Genetics of Coxiella burnetii

伯氏柯克斯体的遗传学

基本信息

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
项目类别：
财政年份：
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&apos 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

项目摘要

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.

Coxiella burnetii是一种无处不在的人畜共患病原体，也是人类急性Q发烧的原因，这是一种残疾的流感样疾病。 Coxiella的前义务细胞内性质显着阻碍了推定的毒力因子的遗传表征。然而，生物体的最新无宿主细胞生长已使穿梭载体，转座子和可诱导基因表达技术的发展，靶向基因失活仍然是一个重要的挑战。为此，我们开发了两种靶向基因缺失的方法，这些方法利用了编码SACB的基于PUC/Cole1 Ori的自杀质粒来进行突变体的阳性选择。作为概念证明，选择了IVB分泌系统（T4BSS）的结构成分的Coxiella dota和dotb被选中以进行删除。第一种方法利用了Cre-Lox介导的重组。将两种带有不同抗生素耐药性标记和LOXP位点的自杀质粒整合到DOTA的5'和3'侧翼区域中。用第三个自杀质粒编码CRE重组酶转化该菌株会导致在蔗糖反选择下删除DOTA。第二种方法利用循环/循环策略删除dota和dotb。首先将单个自杀质粒整合到5'或3'靶基因侧翼区域中。在蔗糖反选择下通过第二次跨界事件解决质粒的综合剂的分辨率导致基因缺失，而PCR和Southern blot证实了基因缺失。 DOTA和DOTB突变体未分泌T4BSS底物并有效地感染了宿主细胞。 Coxiella遗传工具的曲目现在允许传统的突变和毒力因子发现策略。现在已经构建了30多种敲除菌株，包括在编码经过验证的T4BSS底物的其他点/ICM基因和基因中缺失的菌株。这些突变体将极大地帮助分泌仪和分泌效应子蛋白的功能研究。 Coxiella T4BSs将蛋白质分泌为效应函数，直接在宿主细胞胞质中。 Coxiella似乎还直接进入病原体胶水的II型分泌物，其中分泌的蛋白可能会改变腔体微环境以促进病原体的复制。与质谱法结合结合的滑剂染色显示了从对数相培养物中收集的酸化的柠檬酸半胱氨酸培养基（ACCM）中多种coxiella蛋白，其中大多数被注释为含信号肽的假设蛋白。使用质粒转化的细菌，用编码用甲基苯二酚可诱导型启动子表达的质粒化的细菌对蛋白质的积分分泌进行了积极分泌。去除SEC依赖性信号序列后，消除了野生型细菌的分泌。考克斯菌的唯一定义的毒力因子是LPS。具有全长LPS向无毒的II期生物的有毒相的生物体，在重复体外通过后具有严重截断的LPS生物体。鉴于LPS对Coxiella毒力的重要性至关重要，因此重要的是要了解相变的分子基础。我们的库存收集中的高通道II期分离物不是克隆的，并且包含一个仍表达全长I期LPS的Coxiella的少量亚群。所得的混合基因型使识别（插入/缺失）严格与相位变化相关。为了解决这个问题，我们使用微观计算来分离高通道9英里，澳大利亚和加利福尼亚分离株的高通道II期种群。通过将其基因组DNA融合到包含探针集的高密度微阵列，其中包含九英里I期I隔离的整个基因组，可以确定II期生物体中常见的indels，可能会说明有缺陷的LPS生物合成。 Coxiella发病机理的中心是一个细胞内的双相发育循环，产生了两个不同的形态变异，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，表现出凝结的染色质，被认为是细胞外存活形式。 SCV与分散的染色质分化为复制大细胞变体（LCV）。 LCV回到SCV的过渡恰好是将Coxiella进入固定生长阶段的同时，几乎均匀的SCV存在延长的孵育（2至4周）的感染细胞培养物。作为一种可帮助更好地了解Coxiella（分化）生物学相关性的模型，我们确定SCV/LCV转变是由生长在无宿主细胞（Axenic）ACCM中生长的生物概括的。这一发现使对托氏菌发育生物学的研究没有宿主细胞传播的细菌遇到的实验困难。