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: 8336171
• 负责人: robert a heinzen
• 金额: $ 100.04万
• 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/8336171
• 关键词: Affect; Autophagosome; Binding; Biochemical; Biogenesis; Biological; Cell Communication; Cell Survival; Cell membrane; Cell physiology; Cell-Free System; Cells; Cellular biology; Chlamydia; Chlamydia trachomatis; Cholesterol; Cholesterol Homeostasis; Citrates; Collection; Complex; Coxiella; Coxiella burnetii; Culture Media; Cultured Cells; Cysteine; Cytoskeleton; Cytosol; Data; Desmosterol; Development; Developmental Biology; Ectopic Expression; Embryo; Endoplasmic Reticulum; Environment; Epitopes; Fibroblasts; Gene Expression; Generations; Genes; Genetic Transformation; Goals; Growth; Hela Cells; Human; In Vitro; Infection; Invaded; Laboratories; Legionella; Legionella pneumophila; Ligands; Maintenance; Mammalian Cell; Mediating; Membrane; Membrane Microdomains; Metabolism; Methods; Microbiology; Microtubules; Modification; Molecular; Molecular Biology; Mononuclear; Morphogenesis; Mus; Natural History; Organism; Oxidoreductase; Pathogenesis; Pathway interactions; Phagocytes; Phagolysosome; Phenotype; Physiological; Plasmids; Play; Population; Positioning Attribute; Process; Property; Proteins; Q Fever; Reproduction spores; Resistance; Role; Route; Salmonella; Salmonella typhimurium; Signal Transduction; Site; Staging; Sterols; Technology; Type IV Secretion System Pathway; Ubiquitin; Vacuole; Variant; Vesicle; Virulence; Virulence Factors; Work; Yeasts; base; cell type; cholesterol biosynthesis; cholesterol control; extracellular; in vivo; inhibitor/antagonist; interest; knockout gene; macrophage; mutant; overexpression; parasitism; pathogen; rab GTP-Binding Proteins; residence; trafficking; uptake; yeast protein; yeast two hybrid system

Central to human Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, within a large and spacious phagolysosome-like parasitophorous vacuole (PV). Recruitment of membrane required for PV biogenesis is a complex process modulated by host and bacterial factors. We have shown that the PV membrane is cholesterol-rich and that pharmacologic inhibition of host cholesterol metabolism negatively impacts PV generation and pathogen replication. Cholesterol is a critical component of mammalian membranes where it provides structural stability, signaling platforms called lipid rafts, and serves as a precursor of secondary messenger molecules. To better understand the role of cholesterol in Coxiella pathogenesis, and to circumvent potential pleiotropic effects of cholesterol metabolism inhibitors, we developed a cholesterol-free cell system using DHCR24-/- mouse embryonic fibroblasts (MEFs) that lack the mammalian Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis. Membranes of these cells accumulate desmosterol-a sterol unable to form lipid rafts-instead of cholesterol. The ability of Coxiella to colonize DHCR24-/- MEFs was investigated along with colonization by Chlamydia trachomatis and Salmonella typhimurium as control organisms. Uptake of Salmonella and Chlamydia was unaltered in DHCR24-/- MEFs. Moreover, secretion of Salmonella type III effectors, essential for host invasion, was not affected in the absence of cholesterol. In contrast, Coxiella was internalized less efficiently in DHCR24-/- MEFs, suggesting a role for cholesterol-rich lipids rafts in Coxiella host cell entry. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, in DHCR24-/- MEFs, the Coxiella PV lacked the typical multilamellar membranes found in wild type cells, suggesting cholesterol plays a role in vesicle trafficking to the PV. These data indicate cholesterol is not essential for invasion and intracellular replication by Salmonella and Chlamydia, but may play a role in Coxiella-host cell interactions. We have identified 40 Coxiella Dot/Icm Type IV secretion system (T4SS) substrates that represent a treasure trove of potential virulence factors. Elucidation of their cellular activities and targets will provide needed information on the Coxiella/host relationship. Coxiella Dot/Icm substrates were initially identified using Legionella as surrogate host. However, by using new Coxiella genetic transformation methods developed in our laboratory, we have confirmed Dot/Icm dependent secretion of effectors by Coxiella. An interesting subset of six effectors is encoded by the Coxiella cryptic QpH1 plasmid. When ectopically expressed in HeLa cells, plasmid effectors traffic to different subcellular sites, including autophagosomes, ubiquitin-rich compartments, and the endoplasmic reticulum Collectively, these results suggest Coxiella plasmid-encoded T4SS substrates play important roles in subversion of host cell functions, thereby providing a plausible explanation for the absolute maintenance of plasmid genes by this pathogen. Ectopic expression in mammalian cells of chromosomally encoded effectors fused to fluorescent proteins also reveals a variety of subcellular localizations including microtubules and the Coxiella PV membrane. Yeast two-hybrid analysis identified potential eucaryotic binding partners for six Coxiella Dot/Icm substrates. These preliminary results now set the stage for defining effector function. Indeed, we currently have Coxiella transformants expressing T4SS effectors fused to epitope tags for overexpression by Coxiella. The subcellular trafficking of tagged effectors will provide important clues concerning function. We have also generated a Coxiella strain with the icmD gene inactivated with the Himar1 transposon (Tn). This strain was recently used to define the requirements of type IV secretion during Coxiella infection of human macrophages. An intracellular biphasic developmental cycle whereby highly resistant small cell variant (SCV) morphological forms are generated from large cell variant (LCV) morphological forms is considered fundamental to Coxiella virulence. Previous work from our lab revealed that the LCV is the replicative form of Coxiella, and that SCV and LCV are compositionally and antigenically different. Further molecular and biochemical analyses of SCV and LCV morphogenesis is necessary to better understand the physiological relevance of Coxiella biphasic development. However, intracellular growth of Coxiella imposes considerable experimental constraints. Therefore, we investigated development in our new host cell-free growth medium, Acidified Cysteine Citrate Medium (ACCM). SCV to LCV transitions in ACCM are indistinguishable from Coxiella propagated in vivo. The fidelity of Coxiella morphogenesis in ACCM now provides ample pure cell populations for biochemical studies, ultrastructural analyses, and phenotyping.

人类Q热发病机制的核心是病原体伯纳克希菌在一个大而宽敞的吞噬体样寄生液泡内的复制。PV生物发生所需膜的募集是一个受宿主和细菌因素调节的复杂过程。我们已经证明PV膜富含胆固醇，药理抑制宿主胆固醇代谢会对PV生成和病原体复制产生负面影响。胆固醇是哺乳动物细胞膜的重要组成部分，它提供了结构稳定性，称为脂筏的信号平台，并作为二级信使分子的前体。为了更好地了解胆固醇在Coxiella发病机制中的作用，并规避胆固醇代谢抑制剂的潜在多益作用，我们利用DHCR24-/-小鼠胚胎成纤维细胞（MEFs）开发了一种无胆固醇细胞系统，该系统缺乏哺乳动物胆固醇生物合成的最后酶步骤所需的&#916；24固醇还原酶。这些细胞的细胞膜会积累去氨甾醇——一种不能形成脂质筏的甾醇——而不是胆固醇。以沙眼衣原体和鼠伤寒沙门菌为对照，研究了科希氏菌对DHCR24-/- mef的定殖能力。DHCR24-/- mef中沙门氏菌和衣原体的摄取没有改变。此外，在缺乏胆固醇的情况下，沙门氏菌III型效应物的分泌不受影响，这是入侵宿主所必需的。相比之下，Coxiella在DHCR24-/- mef中的内化效率较低，这表明富含胆固醇的脂筏在Coxiella宿主细胞进入过程中起作用。一旦内化，所有三种病原体都建立了各自的空泡生态位并正常复制。然而，在DHCR24-/- mef中，Coxiella PV缺乏野生型细胞中典型的多层膜，这表明胆固醇在向PV转运的囊泡中发挥了作用。这些数据表明，胆固醇对沙门氏菌和衣原体的侵袭和细胞内复制不是必需的，但可能在科西拉-宿主细胞相互作用中发挥作用。