Genetics of Coxiella burnetii

伯氏柯克斯体的遗传学

• 批准号: 8555887
• 负责人: robert a heinzen
• 金额: $ 47.19万
• 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/8555887
• 关键词: 3' Flanking Region; Accounting; Acute; Anabolism; Antibiotic Resistance; Antigens; Australia; Back; Bacteria; Biological; C-terminal; California; Categories; Cell Culture Techniques; Cells; Centers for Disease Control and Prevention (U.S.); Chromatin; Chronic; Citrates; Collection; Complex; Coxiella; Coxiella burnetii; Cysteine; Cytosol; DNA; Development; Developmental Biology; Diagnostic; Endocarditis; Event; Excision; Gene Deletion; Gene Delivery; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Recombination; Genome; Genomics; Genotype; Goals; Growth; Harvest; Human; Immune; In Vitro; Infection; Influenza; Knock-out; Knowledge; Length; Life; Luciferases; Masks; Mass Spectrum Analysis; Mediating; Methods; Micromanipulation; Modeling; Molecular; Molecular Biology; Molecular Probes; Monitor; Mutagenesis; Mutation; Nature; Organism; PAWR protein; Pathogenesis; Peptide Signal Sequences; Phase; Plasmids; Play; Population; Preparation; Production; Proteins; Q Fever; RNA; Reporter; Reporter Genes; Resolution; Role; Sensory; Shuttle Vectors; Site; Southern Blotting; Staining method; Stains; Subunit Vaccines; Sucrose; Suicide; Surface; System; Technology; Testing; Vaccine Antigen; Vacuole; Variant; Virulence; Virulence Factors; Virulent; base; biological adaptation to stress; biothreat; cell type; density; design; extracellular; flu; gene function; genetic manipulation; improved; insertion/deletion mutation; knockout gene; macrophage; mutant; pathogen; programs; promoter; protein function; recombinase; secretion process; suicide vector; tool

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, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. To this end, we developed two methods of targeted gene deletion in Coxiella that exploit pUC/ColE1 ori-based suicide plasmids encoding sacB for positive selection of mutants. As proof of concept, Coxiella dotA and dotB, encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre-lox-mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and a loxP site were integrated into 5' and 3' flanking regions of dotA. Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in deletion of dotA under sucrose counterselection. The second method utilized a loop-in/loop out strategy to delete dotA and dotB. A single suicide plasmid was first integrated into 5' or 3' target gene flanking regions. Resolution of the plasmid co-integrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. dotA and dotB mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire of Coxiella genetic tools now allows traditional mutation and complementation strategies for virulence factor discovery. Over 30 knockout strains have now been constructed, including those with deletions in additional Dot/Icm genes and genes encoding verified T4BSS substrates. These mutants will dramatically aid functional studies of both the secretion apparatus and secreted effector proteins. The Coxiella T4BSS secretes proteins with effector functions directly into the host cell cytosol. Coxiella also appears to engage in type II-like secretion directly into the pathogen-occupied vacuole where secreted proteins likely modify the lumenal microenvironment to promote pathogen replication. Sliver staining combined with mass spectrometry revealed multiple Coxiella proteins in acidified citrate cysteine medium (ACCM) harvested from log phase cultures, most of which are annotated as signal peptide-containing hypothetical proteins. Active secretion of a subset of proteins by Coxiella was confirmed using bacteria transformed with plasmids encoding C-terminal 3x-FLAG-tagged proteins expressed from an anhydrotetracycline-inducible promoter. Secretion by wild type bacteria was eliminated upon removal of the Sec-dependent signal sequence. The only defined virulence factor of Coxiella is LPS. Virulent phase I organisms with full-length LPS transition to avirulent phase II organisms with severely truncated LPS upon repeated in vitro passage. Given the critical importance of LPS to Coxiella virulence, it is important to understand the molecular basis of 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 micromanipulation 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, common indels in phase II organisms were identified that may account for defective LPS biosynthesis. Central to Coxiella pathogenesis is an intracellular biphasic developmental cycle that generates two distinct morphological variants that can be distinguished by ultrastructure and protein composition. Small cell variants (SCV) are non-replicative forms that display condensed chromatin and are considered extracellular survival forms. SCVs differentiate into replicative large cell variants (LCVs) with dispersed chromatin. Transition of LCV back to SCV occurs coincident with entry of Coxiella into stationary growth phase with nearly homogeneous SCVs present with 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 host cell-free (axenic) ACCM. This discovery enables studies of Coxiella developmental biology without experimental difficulties encountered with host cell-propagated bacteria.

贝氏柯克斯体是一种普遍存在的人畜共患细菌病原体，是人类急性Q热（一种致残性流感样疾病）的病因。柯克斯体的前专性细胞内的性质显着阻碍了推定的毒力因子的遗传特征。 然而，最近的宿主无细胞（无菌）生长的生物体已经使穿梭载体，转座子，诱导基因表达技术的发展，与靶向基因失活仍然是一个重要的挑战。 为此，我们开发了两种方法的靶向基因缺失柯克斯体，利用pUC/ColE 1 ori为基础的自杀质粒编码sacB的突变体的积极选择。作为概念证明，选择编码IVB型分泌系统（T4 BSS）的结构组分的Coxiella dotA和dotB进行缺失。 第一种方法利用Cre-lox介导的重组。将携带不同抗生素抗性标记和loxP位点的两个自杀质粒整合到dotA的5'和3'侧翼区。用编码Cre重组酶的第三自杀质粒转化该菌株导致在蔗糖反选择下dotA的缺失。第二种方法利用循环输入/循环输出策略来删除dotA和dotB。首先将单个自杀质粒整合到5'或3'靶基因侧翼区。通过蔗糖反选择下的第二次交换事件对质粒共整合体的解析导致基因缺失，这通过PCR和Southern印迹证实。dotA和dotB突变体不能分泌T4 BSS底物，也不能有效地感染宿主细胞。柯克斯体遗传工具库现在允许传统的突变和互补策略的毒力因子的发现。现在已经构建了超过30种敲除菌株，包括在额外的Dot/Icm基因和编码经验证的T4 BSS底物的基因中具有缺失的那些。 这些突变体将大大有助于分泌装置和分泌的效应蛋白的功能研究。 Coxiella T4 BSS分泌具有效应子功能的蛋白质直接进入宿主细胞胞质溶胶。柯克斯体似乎也参与II型样分泌，直接进入病原体占据的空泡，分泌的蛋白质可能会改变内腔微环境，以促进病原体复制。银染结合质谱分析揭示了从对数期培养物收获的酸化柠檬酸半胱氨酸培养基（ACCM）中的多种柯克斯体蛋白，其中大部分被注释为含有信号肽的假设蛋白。 柯克斯体属的蛋白质子集的主动分泌使用用编码从脱水四环素诱导型启动子表达的C-末端3x-FLAG-标记的蛋白质的质粒转化的细菌来证实。野生型细菌的分泌在除去Sec依赖性信号序列后被消除。 唯一确定的Coxiella毒力因子是LPS。 在体外重复传代后，具有全长LPS的毒力I相微生物转变为具有严重截短的LPS的无毒力II相微生物。由于LPS对Coxiella毒力的关键重要性，了解阶段变异的分子基础是很重要的。在我们的储备库中，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的柯克斯体的小亚群。由此产生的混合基因型使与相位变异严格相关的插入/缺失（插入/缺失）的鉴定复杂化。为了避免这个问题，我们使用显微操作来分离高传代Nine Mile、澳大利亚和加州分离株的克隆II期群体。通过将它们的基因组DNA与包含涵盖Nine Mile I期分离物的整个基因组的探针组的高密度微阵列杂交，鉴定了II期生物体中的共同indel，其可以解释缺陷性LPS生物合成。 柯克斯体发病机制的核心是细胞内双相发育周期，产生两种不同的形态变体，可以通过超微结构和蛋白质组成来区分。 小细胞变体（SCV）是非复制形式，显示浓缩的染色质，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Coxiella进入稳定生长期同时发生，在感染细胞培养物的延长孵育（2至4周）中存在几乎均一的SCV。 作为一个可行的模型，以帮助更好地了解柯克斯体的生物学相关性，分化，我们建立了SCV/LCV转换是由生长在宿主无细胞（纯）ACCM的生物重演。 这一发现使柯克斯体发育生物学的研究没有遇到宿主细胞繁殖的细菌的实验困难。