Genetics of Coxiella burnetii

伯内氏柯克斯体的遗传学

• 批准号: 7964514
• 负责人: robert a heinzen
• 金额: $ 71.67万
• 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/7964514
• 关键词: Accounting; Acute; Affinity; Anabolism; Animals; Ankyrin Repeat; Antibodies; Antibody Formation; Antigens; Attenuated; Autonomous Replication; Bacteria; Biogenesis; Biology; California; Categories; Cell Culture Techniques; Cell division; Cells; Centers for Disease Control and Prevention (U.S.); Characteristics; Chloramphenicol; Chloramphenicol O-Acetyltransferase; Chloramphenicol Resistance; Chromosomal Rearrangement; Chronic; Citrates; Clinical Treatment; Cloning; Collaborations; Collection; Coupled; Coxiella burnetii; Cysteine; Cytopathology; DNA; DNA Resequencing; DNA Sequence; DNA Sequence Rearrangement; Development; Dominant-Negative Mutation; Dose; Endocarditis; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Evolution; Felis catus; Fever; Fluorescence Microscopy; Gene Mutation; Gene Silencing; Gene Targeting; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genetic Transcription; Genetic Transformation; Genetic Variation; Genome; Genomics; Goals; Growth; Heptoses; Human; Hypersensitivity skin testing; IS Elements; Imagery; Immune Sera; Immunodominant Antigens; In Vitro; Individual; Infection; Influenza; Isopropyl Thiogalactoside; Kanamycin; Kanamycin Resistance; Laboratories; Laboratory Animals; Latex Bead; Length; Lesion; Lipopolysaccharides; Membrane; Methods; Microarray Analysis; Micromanipulation; Modeling; Molecular Biology; Molecular Weight; Mus; Mutagenesis; Nature; Open Reading Frames; Organism; PAWR protein; Pathogenicity; Patients; Phagolysosome; Phase; Phenotype; Plasmids; Procedures; Protein Microchips; Proteins; Proteome; Pseudogenes; Pyroxylin; Q Fever; Recombinant Proteins; Recombinants; Replication Origin; Reproduction spores; Resistance; Rodent; Role; Serum; Site; Source; Splenomegaly; Spottings; Staging; Structure; Subunit Vaccines; Suicide; System; T-Lymphocyte; Techniques; Testing; Texas; Time; Transgenes; Translations; Universities; Vaccinated; Vaccination; Vaccines; Variant; Vero Cells; Virulence; Virulence Factors; Virulent; Washington; Zoonoses; axenic culture; base; biothreat; density; design; egg; extracellular; factor C; gene function; genome sequencing; homologous recombination; human disease; immunoreactivity; insertion/deletion mutation; insight; memory CD4 T lymphocyte; mutant; novel; pathogen; porin; prevent; promoter; protein expression; red fluorescent protein; success; tissue culture; tool

C. burnetii is an obligate intracellular bacterium and the causative agent of the zoonosis human Q (query) fever. Acute Q fever normally manifests as a debilitating influenza-like illness. Rare but serious chronic infections can occur that usually present as endocarditis. Adding to the insidious nature of the pathogen is an infective dose approaching one organism and spore-like extracellular stability. These attributes have earned C. burnetii designation as a CDC category B biothreat. Environmental resistance also correlates with resistance to the degradative conditions of the pathogen's intracellular niche: the phagolysosome. Genetically distinct isolates of C. burnetii display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting isolates have distinct virulence characteristics. To provide a more complete understanding of C. burnetii genetic diversity, evolution, and pathogenic potential, we deciphered the whole genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. Cross-genome comparisons that included the previously-sequenced Nine Mile (NM) reference isolate (RSA493) revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates is consistent with genome structures of other bacterial pathogens that have recently emerged from non-pathogens with expanded niches. The observation that the severely attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests this isolate lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages. The lack of methods to genetically manipulate C. burnetii significantly impedes study of the organism. We have successfully transformed C. burnetii to chloramphenicol resistance and mCherry red fluorescent protein expression using the Himar1 transposon (Tn) system. Both chloramphenicol acetyltransferase (CAT) and mCherry were expressed as a single transcriptional unit under control of the C. burnetii Hsp20 promoter p1169. Rescue cloning of the ColE1 origin of replication and DNA sequencing revealed Tn insertion sites scattered throughout the C. burnetii genome. A clone from the transformant mixture was isolated using our micromanipulation cloning method and shown to harbor a Tn insertion within the essential cell division gene ftsZ. Characterization of the FtsZ::Tn mutant revealed a generation time during exponential phase of 19.8 h, almost twice as long as wild type C. burnetii (11.7 h). This is the first description of C. burnetii harboring a defined gene mutation generated by genetic transformation. Importantly, this study shows that the Himar1 transposon system is a robust technique for creating genetic mutations in C. burnetii. While expression of CAT was sufficient to prevent outgrowth of non-transformed bacteria, expression of mCherry was moderate and suboptimal for visualization of transformed organisms by fluorescence microscopy. Therefore, we examined the use of the outer membrane porin P1 (CBU0311) promoter p311 to drive mCherry expression. Moreover, kanamycin resistance as an alternative method of positive selection was tested in addition to host cell-free (axenic) growth of electroporated organisms in acidified citrate cysteine medium (ACCM) as an initial step in expansion of transformants. Expression of mCherry was substantially higher when driven from p311 verses p1169. Indeed, individual organisms were easily visible by fluorescence microscopy. As with infection of Vero cells, C. burnetii transformants initially expanded in ACCM were positive for CAT DNA and resistant to chloramphenicol. However, transformants were detected in 1-2 weeks in ACCM as compared to 4-5 weeks in Vero cells. Kanamycin, which is also not used in the clinical treatment of Q fever, was also tested as an alternative selective marker by transforming C. burnetii with a Himar1 transposon containing the kanamycin resistance gene under control of p1169. Outgrowth of transformants in ACCM containing 250 ug/ml kanamycin was observed with no outgrowth of non-transformed control organisms. Not only does the Himar1 transposon allow random mutagenesis and stable integration of transgenes in C. burnetii, it also provides a tool to test and optimize different aspects of the organisms evolving genetic transformation systems. Moreover, axenic growth of electroporated C. burnetii in ACCM substantially decreases the time of initial outgrowth of transformants in addition to allowing selection of transformants that would otherwise be lethal for growth in host cells. Lipopolysaccharide is the only defined virulence factor of C. burnetii. Virulent phase I organisms, producing full-length LPS, convert to avirulent phase II organisms,synthesizing severely truncated LPS, upon repeated in vitro passages. The genetic lesion(s) accounting for the deep rough phenotype of phase II isolates is unknown. To this end, we generated phase II clones of the high passage Australian and California strains, using our micromanipulation cloning procedure, and hybridized their genomic DNAs to a high-density microarray that contains probe sets encompassing all full-length open reading frames of the Nine Mile phase I strain. These arrays are specifically designed to detect indels (insertions/deletions). A common indel was found within a gene involved in heptose biosynthesis that we believe accounts for phase conversion. A sensitive and specific serodiagnostic test is needed for Q fever that utilizes recombinant C. burnetii protein(s) as antigen. To pursue this goal, we developed a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an E. coli-based cell-free in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated and immunoreactivity confirmed by ELISA. This sensitive and high throughput method for identifying immunoreactive C. burnetii proteins will aid development of Q fever serodiagnostic tests based on recombinant antigen. Moreover, testing of microarray-identified antigens for T-cell antigenicity may identify proteins with efficacy as subunit vaccines against Q fever.

C.burnetii 是一种专性细胞内细菌，也是人畜共患病人类 Q（查询）热的病原体。急性 Q 热通常表现为一种使人衰弱的流感样疾病。罕见但严重的慢性感染可能会发生，通常表现为心内膜炎。接近一种生物体的感染剂量和类似孢子的细胞外稳定性增加了病原体的阴险性质。这些特性使伯氏念珠菌被指定为 CDC B 类生物威胁。环境抵抗力还与病原体细胞内生态位（吞噬溶酶体）的降解条件的抵抗力相关。 遗传上不同的伯内特念珠菌分离株在体外感染性/细胞病理学和对实验动物的致病性方面表现出不同的表型。此外，伯氏念珠菌基因组与人类疾病表现（急性与慢性）之间的相关性已被描述，表明分离株具有独特的毒力特征。为了更全面地了解伯内特隐球菌遗传多样性、进化和致病潜力，我们破译了 K (Q154) 和 G (Q212) 人类慢性心内膜炎分离株和自然减毒 Dugway (5J108-111) 啮齿动物分离株的全基因组序列。 包括之前测序的九英里 (NM) 参考分离株 (RSA493) 在内的跨基因组比较揭示了新的基因内容和不同的假基因集合，这些假基因可能有助于分离株的毒力和其他表型。虽然 C. burnetii 基因组是高度同线性的，但丰富的插入序列 (IS) 元件之间的重组导致了基因组可塑性，表现为同线性块的染色体重排和 DNA 插入/删除。 C.burnetii 分离株的大量 IS 元件、基因组重排和假基因与最近从具有扩大生态位的非病原体中出现的其他细菌病原体的基因组结构一致。观察到严重减毒的 Dugway 分离株具有最大的基因组和最少的假基因和 IS 元件，这表明该分离株谱系比 NM、K 和 G 谱系处于病理适应的早期阶段。 缺乏基因操纵伯内特氏菌的方法严重阻碍了对该生物体的研究。我们使用 Himar1 转座子 (Tn) 系统成功将 C. burnetii 转化为氯霉素抗性并表达 mCherry 红色荧光蛋白。氯霉素乙酰转移酶 (CAT) 和 mCherry 均在 C. burnetii Hsp20 启动子 p1169 的控制下表达为单个转录单位。 ColE1 复制起点的拯救性克隆和 DNA 测序揭示了 Tn 插入位点分散在整个 C. burnetii 基因组中。使用我们的显微操作克隆方法从转化体混合物中分离出一个克隆，并显示其在必需的细胞分裂基因 ftsZ 中包含 Tn 插入。 FtsZ::Tn 突变体的表征显示，指数生长期的世代时间为 19.8 小时，几乎是野生型 C.burnetii（11.7 小时）的两倍。这是对带有通过遗传转化产生的确定基因突变的伯内特 C. burnetii 的首次描述。重要的是，这项研究表明 Himar1 转座子系统是一种在 C. burnetii 中产生基因突变的强大技术。 虽然 CAT 的表达足以防止非转化细菌的生长，但 mCherry 的表达是中等的，对于通过荧光显微镜观察转化的生物体而言不是最佳的。因此，我们检查了外膜孔蛋白 P1 (CBU0311) 启动子 p311 驱动 mCherry 表达的用途。 此外，除了在酸化柠檬酸盐半胱氨酸培养基（ACCM）中电穿孔生物体的宿主无细胞（无菌）生长作为转化体扩增的初始步骤之外，还测试了卡那霉素抗性作为正选择的替代方法。 当从 p311 驱动时，mCherry 的表达明显高于 p1169。事实上，通过荧光显微镜可以很容易地看到单个生物体。与 Vero 细胞的感染一样，最初在 ACCM 中扩增的 C.burnetii 转化体对 CAT DNA 呈阳性并且对氯霉素具有抗性。然而，在 ACCM 中需要 1-2 周才能检测到转化体，而在 Vero 细胞中则需要 4-5 周。 卡那霉素也未用于 Q 热的临床治疗，也通过使用包含 p1169 控制下的卡那霉素抗性基因的 Himar1 转座子转化伯内特衣藻，将其作为替代选择标记进行了测试。观察到转化体在含有250ug/ml卡那霉素的ACCM中生长，而未转化的对照生物体没有生长。 Himar1 转座子不仅允许在 C. burnetii 中进行随机突变和转基因的稳定整合，而且还提供了测试和优化生物体进化遗传转化系统的不同方面的工具。 此外，电穿孔伯氏梭菌在 ACCM 中的无菌生长除了允许选择否则在宿主细胞中生长会致命的转化体之外，还大大减少了转化体的初始生长时间。 脂多糖是伯氏念珠菌唯一确定的毒力因子。产生全长 LPS 的毒力 I 期生物体在体外反复传代后转化为无毒力 II 期生物体，合成严重截短的 LPS。导致 II 期分离株深部粗糙表型的遗传损伤尚不清楚。为此，我们使用显微操作克隆程序生成了高传代澳大利亚和加利福尼亚菌株的 II 期克隆，并将其基因组 DNA 与高密度微阵列杂交，该芯片包含包含九英里 I 期菌株所有全长开放阅读框的探针组。这些数组专门设计用于检测 indel（插入/删除）。在参与庚糖生物合成的基因中发现了一个常见的插入缺失，我们认为它是相转换的原因。 Q 热需要利用重组伯氏梭菌蛋白作为抗原进行灵敏且特异的血清诊断测试。为了实现这一目标，我们开发了伯内特衣原体蛋白微阵列，以全面鉴定在人类伯内特衣原体感染或疫苗接种情况下抗体识别的免疫显性抗原。生成了对应于 1988 C. burnetii 开放阅读框 (ORF) 的转录活性 PCR 产物。使用基于大肠杆菌的无细胞体外转录和翻译系统 (IVTT)，成功从 75% 的 ORF 合成了全长蛋白质。用粗 IVTT 裂解物点样硝酸纤维素微阵列，并用急性 Q 热患者和接种 Q-Vax 疫苗的个体的血清进行探测。免疫血清与大约 50 种伯氏梭菌蛋白发生强烈反应，包括先前鉴定的免疫原、含有锚蛋白重复结构域的蛋白和多种假设蛋白。生成了与所选阵列反应性抗原相对应的重组蛋白，并通过 ELISA 确认了免疫反应性。这种用于鉴定免疫反应性伯内特念珠菌蛋白的灵敏且高通量的方法将有助于开发基于重组抗原的 Q 热血清诊断测试。此外，测试微阵列识别的抗原的 T 细胞抗原性可能会识别出具有抗 Q 热亚单位疫苗功效的蛋白质。