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.

伯内特柯克斯体是一种普遍存在的人畜共患细菌病原体，也是人类急性 Q 热（一种致残性流感样疾病）的病因。柯克斯体以前的专性细胞内性质显着阻碍了假定毒力因子的遗传表征。然而，最近生物体的宿主无细胞（无菌）生长使得穿梭载体、转座子和诱导型基因表达技术的发展成为可能，而靶向基因失活仍然是一个重要的挑战。为此，我们开发了两种柯克斯体定向基因删除方法，利用编码 sacB 的基于 pUC/ColE1 ori 的自杀质粒来阳性选择突变体。作为概念证明，选择删除编码 IVB 型分泌系统 (T4BSS) 结构成分的 Coxiella dotA 和 dotB。第一种方法利用 Cre-lox 介导的重组。将两个携带不同抗生素抗性标记和 loxP 位点的自杀质粒整合到 dotA 的 5' 和 3' 侧翼区域中。用编码 Cre 重组酶的第三个自杀质粒转化该菌株，导致在蔗糖反选择下 dotA 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' 靶基因侧翼区域。在蔗糖反选择下通过第二次交叉事件解析质粒共整合体导致基因缺失，这通过 PCR 和 Southern blot 得到证实。 dotA 和 dotB 突变体无法分泌 T4BSS 底物并有效感染宿主细胞。柯克斯体遗传工具的全部功能现在允许使用传统的突变和互补策略来发现毒力因子。目前已构建了 30 多个敲除菌株，包括删除了额外 Dot/Icm 基因和编码已验证 T4BSS 底物的基因的菌株。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。 Coxiella T4BSS 将具有效应功能的蛋白质直接分泌到宿主细胞胞质中。柯克斯体似乎还直接参与病原体占据的液泡中的 II 型分泌，其中分泌的蛋白质可能会改变腔微环境以促进病原体复制。银染色结合质谱分析揭示了从对数期培养物收获的酸化柠檬酸盐半胱氨酸培养基（ACCM）中存在多种柯克斯体蛋白，其中大多数被注释为含有信号肽的假设蛋白。使用用编码由无水四环素诱导型启动子表达的 C 端 3x-FLAG 标记蛋白的质粒转化的细菌，证实了柯克斯体对蛋白质子集的主动分泌。去除Sec依赖性信号序列后，野生型细菌的分泌被消除。柯克斯体唯一确定的毒力因子是 LPS。具有全长 LPS 的毒力 I 期生物体在体外重复传代后转变为具有严重截短的 LPS 的无毒力 II 期生物体。鉴于 LPS 对柯克斯体毒力至关重要，了解相变的分子基础非常重要。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。通过将其基因组 DNA 与包含包含九英里 I 期分离物整个基因组的探针组的高密度微阵列杂交，鉴定出 II 期生物体中的常见插入缺失，这些插入缺失可能是 LPS 生物合成缺陷的原因。柯克斯体发病机制的核心是细胞内双相发育周期，它产生两种不同的形态变异，可以通过超微结构和蛋白质组成来区分。小细胞变异体 (SCV) 是显示浓缩染色质的非复制形式，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 的转变与柯克斯体进入静止生长期同时发生，随着受感染细胞培养物的延长孵育（2 至 4 周），存在几乎同质的 SCV。作为一个有助于更好地理解柯克斯体、分化的生物学相关性的可靠模型，我们确定了在宿主无细胞（无菌）ACCM 中生长的生物体可以重现 SCV/LCV 转变。这一发现使得研究柯克斯体发育生物学成为可能，而不会遇到宿主细胞繁殖细菌的实验困难。