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是一种强制性细胞内细菌，是人动物病（查询）发烧的病因。急性Q发烧通常表现为一种使人衰弱的流感样疾病。通常会出现罕见但严重的慢性感染，通常会作为心内膜炎。病原体的阴险性质是一种感染剂量，接近一种生物体和类似孢子的细胞外稳定性。这些属性已将C. burnetii C. c。c。c. buothreat均为CDC类。环境抗性还与对病原体细胞内生态裂市场的降解条件的抗性相关。 对于体外感染性/细胞病理学和实验动物的致病性，植物上的遗传学不同的分离株显示出不同的表型。此外，已经描述了C. burnetii基因组群与人类疾病表现（急性与慢性）之间的相关性，这表明分离株具有独特的毒力特征。为了更完整地了解C. burnetii遗传多样性，进化和致病潜力，我们破译了K（Q154）和G（Q154）和G（Q212）人类慢性心内膜炎的整个基因组序列，并自然减弱了Dugway（5J108-111）Rodent rodent rodent孤立孤立。 跨基因组比较包括先前序列的九英里（NM）参考分离株（RSA493），揭示了新型基因含量和伪基的不同集合，这可能有助于分离毒力和其他表型。虽然C. burnetii基因组高度同步，但丰富的插入序列（IS）元素之间的重组导致基因组可塑性表现为同义块的染色体重排和DNA插入/缺失。众多的元素，基因组重排和burnetii分离株的假基因与其他细菌病原体的基因组结构一致，这些细菌病原体最近来自非pathogens，这些病原体来自具有扩展的壁nikes的非pathogens。严重减弱的dugway分离株具有最大的伪基因基因组，并且是元素表明该分离株谱系比NM，K和G谱系的病原体阶段更早。 缺乏遗传操纵C. burnetii的方法显着阻碍了对生物体的研究。我们使用HIMAR1转座子（TN）系统成功地将C. burnetii转化为氯霉素耐药性和MCHERRY红色荧光蛋白表达。在C. burnetii HSP20启动子P1169的控制下，氯霉素乙酰转移酶（CAT）和MCHERRY均以单个转录单位表示。 COLE1复制和DNA测序的救援克隆揭示了散布在整个C. burnetii基因组中的TN插入位点。使用我们的微观液化克隆方法分离了来自转化的混合物的克隆，并显示出在必需细胞分裂基因FTSZ中插入TN插入。 FTSZ :: TN突变体的表征显示了在19.8小时的指数期间的一代时间，几乎是C. burnetii（11.7 h）的两倍。这是burnetii的第一个描述，该描述具有由遗传转化产生的定义基因突变。重要的是，这项研究表明，HIMAR1转座子系统是在C. burnetii中创建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，次优于通过荧光显微镜可视化转化的生物体。因此，我们检查了外膜Porin P1（CBU0311）启动子P311的使用来驱动麦克利表达。 此外，除了酸化的柠檬酸酸性半胱氨酸培养基（ACCM）中电穿孔生物的无宿主细胞生长外，还测试了卡纳霉素作为阳性选择的替代方法。 从p311 verses p1169驱动时，麦克里的表达明显更高。实际上，通过荧光显微镜很容易看到单个生物。与Vero细胞的感染一样，最初在ACCM中膨胀的C. burnetii转化子对CAT DNA呈阳性，并且对氯霉素的抗性。但是，在ACCM中，在1-2周内检测到转化体，而Vero细胞中的4-5周则检测到转化体。 卡纳米霉素也未用于Q热的临床治疗中，也通过用含有卡纳霉素耐药基因在P1169控制下的himar1转座子转化C. burnetii，作为替代选择标记。观察到含有250 ug/ml卡纳米霉素的ACCM中转化体的生长，没有非转化的对照生物的生长。 HIMAR1转座子不仅允许随机诱变和转基因在C. burnetii中的稳定整合，而且还提供了一种工具来测试和优化生物体不断发展的遗传转化系统的不同方面。 此外，除了允许选择转化体的选择外，ACCM中电穿孔C. burnetii的轴突生长显着降低了转化体的初始生长时间，而转化体对宿主细胞的生长却是致命的。 脂多糖是C. burnetii的唯一定义的毒力因子。具有全长LP的有毒的I期生物，转化为无毒的II期生物，并在重复的体外传递后合成严重截短的LPS。考虑到II期分离株的深度粗糙表型的遗传病变尚不清楚。为此，我们使用微量操纵克隆程序生成了高通道澳大利亚和加利福尼亚菌株的II期克隆，并将其基因组DNA杂交到了一个高密度的微阵列，其中包含探头集，其中包含所有九英里I期应变的所有全长读取框架。这些阵列是专门设计用于检测indels（插入/删除）的。在参与七糖生物合成的基因中发现了一个共同的indel，我们认为这是相关的造成的。 Q热需要进行敏感且特异性的血清诊断测试，该测试利用重组C. burnetii蛋白作为抗原。为了实现这一目标，我们开发了一种burnetii C. burnetii蛋白微阵列，以全面鉴定在人杆菌感染或疫苗接种的背景下，抗体识别的免疫主导抗原。与1988年C. burnetii开放式阅读框（ORF）相对应的具有转录活性的PCR产品。通过使用基于大肠杆菌的细胞体外转录和翻译系统（IVTT），通过75％的ORF成功合成了全长蛋白。发现硝酸纤维素微阵列用粗糙的IVTT裂解物发现，并用急性Q热患者的血清和Q-VAX疫苗接种的个体进行探测。免疫血清与大约50 C. burnetii蛋白（包括先前鉴定出的免疫原子，含有蛋白质的脚踝重复域）和多种假设蛋白质的burnetii蛋白强烈反应。产生了与选定的阵列反应性抗原相对应的重组蛋白，ELISA证实了免疫反应性。这种敏感且高的吞吐量方法用于鉴定免疫反应性C. burnetii蛋白将有助于基于重组抗原的Q发烧血清诊断测试。此外，对微阵列识别抗原的T细胞抗原性测试可能会鉴定出有效性的蛋白质，作为抗Q发烧的亚基疫苗。