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.

伯纳蒂克希菌是一种普遍存在的人畜共患细菌病原体，是人类急性Q热（一种致残的流感样疾病）的病因。Coxiella的前专性细胞内性质显著阻碍了假定的毒力因子的遗传表征。然而，近年来无宿主细胞（无菌）生长的生物体使得穿梭载体，转座子和诱导基因表达技术的发展成为可能，靶向基因失活仍然是一个重要的挑战。为此，我们开发了两种针对Coxiella基因缺失的方法，利用pUC/ColE1 ori-based编码sacB的自杀质粒进行突变体的阳性选择。为了证明这一概念，我们选择了编码IVB型分泌系统（T4BSS）结构成分的Coxiella dotA和dotB进行删除。第一种方法利用cre -lox介导的重组。两个携带不同抗生素耐药标记的自杀质粒和一个loxP位点被整合到dotA的5‘和3’侧区。用编码Cre重组酶的第三个自杀质粒转化该菌株，在蔗糖反选择下导致dotA缺失。第二种方法利用循环入/循环出策略来删除dotA和dotB。单个自杀质粒首先被整合到5‘或3’靶基因的侧翼区域。在蔗糖反选择条件下，通过第二次交叉事件分离质粒协整性，导致基因缺失，这一点经PCR和Southern blot证实。dotA和dotB突变体不能分泌T4BSS底物，也不能有效感染宿主细胞。科希氏菌遗传工具的曲目现在允许传统的突变和互补策略的毒力因子发现。目前已经构建了30多个敲除菌株，其中包括额外Dot/Icm基因缺失的菌株和编码经验证的T4BSS底物的基因。这些突变体将极大地帮助分泌装置和分泌效应蛋白的功能研究。