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.

Coxiella burnetii是一种无处不在的人畜共患病原体，也是人类急性Q发烧的原因，这是一种残疾的流感样疾病。 Coxiella的前义务细胞内性质显着阻碍了推定的毒力因子的遗传表征。然而，我们在酸化的柠檬酸酸性半胱氨酸培养基（ACCM）中无宿主细胞（轴突）生长的开创性使我们能够快速开发一个完整的遗传学工具盒。最近，我们开发了一种定义的培养基，该培养基支持称为ACCM-D的Coxiella稳健生长，该培养基包含氨基酸作为唯一的碳和能源。 Coxiella缺乏生物合成中的最终酶，是Arg，Lys，Pro和Tyr的可营养性。 Coxiella的异源表达分别为Arg，Lys，Lys，Pro和Tyr Accm-D辍学培养基中的生长，从而提供了四种方法，从而提供了四种方法，可为Coxiella转化剂的营养选择提供四种方法。考虑到基于抗生素耐药性的可选标记，该精选药物的可选标记物的强度，非抗生素的选择是重要的进步。 总的来说，我们的Coxiella遗传工具的曲目现在允许发现传统的突变和互补策略。实际上，我们已经在毒和无毒的考氏菌中构建了基因敲除菌株，包括在编码DOT/ICM型IVB分泌系统（T4BSS）和分泌蛋白质的基因中缺失的淘汰菌株。这些研究已经证实，T4BSS功能对于巨噬细胞中的考克斯菌生长至关重要。此外，使用Cre-lox，我们创建了一个32.4 kb的点/ICM突变体的毒气9英里I期菌株，该菌株缺乏合成T4BSS所需的整个点/ICM基因座。突变体在合成介质中显示出剧烈的生长，但不能细胞内生长。 突变分析还确定了与毒力相关的LPS相变的遗传机制。 所有测序的coxiella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。 QPH1质粒上的七个基因，由参考九英里菌株携带，编码4B型分泌系统效应子蛋白，涉嫌介导毒力。这些基因中只有两个在coxiella质粒或IP之间保守。 我们开发了一种新的大肠杆菌驾驶汽车载体（PBR322-CAT-SACB-TYRB-QPH1ORI），该载体包含复制的QPH1起源，该复制的起源克隆到包含Tyrb基因的大肠杆菌质粒中。在存在酪氨酸前体4-羟基苯基丙酮酸（4-HPA）的情况下，Tyrb的Coxiella生产可挽救细菌的天然合物用于酪氨酸。还为Coxiella开发了一种用于基因沉默的CRISPRI系统，该系统利用了从Legionella的探针基因来补充Coxiella的天然合物为脯氨酸。将PBR322-CAT-SACB-TYRB-QPH1ORI引入到C. burnetii 9英里（II期）中，然后在补充了4-HPA的酪氨酸缺陷型ACCM-D中生长，导致驱逐天然QPH1质粒。突变菌株通常在轴突培养基中生长，但在宿主细胞中存在严重的生长缺陷。使用QPH1的大片段的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coxiella生长至关重要的单个QPH1基因。这项研究确定了两种新型的coxiella遗传转化物的营养选择方法，并建立了一个CRISPRI系统，用于有条件地敲低基因表达。 Coxiella编码了可能对噬菌体内存活和/或发育过渡至关重要的转录调节剂的缺乏。 Coxiella的PhOBR两组分系统（TC）在其他细菌中特别吸引了同源系统调节毒力基因的表达。使用基因敲除，报告基因测定，RNASEG和整个细菌蛋白质组质谱法，我们解决了PHOBR的调节级联。阐明PHOBR和其他TCS的调节网络将确定重要的毒力决定因素。 Coxiella经历了一个细胞内双相发育周期，该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。小细胞变体（SCV）不复制，含有冷凝的染色质，被认为是细胞外存活形式。 SCV用分散的染色质分化为复制的大细胞变体（LCV）。 LCV回到SCV的过渡是与Coxiella进入固定生长阶段的同时发生的，在延长的孵育（2至4周）的感染细胞培养物中，几乎均匀的SCV存在。作为一个可帮助更好地了解Coxiella分化的生物学相关性的可拟合模型，我们确定SCV/LCV转变是由第三代轴突介质中生长的生物ACCM-D概括的。这一发现使对托氏菌发育生物学的研究没有宿主细胞传播的细菌遇到的实验困难。 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，这可能有助于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表现出突变体。 当前的人类Q发烧疫苗Q-VAX是一种固定的全细胞疫苗（WCV），仅在澳大利亚使用许可。虽然高度有效，但Coxiella wcvs与先前对Coxiella免疫力的患者具有潜在的严重疫苗接种后皮肤超敏反应有关，这限制了其更广泛的使用。因此，需要较少的反应疫苗。我们研究了Coxiella Dot/ICM IVB分泌系统（T4BSS）和脂多糖（LPS）在固定WCV的保护和反应生成性方面的贡献。在有毒的9英里I期（NMI）菌株中删除了一个包含23个点/ICM基因的32.5 Kb区域，并在Q热感染，疫苗接种和挑战的豚鼠模型中评估了所得突变体，并在疫苗接种后超敏。 NMI DOT/ICM菌株是无毒的，是针对强大的Coxiella挑战的WCV的保护性，与野生型Coxiella相比，反应生成可能改变。类似测试了NMI和等源性九英里II期Coxiella菌株，分别产生光滑且粗糙的LPS。 NMI比NMII作为WCV更具保护性。但是，两种疫苗都表现出相似的反应性。总体而言，我们的结果表明，像I期LPS一样，Coxiella完全毒力需要T4BS。相反，与I期LP不同，疫苗诱导的保护不需要T4BS。 LPS的长度似乎也没有导致真皮超敏反应，而T4BSS可能会导致这种反应。 NMI DOT/ICM代表具有全疫苗功效的无毒性I期菌株，并说明了将转基因的Coxiella用作改进的WCV的潜力。 作为NIAID/RMLS对COVID-19大流行的响应的一部分，CPS与Sonja Best Lab（LV）合作生成了慢病毒构建体，以表达SARS-COV-2受体ACE2和相关的丝氨酸蛋白酶蛋白酶TMPRSS2，在哺乳动物细胞中。 目的是生成支持SARS-COV-2稳健感染的细胞系，以生产高滴度病毒量。还使用慢病毒系统生成了敏感的细胞培养系统，以研究与先天免疫系统的病毒相互作用。