Genetics of Coxiella burnetii

伯内氏柯克斯体的遗传学

• 批准号: 10272106
• 负责人: robert a heinzen
• 金额: $ 35.23万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272106
• 关键词: 2019-nCoV; Acids; Acute; Acute Disease; Amino Acids; Anabolism; Animals; Antibiotic Resistance; Australia; Back; Bacteria; Behavior; Biologic Characteristic; Biological; Biological Assay; COVID-19 pandemic; CRISPR interference; Carbon; Cavia; Cell Culture System; Cell Culture Techniques; Cell Line; Cell Wall; Cells; Characteristics; Chromatin; Chromosome Deletion; Chromosomes; Citrates; Complement; Coxiella; Coxiella burnetii; Cysteine; Defect; Dermal; Development; Developmental Biology; Developmental Gene; Disease; Disease Outcome; Dropout; Energy-Generating Resources; Enzymes; Escherichia coli; Exhibits; Gene Expression; Gene Silencing; Generations; Genes; Genetic; Genetic Transcription; Genomics; Growth; Human; Hypersensitivity; Immunity; Individual; Infection; Influenza; Innate Immune System; Knock-out; Laboratory Animals; Legionella; Legionella pneumophila; Length; Lipopolysaccharides; Mammalian Cell; Mass Spectrum Analysis; Mediating; Methods; Modeling; Molecular; Morphology; Mutation; Mutation Analysis; National Institute of Allergy and Infectious Disease; Nature; Nutritional; O Antigens; Organism; Phase; Phenotype; Plasmids; Production; Proline; Property; Proteins; Proteome; Proteomics; Q Fever; Reaction; Replication Origin; Reporter; Reproduction spores; Role; Seminal; Serine Protease; Shuttle Vectors; System; TMPRSS2 gene; Testing; Thick; Tropism; Tyrosine; Vaccination; Vaccines; Variant; Viral; Virulence; Virulence Factors; Virulent; Whole Cell Vaccine; Zoonoses; arginyllysine; auxotrophy; base; comparative; extracellular; genetic manipulation; genetic selection; improved; inorganic phosphate; knock-down; knockout gene; macrophage; mutant; novel; pathogen; pathogenic bacteria; programs; prolyl-tyrosine; receptor; response; tool; transcription factor; transcriptomics; vaccine development; vaccine efficacy; vaccine-induced immunity

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. 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. All Coxiella strains sequenced to date carry a large (32-54 kb), autonomously replicating plasmid or have chromosomally integrated plasmid-like sequences, suggesting that plasmid genes are important for infection. Seven genes on the QpH1 plasmid, carried by the reference Nine Mile strain, encode type 4B secretion system effector proteins suspected in mediating virulence. Only two of these genes are conserved between Coxiella plasmids or IPS. We developed a new E. coli-Coxiella shuttle vector (pBR322-CAT-sacB-tyrB-QpH1ori) that contains the QpH1 origin of replication cloned into an E. coli plasmid containing the tyrB gene. Coxiella production of TyrB in the presence of the tyrosine precursor 4-hydroxyphenylpyruvic acid (4-HPA) rescues the bacterium's natural auxotrophy for tyrosine. A CRISPRi system for gene silencing was also developed for Coxiella that utilizes proBA genes from Legionella to complement Coxiella's natural auxotrophy for proline. Introduction of pBR322-CAT-sacB-tyrB-QpH1ori into C. burnetii Nine Mile (phase II), followed by growth in tyrosine-deficient ACCM-D supplemented with 4-HPA, resulted in expulsion of the native QpH1 plasmid. The mutant strain grew normally in axenic medium but had a severe growth defect in host cells. Complementation using large fragments of QpH1 identified general regions required for intracellular growth. CRISPRi gene silencing is being used to identify individual QpH1 genes essential for Coxiella growth in host cells. This study identified two novel methods of nutritional selection of genetic transformants of Coxiella and established a CRISPRi system for robust conditional knockdown of gene expression. 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. Using gene knockouts, reporter assays, RNAseg, and whole bacterial proteome mass spectrometry, we resolved the regulatory cascade of PhoBR. Unraveling the regulatory networks of PhoBR and other TCS's will identify important virulence determinants. 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. 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. NMI and isogenic Nine Mile phase II (NMII) strains of Coxiella that produce smooth and rough LPS respectively, were similarly tested. NMI was significantly more protective than NMII as a WCV; however, both vaccines exhibited similar reactogenicity. Collectively, our results indicate that, like phase I LPS, the T4BSS is required for full virulence by Coxiella. Conversely, unlike phase I LPS, the T4BSS is not required for vaccine-induced protection. LPS length also does not appear to contribute to the dermal hypersensitivity reaction while the T4BSS may contribute to this response. NMI dot/icm represents an avirulent phase I strain with full vaccine efficacy and illustrates the potential of using genetically modified Coxiella as improved WCVs. As part of NIAID/RMLs response to the COVID-19 pandemic, the CPS collaborated with the Sonja Best lab (LV) to generate lentiviral constructs for expressing the SARS-CoV-2 receptor ACE2, and associated serine protease TMPRSS2, in mammalian cells. The purpose was to generate cell lines that support robust infection of SARS-CoV-2 for production of high titer viral stocks. Sensitive cell culture systems to investigate viral interactions with the innate immune system were also generated using lentiviral systems.

伯内特柯克斯体是一种普遍存在的人畜共患细菌病原体，也是人类急性 Q 热（一种致残性流感样疾病）的病因。柯克斯体以前的专性细胞内性质显着阻碍了假定毒力因子的遗传表征。然而，我们在酸性柠檬酸盐半胱氨酸培养基 (ACCM) 中实现柯克斯体宿主无细胞（无菌）生长的开创性进展使我们能够快速开发出完整的遗传学工具箱。最近，我们开发了一种名为 ACCM-D 的成分确定培养基，可支持 Coxiella 的强劲生长，其中含有氨基酸作为唯一的碳和能源。 由于缺乏生物合成中的最终酶，柯克斯体对精氨酸、赖氨酸、脯氨酸和酪氨酸是营养缺陷型的。嗜肺军团菌 argGH、lysA 和 proAB 以及大肠杆菌 tyrB 的柯克斯体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，从而为柯克斯体转化体的营养选择提供了四种方法。考虑到基于抗生素抗性的选择标记对于这种选择剂来说是有限的，对遗传转化体的强的、非抗生素的选择是一个重要的进步。 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来发现毒力因子。事实上，我们已经在强毒力和无毒力柯克斯体中构建了敲除菌株，包括编码 Dot/Icm 型 IVB 分泌系统 (T4BSS) 和分泌蛋白的基因缺失的菌株。这些研究证实 T4BSS 功能对于巨噬细胞中柯克斯体的生长至关重要。此外，使用 Cre-lox，我们创建了有毒力九英里 I 期菌株的 32.4 kb dot/icm 突变体，该突变体缺乏合成 T4BSS 所需的整个 dot/icm 基因座。该突变体在合成培养基中表现出旺盛的生长，但不能在细胞内生长。 突变分析还确定了与毒力相关的 LPS 相位变异的遗传机制。 迄今为止，所有被测序的柯克斯体菌株都携带一个大的（32-54 kb）自主复制质粒或具有染色体整合的质粒样序列，这表明质粒基因对于感染很重要。参考九英里菌株携带的 QpH1 质粒上的七个基因编码疑似介导毒力的 4B 型分泌系统效应蛋白。这些基因中只有两个在 Coxiella 质粒或 IPS 之间是保守的。 我们开发了一种新的大肠杆菌-柯克斯体穿梭载体 (pBR322-CAT-sacB-tyrB-QpH1ori)，其中包含克隆到含有 tyrB 基因的大肠杆菌质粒中的 QpH1 复制起点。在酪氨酸前体 4-羟基苯基丙酮酸 (4-HPA) 存在的情况下，柯克斯体产生 TyrB，挽救了细菌的天然酪氨酸营养缺陷型。还为 Coxiella 开发了用于基因沉默的 CRISPRi 系统，该系统利用来自军团菌的 proBA 基因来补充 Coxiella 的天然脯氨酸营养缺陷型。将 pBR322-CAT-sacB-tyrB-QpH1ori 引入 C.burnetii Nine Mile（II 期），然后在补充有 4-HPA 的酪氨酸缺陷 ACCM-D 中生长，导致天然 QpH1 质粒的排出。该突变株在无菌培养基中生长正常，但在宿主细胞中存在严重的生长缺陷。使用 QpH1 大片段进行互补，确定了细胞内生长所需的一般区域。 CRISPRi 基因沉默被用来识别宿主细胞中柯克斯体生长所必需的单个 QpH1 基因。这项研究确定了两种对柯克斯体遗传转化体进行营养选择的新方法，并建立了一个 CRISPRi 系统，用于强有力的条件性基因表达敲低。 柯克斯体编码少量转录调节因子，这些调节因子可能对巨噬细胞内的存活和/或发育转变至关重要。柯克斯体的 PhoBR 双组分系统 (TCS) 特别令人感兴趣，因为其他细菌中的同源系统调节毒力基因表达。利用基因敲除、报告基因检测、RNAseg 和全细菌蛋白质组质谱分析，我们解决了 PhoBR 的调控级联问题。解开 PhoBR 和其他 TCS 的监管网络将确定重要的毒力决定因素。 柯克斯体经历细胞内双相发育周期，产生两种不同的形态变异，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 的转变与柯克斯体进入静止生长期同时发生，在受感染细胞培养物的延长孵育（2 至 4 周）后出现几乎均质的 SCV。作为一个有助于更好地理解柯克斯体分化的生物学相关性的可靠模型，我们确定了在第三代无菌培养基 ACCM-D 中生长的生物体可以重现 SCV/LCV 转变。这一发现使得研究柯克斯体发育生物学成为可能，而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已揭示了形态分化的分子决定因素，这些因素可能有助于形成细胞形式的独特生物学特征。 与分化相关的基因现在被灭活，并对突变体进行表型分析。 目前的人类 Q 热疫苗 Q-VAX 是一种固定全细胞疫苗 (WCV)，仅获准在澳大利亚使用。虽然柯克斯体 WCV 非常有效，但对于已有柯克斯体免疫力的人来说，疫苗接种后可能会出现严重的皮肤过敏反应，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贡献。在强毒力九英里 I 期 (NMI) 菌株中删除了包含 23 个点/icm 基因的 32.5 kb 区域，并在 Q 热感染、疫苗接种和攻击以及疫苗后超敏反应的豚鼠模型中评估了所得突变体。 NMI dot/icm 菌株无毒，可像 WCV 一样抵抗强烈的柯克斯体挑战，并且与野生型柯克斯体相比，表现出潜在改变的反应原性。分别产生平滑和粗糙 LPS 的 NMI 和同基因九英里 II 期 (NMII) 柯克斯体菌株也进行了类似测试。作为 WCV，NMI 比 NMII 具有更强的保护作用；然而，两种疫苗表现出相似的反应原性。总的来说，我们的结果表明，与 I 期 LPS 一样，T4BSS 是柯克斯体完全毒力所必需的。相反，与 I 期 LPS 不同，疫苗诱导的保护不需要 T4BSS。 LPS 长度似乎也不会导致皮肤过敏反应，而 T4BSS 可能会导致这种反应。 NMI dot/icm 代表一种具有完整疫苗功效的无毒力 I 期菌株，并说明了使用转基因柯克斯体作为改良 WCV 的潜力。 作为 NIAID/RML 应对 COVID-19 大流行的一部分，CPS 与 Sonja Best 实验室 (LV) 合作生成慢病毒构建体，用于在哺乳动物细胞中表达 SARS-CoV-2 受体 ACE2 和相关丝氨酸蛋白酶 TMPRSS2。 目的是产生支持 SARS-CoV-2 强力感染的细胞系，以生产高效价病毒原种。还使用慢病毒系统生成了用于研究病毒与先天免疫系统相互作用的敏感细胞培养系统。