了一种可诱导的CRISPR干扰系统，使用第二个营养选择标记(Pro)来检测单个QPH1质粒基因的作用。单独敲除QPH1编码的8种Dot/ICM底物不影响无菌培养液和THP1细胞的生长，而cbuA0027基因敲除则导致无菌培养液和THP1细胞的生长缺陷。CbuA0027基因是cbuA0028双基因操纵子的下游组成部分。CBUA0028和CBUA0027分别与霍乱弧菌的HIGB毒素和HIGA抗毒素同源，霍乱弧菌属于Rele类毒素-抗毒素系统的一部分。CbuA0028的过表达导致了细胞内生长的严重缺陷，这可以通过与cbuA0027的共同表达来挽救。CBUA0028在无细胞系统中的产生以剂量依赖的方式抑制控制蛋白的产生。与其他毒素-抗毒素系统一样，CBUA0027直接与CBUA0028结合，生成的复合体可以与cbuA0028启动子(PcbuA0028)结合，CBUA0027也可以与PcbuA0028结合。还鉴定了cbuA0028内部的两个cbuA0027启动子。综上所述，我们的数据表明，伯氏梭菌分离株保持自主复制质粒的原因是由于存在基于质粒的毒素-抗毒素系统。 为了确保宿主细胞和哺乳动物宿主之间的生存，伯氏梭菌在复制的、代谢活性的大细胞变体(LCV)和孢子样静止的小细胞变体(SCV)之间进行转换。伯氏梭菌编码3个典型的双组分系统、4个孤儿杂合组氨酸激酶、5个孤儿反应调节蛋白和一个组氨酸磷酸转移蛋白，这些蛋白被推测在伯氏梭菌形态发生和毒力所需的信号转导中发挥作用，但这些系统很少被描述。通过使用CRISPR干扰系统对伯氏梭菌进行遗传操作，我们创造了涉及大多数这些信号基因的单基因和多基因转录下调菌株。通过这一结果，我们确定了CBU_0780/CBU_0955/CBU_1043孤儿反应调节子协同和不同地调控SCV相关基因在伯氏梭菌LCV中的表达。我们还揭示了Burnetii PhoBR典型的两组分系统在毒力、PI维持和PI运输方面的潜在作用。我们还概述了一种新的机制，通过该机制，PhoBR的功能可能受到非典型的PhoQ样蛋白的调节。这些基础性的结果将为未来研究伯氏梭菌的两个组分调节因子在毒力和形态发生中的作用提供信息。 目前的人类Q热疫苗Q-VAX是一种固定的全细胞疫苗(WCV)，仅在澳大利亚获得使用许可。虽然柯克斯体WCVS非常有效，但在对柯克斯体有免疫力的人中，接种后可能会出现严重的皮肤超敏反应，这限制了它们的广泛使用。因此，需要一种反应性较低的疫苗。我们研究了柯克斯氏菌Dot/ICM型IVB分泌系统(T4BSS)和脂多糖(LPS)在固定WCVS保护和反应性中的作用。在9英里I期(NMI)强毒株中，缺失了一个包含23个Dot/ICM基因的32.5kb区域，并在Q热感染、疫苗接种和攻击以及疫苗接种后超敏反应的豚鼠模型中进行了评估。NMI DOT/ICM株是无毒的，作为一种WCV对强大的Coxiella攻击具有保护作用，并且与野生型Coxiella相比，显示出潜在的反应原性改变。

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 enzymes in biosynthesis. Heterologous expression by Coxiella of Legionella pneumophila argGH, lysA and proAB and E.coli tyrB rescues growth in Arg, Lys, Pro and Tyr ACCM-D dropout media, respectively, thus providing four methods for nutritional selection of Coxiella transformants. Strong, non-antibiotic-based selection of genetic transformants is an important advance considering selectable markers based on antibiotic resistance are limited for this select agent. All C. burnetii isolates sequenced to date harbor an autonomous replicating plasmid (QpH1, QpDG,, QPRS QpRS, or QpDV) or have QpRS-like plasmid sequences integrated into the chromosome. The roles of the genes present on these sequences are unknown but some encode Dot/Icm type IVB secretion substrates. The role of the genes encoded on QpH1 were investigated. Using a new C. burnetii shuttle vector (pBR322-TyrB-QpH1ori) containing the C. burnetii QpH1 origin of replication and new nutritional-based selection marker (tyrosine) in C. burnetii, we were able to cure the QpH1 plasmid from the C. burnetii Nine Mile Phase II strain. The QpH1 strain grew normally in axenic media but had a significant growth defect in Vero cells, suggesting the plasmid is critical for C. burnetii virulence. We developed an inducible CRISPR interference system using a second nutritional selection marker (proline) to examine the role of individual QpH1 plasmid genes. Individual knockdown of the 8 Dot/Icm substrates encoded by QpH1 did not affect growth in axenic media or THP1 cells, while knockdown of cbuA0027 resulted in significant growth defects in axenic media and THP1 cells. The cbuA0027 gene is the downstream component of a two-gene operon with cbuA0028. CBUA0028 and CBUA0027 are homologous to the HigB toxin and HigA anti-toxin, respectively, from Vibrio cholerae that are part of the RelE-like family of toxin-antitoxin systems. Overexpression of cbuA0028 resulted in a severe defect in intracellular growth that could be rescued by coexpression with cbuA0027. Production of CBUA0028 in a cell free system inhibited production of a control protein in a dose-dependent manner. Consistent with other toxin-antitoxin systems, CBUA0027 bound directly to CBUA0028 and the resulting complex could bind the cbuA0028 promoter (PcbuA0028), CBUA0027 could also bind PcbuA0028. Two cbuA0027 promoters internal to cbuA0028 were also identified. In summary, our data indicate that the reason C. burnetii isolates maintain an autonomously replicating plasmid is due to the presence of a plasmid-based toxin-antitoxin system. C. burnetii transitions between a replicative, metabolically-active large-cell variant (LCV), and a spore-like quiescent small-cell variant (SCV) in order to ensure survival between host cells and mammalian hosts. C. burnetii encodes three canonical two-component systems, four orphan hybrid histidine kinases, five orphan response regulators, and a histidine phosphotransfer protein that have been speculated to play roles in the signaling required for C. burnetii morphogenesis and virulence, but very few of these systems have been characterized. By employing a CRISPR interference system for genetic manipulation of C. burnetii, we have created single and multi-gene transcriptional knock-down strains involving most of these signaling genes. Through this, we have determined that the CBU_0780/CBU_0955/CBU_1043 orphan response regulators coordinately and disparately regulate expression of SCV-associated genes in C. burnetii LCVs. We also revealed a potential role for the C. burnetii PhoBR canonical two component system in virulence, regulation of Pi maintenance, and Pi transport. We also outline a novel mechanism by which PhoBR function may be regulated by an atypical PhoQ-like protein. These foundational results will inform future studies on the role of C. burnetii two component regulators in virulence and morphogenesis. The current human Q fever vaccine, Q-VAX, is a fixed, whole cell vaccine (WCV) and is licensed solely for use in Australia. While highly efficacious, Coxiella WCVs are associated with a potentially severe postvaccination dermal hypersensitivity reaction in people with pre-existing immunity to Coxiella, which limits their wider use. Consequently, a less reactogenic vaccine is needed. We investigated contributions of the Coxiella Dot/Icm type IVB secretion system (T4BSS) and lipopolysaccharide (LPS) in protection and reactogenicity of fixed WCVs. A 32.5 kb region containing 23 dot/icm genes was deleted in the virulent Nine Mile phase I (NMI) strain and the resulting mutant was evaluated in guinea pig models of Q fever infection, vaccination and challenge, and post-vaccination hypersensitivity. The NMI dot/icm strain was avirulent, protective as a WCV against a robust Coxiella challenge, and displayed potentially altered reactogenicity compared to wild type Coxiella.