Cellular and Developmental Biology of Coxiella burnetii

伯内氏柯克斯体的细胞和发育生物学

• 批准号: 9161549
• 负责人: robert a heinzen
• 金额: $ 72.55万
• 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/9161549
• 关键词: Appearance; Arginine; Bacteria; Binding; Biochemical; Biogenesis; Biological; Biology; Cardiolipins; Cell Wall; Cell physiology; Cells; Cellular biology; Cereals; Clathrin-Coated Vesicles; Complex; Coxiella; Coxiella burnetii; Cryoelectron Microscopy; Cytosol; Defect; Development; Developmental Biology; Employee Strikes; Epithelial Cells; Escherichia coli; Ethanolamines; Event; Exhibits; Gene Expression Profile; Gene Silencing; Generations; Genes; Genetic Determinism; Goals; Growth; Homologous Gene; Human; Individual; Infection; Laboratories; Lactams; Legionella pneumophila; Lipids; Mass Spectrum Analysis; Membrane; Membrane Fusion; Metabolism; Methods; Molecular; Molecular Biology; Natural History; Nonesterified Fatty Acids; Nutrient; Organism; Oxidative Stress; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Peptides; Peptidoglycan; Peptidyltransferase; Phagolysosome; Phagosomes; Phase; Phosphatidylethanolamine; Phosphatidylglycerols; Phospholipases A; Plasmalogens; Process; Property; Protein Biosynthesis; Proteins; Proteome; Q Fever; Regulation; Relative (related person); Resistance; Role; System; Technology; Thick; Thin Layer Chromatography; Up-Regulation; Vacuole; Variant; Vesicle; Virulence; adapter protein; base; biological adaptation to stress; cell envelope; cohort; crosslink; extracellular; genetic manipulation; genetic technology; insight; macrophage; mutant; neurotensin mimic 2; pathogen; periplasm; physical property; protein transport; residence; tool; trafficking; uptake

Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, in a large and spacious phagolysosome-like parasitophorous vacuole (PV). Recruitment of membrane during PV biogenesis is a complex process modulated by both host and bacterial factors. Coxiella encodes a specialized Dot/Icm type IVB secretion system (T4BSS) that secretes proteins with effector functions directly into the host cell cytosol. Effector proteins are predicted to modulate an array of host cell processes, such as vesicular trafficking, that promote pathogen growth. Coxiella Dot/Icm function was initially studied using Legionella pneumophila as surrogate host. However, by using new gene inactivation technologies developed in our laboratory, we have recently confirmed that a functional T4BSS is required for productive infection of human macrophages by Coxiella. Furthermore, we have verified Dot/Icm-dependent secretion by Coxiella of over 30 proteins. Coxiella must co-opt vesicular trafficking pathways to promote PV development. We are currently elucidating the activities of five effector proteins that traffic to the PV membrane termed CvpA (Coxiella vacuolar protein A), CvpB, CvpC, CvpD, and CvpE that are speculated to modulate membrane fusion events. Mutants in individual cvp genes all display significant defects in replication and PV development. Particular insight into the function of CvpA has been grained by showing the protein subverts clathrin-coated vesicle trafficking. Regulation of the Coxiella T4BSS is poorly defined. IcmS is a predicted cytoplasmic adapter protein that facilitates translocation of certain T4BSS effectors by binding an internal signal sequence(s). We examined the function of Coxiella IcmS by generating an icmS deletion mutant. Coxiella ΔicmS grows normally in axenic media while having a pronounced growth defect in host cells that is rescued with a single chromosomal copy of icmS. Optimal secretion of individual substrates is either IcmS-dependent or independent. Additionally, a subset of substrates display hyper-secretion in Coxiella ΔicmS, suggesting IcmS may also suppress secretion of some Dot/Icm substrates. Thus, regulation by IcmS appears complex with the growth defect of Coxiella ΔicmS potentially explained by both deficient and aberrant secretion of effector proteins. A hallmark of Coxiella is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCV are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. Although the developmental cycle is considered fundamental to Coxiella virulence, the molecular biology of this process is poorly understood. Recently, we discovered that Coxiella developmental transitions and viability in the synthetic medium ACCM-2 mimic host cell-cultivated organisms. Axenic cultivation of Coxiella in ACCM-2, along with new methods for genetic manipulation, now provides powerful tools to investigate the molecular basis and biological relevance of Coxiella biphasic development. Ultrastructural studies show marked differences in the cell envelope between cell variants, but little is known about biochemical differences between SCV and LCV that confer their distinct biological and physical properties. We analyzed the lipid composition of Coxiella after 4 (LCV), 7 (intermediate forms) and 14 (SCV) days of growth in synthetic medium, using thin layer chromatography and mass spectrometry. Similar to Escherichia coli, Coxiella contains cardiolipin, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), with some PE in an unusual plasmalogen form. PE and PG are present in lower quantities in the SCV relative to the LCV. However, three additional major lipid species are present in higher quantities in the SCV: lyso-phosphatidylethanolamine, a breakdown product of PE; glycerophospho-N-acyl-ethanolamine, a lipid previously not found in bacteria; and free fatty acids, which are normally toxic for bacteria. Mutational analysis indicates that these three lipids are generated via the activity of a Coxiella outer membrane phospholipase A homolog (CBU0489). A cbu0489 mutant exhibits a significant growth defect in THP-1 macrophage-like cells, suggesting developmentally regulated lipid synthesis is required for optimal intracellular growth and could contribute to the distinct properties of LCV and SCV. To further identify genetic determinants of LCV to SCV transition, we profiled the Coxiella transcriptome by microarray at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV) and 21 (late SCV) days post-infection (dpi) of Vero epithelial cells. Transcriptional signatures of SCV are up-regulation of genes involved in oxidative stress responses, arginine metabolism, and cell wall remodeling. Genes down-regulated in SCV are primarily associated with intermediary metabolism. A striking transcriptional signature of the SCV is induction (10-fold) of five genes encoding predicted L,D transpeptidases that catalyze β-lactam resistant 3-3 peptide crosslinks typically found in the peptidoglycan (PG) of stationary phase bacteria. Cryo-electron microscopy reveals an unusually thick and dense periplasmic layer specific to SCV, suggestive of PG, whereas the periplasm and inner and outer membranes of LCV exhibits a more typical gram-negative appearance. Muropeptide analysis of Coxiella PG shows an increasing percentage of 3-3 cross-links as LCV transition to SCV raising the possibility that the Coxiella L,D transpeptidase homologs up-regulated by microarray may be important in cross-linking the PG of SCV. Collectively, these results indicate the SCV produces a unique transcriptome with a major subset of these genes directed towards remodeling a PG layer that may contribute to Coxiellas environmental resistance.

Q发烧发病机理的中心是在宽敞而宽敞的吞噬体样寄生虫液泡（PV）中复制Coxiella burnetii的复制。 PV生物发生过程中膜的募集是由宿主和细菌因子调节的复杂过程。 Coxiella编码一个专门的DOT/ICM类型IVB分泌系统（T4BS），该系统将蛋白质直接函数直接分泌到宿主细胞细胞质中。预计效应子蛋白会调节促进病原体生长的一系列宿主细胞过程，例如囊泡运输。最初使用肺炎军团菌作为替代宿主研究了Coxiella dot/ICM功能。但是，通过使用在我们的实验室中开发的新基因灭活技术，我们最近确认，Coxiella对人类巨噬细胞的生产性感染需要功能性T4BS。此外，我们已经验证了30多个蛋白质的Coxiella依赖DOT/ICM依赖性分泌。 Coxiella必须选择囊泡贩运途径以促进PV的发展。目前，我们正在阐明五种效应子蛋白的活性，这些蛋白质的活性被称为CVPA（Coxiella acciellaal Protina），CVPB，CVPC，CVPD和CVPE，这些活动被推测用于调节膜融合事件。单个CVP基因中的突变体在复制和PV发育中均显示出明显的缺陷。通过显示蛋白质颠覆网状蛋白包被的囊泡运输，特别了解CVPA的功能。 Coxiella T4BSS的调节定义很差。 ICMS是一种预测的细胞质衔接蛋白，通过结合内部信号序列（S）来促进某些T4BSS效应子的易位。我们通过产生ICMS缺失突变体来检查Coxiella ICM的功能。 coxiellaΔICM在轴突培养基中正常生长，同时在宿主细胞中具有明显的生长缺陷，该缺陷被ICM的单个染色体拷贝救出。单个底物的最佳分泌是ICMS依赖性或独立的。 另外，底物的一个子集显示在CoxiellaΔICMS中的过度分泌，这表明ICM还可以抑制某些点/ICM底物的分泌。因此，ICMS的调节似乎与CoxiellaΔICMS的生长缺陷有关，这可能通过效应蛋白的分泌不足和异常分泌可能解释。 Coxiella的标志是一种双相发育循环，可在生物学，超微结构和组成上不同的大细胞变体（LCV）和小细胞变体（SCV）形式产生。 LCV正在复制指数相的形式，而SCV则是不复制的，固定相的形式。 SCV具有多种特性，例如凝结的核苷和异常细胞包膜，涉嫌赋予增强的环境稳定性。尽管发育周期被认为是毒力性的基础，但该过程的分子生物学知识很少。最近，我们发现合成培养基ACCM-2模拟宿主细胞培养的生物中的Coxiella发育过渡和生存能力。 Coxiella在ACCM-2中的轴承培养，以及用于基因操纵的新方法，现在提供了研究Coxiella Biphasic发育的分子基础和生物学相关性的强大工具。 超微结构研究表明，细胞变体之间细胞膜的差异很大，但对SCV和LCV之间的生化差异知之甚少，从而赋予其独特的生物学和物理性质。我们使用薄层色谱法和质谱法分析了4（LCV），7（中间形式）和14（SCV）生长天的脂质组成的脂质组成。 与大肠杆菌类似，柯西埃拉含有心氨基脂蛋白，磷脂酰甘油（Pg）和磷脂酰乙醇胺（PE），其中一些PE的PE形式不寻常。 相对于LCV，SCV中的PE和PG数量较低。 然而，在SCV中，其他三种主要脂质物种以较高的数量存在：溶酶 - 磷脂酰乙醇胺，PE的分解产物。甘油磷酸-N-酰基乙醇胺，以前在细菌中未发现的脂质；和游离脂肪酸，通常对细菌有毒。 突变分析表明，这三种脂质是通过Coxiella外膜磷脂酶A同源物（CBU0489）的活性产生的。 CBU0489突变体在Thp-1巨噬细胞样细胞中表现出显着的生长缺陷，这表明在发育中调节的脂质合成是最佳细胞内生长所必需的，并且可能有助于LCV和SCV的不同特性。 为了进一步识别LCV对SCV转变的遗传决定因素，我们通过微阵列在3（早期LCV），5（LCV晚期），7（中间形式），14（SCV早期），21（SCV）和21（SCV晚期）和21（SCV晚期）天数（DPI）后（DPI）（DPI）（dpi）介绍了Coxiella转录组。 SCV的转录特征是与氧化应激反应，精氨酸代谢和细胞壁重塑有关的基因上调。 SCV中下调的基因主要与中介代谢有关。 SCV的引人注目的转录特征是诱导（10倍）的五个基因，这些基因编码了预测的L，D转肽酶，这些基因催化通常在固定阶段细菌的肽聚糖（PG）中催化β-莱顿酰胺耐药3-3肽交联。冷冻电子显微镜揭示了特定于SCV的异常厚且密集的周质层，暗示了PG，而LCV的周期和内膜和外膜表现出更典型的革兰氏阴性外观。 Coxiella PG的杂肽分析显示，随着LCV向SCV的转变，coxiella l，d转肽酶同系物的可能性增加了3-3个交联的比例增加，这对于交联SCV的PG可能很重要。 总的来说，这些结果表明SCV产生独特的转录组，其中这些基因的主要子集用于重塑PG层，该PG层可能有助于Coxiellas环境抗性。