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。 柯克斯体是精氨酸、赖氨酸、脯氨酸和酪氨酸的营养缺陷型，缺乏生物合成中的最终酶。嗜肺军团菌argGH、lysA和proAB的柯克斯体和大肠杆菌tyrB的异源表达分别挽救了Arg、Lys、Pro和Tyr ACCM-D缺失培养基中的生长，从而提供了用于柯克斯体转化体的营养选择的四种方法。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，遗传转化体的强的、非基于抗生素的选择是重要的进步。 全是C。迄今为止测序的贝氏体分离物含有自主复制质粒（QpH1、QpDG、QPRSQpRS或QpDV）或具有整合到染色体中的QpRS样质粒序列。存在于这些序列上的基因的作用是未知的，但一些编码Dot/Icm型IVB分泌底物。研究了QpH1上编码的基因的作用。使用新的C。贝氏梭菌穿梭载体pBR322-TyrB-QpH1ori。Burnetii QpH1复制起点和新的营养选择标记（酪氨酸）。在Burnettii中，我们能够从C.贝氏九英里II期菌株。QpH1菌株在无菌培养基中生长正常，但在Vero细胞中有明显的生长缺陷，表明该质粒对C.贝氏菌毒力我们开发了一种诱导型CRISPR干扰系统，使用第二种营养选择标记（脯氨酸）来检查单个QpH1质粒基因的作用。QpH1编码的8个Dot/Icm底物的单独敲低不影响在无菌培养基或THP 1细胞中的生长，而cbuA0027的敲低导致在无菌培养基和THP 1细胞中的显著生长缺陷。cbuA0027基因是具有cbuA0028的双基因操纵子的下游组分。CBUA0028和CBUA0027分别与来自霍乱弧菌的HigB毒素和HigA抗毒素同源，它们是毒素-抗毒素系统的RelE样家族的一部分。cbuA0028的过表达导致细胞内生长的严重缺陷，可以通过与cbuA0027的共表达来挽救。在无细胞系统中产生CBUA 0028以剂量依赖性方式抑制对照蛋白的产生。与其他毒素-抗毒素系统一致，CBUA0027直接结合CBUA0028，所得复合物可结合cbuA0028启动子（PcbuA0028），CBUA0027也可结合PcbuA0028。还鉴定了cbuA0028内部的两个cbuA0027启动子。总之，我们的数据表明，原因C。贝氏分离物保持自主复制质粒是由于存在基于质粒的毒素-抗毒素系统。 C.贝氏体在复制型代谢活性大细胞变体（LCV）和孢子样静止小细胞变体（SCV）之间转换，以确保宿主细胞和哺乳动物宿主之间的存活。C. Burnetii编码三个典型的双组分系统，四个孤儿杂合组氨酸激酶，五个孤儿反应调节因子，和一个组氨酸磷酸转移蛋白，推测它们在C. burnetii形态发生和毒力，但很少有这些系统的特点。通过使用CRISPR干扰系统对C. Burnetii中，我们已经创建了涉及大多数这些信号传导基因的单基因和多基因转录敲低菌株。通过这一点，我们已经确定CBU_0780/CBU_0955/CBU_1043孤儿应答调节因子协调且独立地调节C.贝氏菌我们还揭示了C. BurnetiiPhoBR典型的双组分系统在毒力、Pi维持和Pi运输的调节中的作用。我们还概述了一种新的机制，通过这种机制，PhoBR功能可能受到非典型PhoQ样蛋白的调节。这些基础性结果将为未来关于C的作用的研究提供信息。贝氏双组分调节剂在毒力和形态发生中的作用。 目前的人Q热疫苗Q-VAX是一种固定的全细胞疫苗（WCV），仅在澳大利亚获得许可使用。虽然非常有效，但Coxiella WCV与预先存在Coxiella免疫力的人群中潜在的严重接种后皮肤超敏反应相关，这限制了其更广泛的使用。因此，需要一种反应原性较低的疫苗。我们研究了Coxiella Dot/Icm型IVB分泌系统（T4BSS）和脂多糖（LPS）在固定WCV的保护和反应原性中的作用。一个32.5 kb的区域，含有23个点/icm基因删除的毒力九里I期（NMI）菌株和产生的突变体进行了评价，在豚鼠模型的Q热感染，疫苗接种和挑战，接种后超敏反应。NMI dot/icm菌株是无毒力的，作为WCV对强Coxiella攻击具有保护性，并且与野生型Coxiella相比显示出潜在改变的反应原性。

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.