Genetics of Coxiella burnetii

伯氏柯克斯体的遗传学

• 批准号: 8336184
• 负责人: robert a heinzen
• 金额: $ 68.74万
• 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/8336184
• 关键词: Accounting; Acute; Anabolism; Antibiotics; Antigens; Archives; Australia; Bacteria; Bacteriophage P1; Biochemical; Biological Assay; C-terminal; California; Categories; Cell Communication; Cell Culture Techniques; Cells; Centers for Disease Control and Prevention (U.S.); Chloramphenicol; Chloramphenicol Resistance; Chromosomes; Chronic; Citrates; Cloning; Collection; Complex; Coxiella; Coxiella burnetii; Culture Media; Culture Techniques; Cysteine; Cytosol; DNA; Defect; Development; Diagnostic; Dominant-Negative Mutation; Electroporation; Endocarditis; Epitopes; Event; Excision; Exhibits; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Frequencies; Gene Deletion; Gene Dosage; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genetic Transformation; Genome; Genomics; Genotype; Glucosamine; Goals; Gram-Negative Bacteria; Growth; Heat shock proteins; Immune; Individual; Infection; Intercistronic Region; Kanamycin; Knowledge; Lactamase; Length; Libraries; Life; Ligase; Liquid substance; Masks; Mediating; Membrane; Methods; Micromanipulation; Modeling; Molecular; Mutagenesis; Nutrient; Organism; PAWR protein; Pathogenesis; Patients; Phase; Pilot Projects; Plasmids; Population; Preparation; Procedures; Production; Prokaryotic Cells; Protein Binding; Protein Biosynthesis; Proteins; Pseudomonas aeruginosa; Q Fever; Relative (related person); Reporter; Resistance; SCID Mice; Sepharose; Serum; Shuttle Vectors; Signal Transduction; Site; Solid; Subunit Vaccines; Sucrose; Suicide; Surface; System; Techniques; Testing; Tissues; Transposase; Vaccine Antigen; Variant; Vero Cells; Vertebral column; Virulence; Virulence Factors; Virulent; base; biothreat; commercial application; density; design; flu; genetic manipulation; improved; insertion/deletion mutation; interest; macrophage; mutant; parasitism; pathogen; porin; promoter; recombinase; red fluorescent protein; tool; trafficking; vector

Acidified Citrate Cysteine Medium (ACCM) that supports approximately 3 logs of Coxiella growth under microaerobic (2.5% O2) conditions. We have now developed a serum-free version of ACCM termed ACCM-2 that supports improved growth of both virulent and avirulent strains of Coxiella in both liquid and solid media. For example, we now routinely obtain > 4 logs of growth in liquid media. Moreover, we have increased the colony size on agarose plates by roughly 5-fold to approximately 0.5 mm, greatly aiding clonal isolation by colony picking. Coxiella was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice, providing a sensitive means of primary isolation of the pathogen from Q fever patients. A completely axenic Coxiella genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 weeks. Transformation using RF1010 ori-based shuttle demonstrated a transformation frequency of approximately 5 x 10-5. Moreover, both CAT and Kan genes are excellent selectable markers for Coxiella transformants in both ACCM as well as cell culture. Both antibiotics are Select Agent compliant, allowing two transformation procedures in a single strain. ACCM-2 will facilitate biochemical studies and, as described below, development of Coxiella genetic systems. Coxiella plates with high efficiency on ACCM-2 agarose ( 1 to 2 genome equivalents per colony) and the resulting colonies contain clonal populations. Colonies can be expanded by direct transfer into ACCM-2 and resultant liquid cultures stably archived. 2) Development of new tools for Coxiella genetic manipulation ACCM culture techniques were used to develop an optimized Himar1-based transposon (Tn) mutagenesis system for Coxiella. In our original Himar1 system, the promoter from CBU1169 (1169P), which encodes the small heat shock protein Hsp20, was used to drive expression of CAT and mCherry red fluorescent protein genes as a single transcriptional unit. Expression was sufficient to confer resistance to 5 μg/ml of chloramphenicol, but mCherry fluorescence was low. Therefore, a new Himar1 transposon plasmid (pITR-CAT:: 311P-MC) was constructed that employs the CBU0311 (outer membrane porin P1) promoter (311P) to drive expression of the mCherry red fluorescent protein gene independent of 1169P-driven CAT. CBU0311 is constitutively expressed at an approximately 6-fold higher level than CBU1169. By both fluorescence microscopy and flow cytometry, transformants harboring this Tn show significantly enhanced mCherry red protein fluorescence and are useful in pathogen-host cell interaction studies. With the optimized Himar1 Tn, we are now constructed ordered libraries of random Coxiella Tn mutants for wholesale testing of individual mutant clones for defects in macrophage invasion and/or replication. An important recent discovery aided by ACCM culture is that vectors derived from the IncQ plasmid RSF1010 autonomously replicate in Coxiella. Shuttle vectors containing the RSF1010 ori are compatible with the endogenous Coxiella plasmid and have a copy number of roughly 3 to 6. This discovery allowed construction of reporter plasmids for identification of proteins secreted by Coxiella into the host cell cytosol. The secretion assay detects cytosolic delivery of β-lactamase (BlaM) or camodulin-activated andenylate cyclase (CyaA) C-terminally fused in-frame to a Coxiella protein containing a translocation signal. In pJB-CAT-BlaM, blaM and CAT are driven independently by constitutive 1169P. Use of each vector verified cytoplasmic translocation of a unique set of 6 suspected C. burnetii secreted proteins. We have modified the backbone of pJB-CAT-BlaM (i. e., pJB-CAT) to contain a cloning site that allows 1169P-directed synthesis of proteins with N- or C-terminal epitope tags of 2xHA or 3xFLAG. A version encoding kanamycin instead of chloramphenicol resistance (pJB-KAN) has also been constructed. Applications of these vectors include expression of dominant/negative proteins for functional studies, expression of epitope tagged proteins to assess protein binding partners and intracellular trafficking, and in trans complementation of mutants generated by Tn or other mutagenesis techniques to fulfill molecular Kochs postulates. The establishment of pJB-CAT/KAN and other RSF1010 ori-based based shuttle vectors is a significant advance in Coxiella genetic manipulation. However, a potential downside of their use is unwanted gene dosage effects due to multiple plasmid copies. Therefore, we developed a system for site-specific single-copy Coxiella gene knock-ins using Tn7. The Tn7 transposase, encoded by tnsABCD, recognizes a specific 30 bp site termed attTn7 that, in Pseudomonas aeruginosa and other gram-negative bacteria, is located in the extreme 3 end of glmS encoding glucosamine-6-phophosphate synthetase. Transposition is orientation specific and occurs 36 bp downstream of attTn7 in an intergenic region. Analysis of the 3 end C. burnetii glmS (CBU1787) revealed a putative attTn7 site. To test whether Tn7 can utilize this site, a two-suicide plasmid Tn7 system was constructed. The plasmid pTNS2::1169P-tnsABCD carries the transposase genes tnsABCD under control of 1169P. The plasmid pMiniTn7T-CAT::MC carries Tn7 with CAT and mCherry red fluorescent protein genes driven independently by 1169P and 311P, respectively. Co-electroporation with both plasmids and selection of chloramphenicol resistant Coxiella in ACCM yielded transformants with Tn7 inserted 36 bp downstream of the predicted attTn7 site in an intergenic region between glmS (CBU1787) and CBU1788, which encodes a hypothetical protein. The transformant exhibits bright mCherry red protein fluorescence in infected Vero cells at 5 days post infection with no obvious growth defect relative to wild-type bacteria. Tn7 was used in extensively in complementation studies of a Coxiella mutant containing a Himar1 insertion in icmD (Project AI000931-09). The mutant exhibits deffective type IV secretion of effector proteins and fails to replicate in macrophages. We also developed a Tn7-based system of anhydrotetracycline inducible gene expression to evaluate the temporal requirements of type IV secretion in Coxiella infection. Stringent control of gene expression by Coxiella growing in ACCM or macrophage host cells was achieved by using anhydrotetracycline induction of the TetRA repressor-promoter fragment. 3) Re-sequencing microarrays reveal genetic polymorphisms of clonal phase II isolates likely responsible for LPS phase variation. The high passage phase II isolates in our stock collection are not clonal and contain a small subpopulation of Coxiella still expressing full-length phase I LPS. The resulting mixed genotype complicates identification of indels (insertions/deletions) strictly associated with phase variation. To circumvent this problem, we used our micromanipulation cloning procedure to isolate clonal phase II populations of high passage Nine Mile, Australia and California isolates. By hybridizing their genomic DNA to a high-density microarray that contains probe sets encompassing the entire genome of the Nine Mile phase I isolate, we identified common indels in phase II organisms that may account for defective LPS biosynthesis.

酸化柠檬酸盐半胱氨酸培养基 (ACCM) 在微氧 (2.5% O2) 条件下支持约 3 个对数的柯克斯体生长。我们现在开发了一种称为 ACCM-2 的无血清版本的 ACCM，它支持在液体和固体培养基中改善有毒力和无毒力的柯克斯体菌株的生长。例如，我们现在通常在液体培养基中获得 > 4 对数的生长。此外，我们将琼脂糖平板上的菌落大小增加了约 5 倍，达到约 0.5 毫米，极大地有助于通过菌落挑取进行克隆分离。当培养基中接种了受感染 SCID 小鼠的组织匀浆中含有的少至 10 个基因组当量时，柯克斯体在 ACCM-2 中成功培养，为从 Q 热患者中初步分离病原体提供了一种灵敏的方法。 使用 ACCM-2 开发了完​​全无菌的柯克斯体遗传转化系统，可以在大约 2 周内分离出转化体。使用基于 RF1010 ori 的穿梭机进行的转化显示出大约 5 x 10-5 的转化频率。此外，CAT 和 Kan 基因都是 ACCM 和细胞培养物中柯克斯体转化体的优异选择标记。两种抗生素均符合 Select Agent 标准，允许在单一菌株中进行两种转化程序。 ACCM-2 将促进生化研究以及 Coxiella 遗传系统的开发（如下所述）。柯克斯体平板在 ACCM-2 琼脂糖上具有高效率（每个菌落 1 至 2 个基因组当量），所得菌落包含克隆群体。可以通过直接转移到 ACCM-2 中来扩展集落，并将所得液体培养物稳定存档。 2) 柯克斯体基因操作新工具的开发 ACCM 培养技术用于开发基于 Himar1 的优化 Coxiella 转座子 (Tn) 诱变系统。 在我们最初的 Himar1 系统中，编码小型热休克蛋白 Hsp20 的 CBU1169 (1169P) 启动子被用来驱动 CAT 和 mCherry 红色荧光蛋白基因作为单个转录单元的表达。表达足以赋予对 5 μg/ml 氯霉素的抗性，但 mCherry 荧光较低。因此，构建了新的Himar1转座子质粒（pITR-CAT::311P-MC），其采用CBU0311（外膜孔蛋白P1）启动子（311P）来驱动mCherry红色荧光蛋白基因的表达，不依赖于1169P驱动的CAT。 CBU0311 的组成型表达水平比 CBU1169 高约 6 倍。通过荧光显微镜和流式细胞术，携带该 Tn 的转化子显示出显着增强的 mCherry 红色蛋白荧光，可用于病原体-宿主细胞相互作用研究。通过优化的 Himar1 Tn，我们现在构建了随机 Coxiella Tn 突变体的有序文库，用于批量测试单个突变体克隆的巨噬细胞入侵和/或复制缺陷。 最近由 ACCM 培养辅助的一项重要发现是，源自 IncQ 质粒 RSF1010 的载体可在柯克斯体中自主复制。含有 RSF1010 ori 的穿梭载体与内源柯克斯体质粒相容，拷贝数约为 3 至 6。这一发现允许构建报告质粒，以鉴定柯克斯体分泌到宿主细胞胞质中的蛋白质。分泌测定可检测 β-内酰胺酶 (BlaM) 或卡调素激活的安烯酸环化酶 (CyaA) C 端与含有易位信号的柯克斯体蛋白框内融合的胞质递送。在pJB-CAT-BlaM中，blaM和CAT由本构1169P独立驱动。每个载体的使用都验证了一组独特的 6 种疑似伯氏念珠菌分泌蛋白的细胞质易位。我们修改了 pJB-CAT-BlaM（即 pJB-CAT）的主链，以包含一个克隆位点，该位点允许 1169P 定向合成带有 2xHA 或 3xFLAG 的 N 或 C 末端表位标签的蛋白质。还构建了编码卡那霉素而不是氯霉素抗性的版本（pJB-KAN）。这些载体的应用包括用于功能研究的显性/阴性蛋白的表达、用于评估蛋白结合配偶体和细胞内运输的表位标记蛋白的表达，以及通过Tn或其他诱变技术产生的突变体的反式互补以满足分子科赫斯假设。 pJB-CAT/KAN 和其他基于 RSF1010 ori 的穿梭载体的建立是柯克斯体遗传操作的重大进展。然而，它们的使用的一个潜在缺点是由于多个质粒拷贝而导致不必要的基因剂量效应。 因此，我们开发了一种使用 Tn7 进行位点特异性单拷贝 Coxiella 基因敲入的系统。 Tn7 转座酶由 tnsABCD 编码，可识别称为 attTn7 的特定 30 bp 位点，在铜绿假单胞菌和其他革兰氏阴性细菌中，该位点位于编码 6-磷酸葡萄糖胺合成酶的 glmS 的最末端 3 端。 转座具有方向特异性，发生在 attTn7 基因间区域下游 36 bp 处。 对 C.burnetii glmS (CBU1787) 3 端的分析揭示了假定的 attTn7 位点。为了测试Tn7是否可以利用该位点，构建了双自杀质粒Tn7系统。 质粒pTNS2::1169P-tnsABCD携带受1169P控制的转座酶基因tnsABCD。 质粒pMiniTn7T-CAT::MC携带分别由1169P和311P独立驱动的具有CAT和mCherry红色荧光蛋白基因的Tn7。 与两种质粒共电穿孔并在 ACCM 中选择氯霉素抗性柯克斯体，产生了 Tn7 插入到 glmS (CBU1787) 和 CBU1788 之间基因间区域中预测 attTn7 位点下游 36 bp 的转化体，该区域编码假设的蛋白质。 感染后 5 天，转化体在受感染的 Vero 细胞中表现出明亮的 mCherry 红色蛋白荧光，与野生型细菌相比没有明显的生长缺陷。 Tn7 广泛用于对在 icmD 中包含 Himar1 插入的 Coxiella 突变体的互补研究（项目 AI000931-09）。 该突变体表现出无效的 IV 型效应蛋白分泌，并且无法在巨噬细胞中复制。我们还开发了基于 Tn7 的脱水四环素诱导基因表达系统，以评估柯克斯体感染中 IV 型分泌的时间要求。通过使用无水四环素诱导 TetRA 阻遏物-启动子片段，实现了在 ACCM 或巨噬细胞宿主细胞中生长的柯克斯体对基因表达的严格控制。 3) 重测序微阵列揭示了可能导致LPS相位变异的克隆II期分离株的遗传多态性。 我们的库存中的高传代 II 期分离株不是克隆的，并且含有仍表达全长 I 期 LPS 的柯克斯体小亚群。由此产生的混合基因型使与相位变化严格相关的插入缺失（插入/删除）的识别变得复杂。为了解决这个问题，我们使用显微操作克隆程序来分离高传代九英里、澳大利亚和加利福尼亚分离株的克隆 II 期群体。通过将它们的基因组 DNA 与包含涵盖九英里 I 期分离物整个基因组的探针组的高密度微阵列杂交，我们鉴定了 II 期生物体中可能导致 LPS 生物合成缺陷的常见插入缺失。