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伯氏柯克斯体在一个巨大而宽敞的吞噬酶体样寄生虫空泡(PV)内复制。光伏生物发生所需的膜的募集是一个受宿主和细菌因素调控的复杂过程。我们已经证明，PV膜富含胆固醇，对宿主胆固醇代谢的药物抑制对PV的产生和病原体复制具有负面影响。胆固醇是哺乳动物细胞膜的关键成分，在那里它提供结构稳定的信号平台，称为脂筏，并作为二级信使分子的前体。为了更好地了解胆固醇在柯克斯氏菌发病机制中的作用，并避免胆固醇代谢抑制剂的潜在多效性，我们开发了一种无胆固醇细胞系统，使用的是缺乏哺乳动物胆固醇生物合成最后酶步骤所需的甾醇还原酶的DHCR24-/-小鼠胚胎成纤维细胞(MEF)。这些细胞膜上堆积的是一种不能形成脂筏的类固醇，而不是胆固醇。以沙眼衣原体和鼠伤寒沙门氏菌为对照菌，研究了柯克斯体定植DHR24-/-MEF的能力。在DHCR24-/-MEF中，沙门氏菌和衣原体的摄取没有改变。此外，在没有胆固醇的情况下，对宿主入侵至关重要的III型沙门氏菌效应器的分泌不受影响。相反，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宿主细胞进入中发挥了作用。一旦内化，所有三种病原体都建立了各自的液泡生态位，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，这表明胆固醇在囊泡向PV的运输中发挥作用。这些数据表明，胆固醇对沙门氏菌和衣原体的入侵和细胞内复制并不是必不可少的，但可能在柯克斯体与宿主细胞的相互作用中发挥作用。 我们已经确定了40种柯克斯体Dot/ICM IV型分泌系统(T4SS)底物，它们代表了潜在毒力因子的宝库。阐明它们的细胞活动和靶标将提供有关Coxiella/宿主关系的必要信息。以军团菌为替代宿主初步鉴定了柯克斯氏菌Dot/ICM底物。然而，通过我们实验室开发的新的Coxiella遗传转化方法，我们已经证实了Coxiella对Dot/ICM依赖的效应物的分泌。一个有趣的六个效应器的子集是由柯克斯体隐蔽的QpH1质粒编码的。当在HeLa细胞中异位表达时，质粒效应器运输到不同的亚细胞位置，包括自噬小体、富含泛素的隔室和共同的内质网，这些结果表明柯克斯体编码的T4SS底物在颠覆宿主细胞功能方面发挥着重要作用，从而为这种病原体绝对维持质粒基因提供了合理的解释。融合到荧光蛋白上的染色体编码的效应器在哺乳动物细胞中的异位表达也揭示了包括微管和柯克斯体PV膜在内的各种亚细胞定位。酵母双杂交分析确定了六种柯克斯氏菌Dot/ICM底物的潜在真核结合伙伴。这些初步结果现在为定义效应器功能奠定了基础。事实上，我们目前有表达T4SS效应器的Coxiella转化子融合到表位标签以供Coxiella过度表达。标记效应器的亚细胞运输将提供有关功能的重要线索。我们还产生了一株icmD基因被Himar1转座子(TN)灭活的Coxiella菌株。该菌株最近被用来确定柯克斯体感染人巨噬细胞时对IV型分泌物的要求。 由大细胞变异(LCV)形态产生高度抗性小细胞变异(SCV)形态的细胞内两相发育周期被认为是柯克斯体毒力的基础。我们实验室以前的工作表明，LCV是柯克斯体的复制形式，SCV和LCV在成分和抗原性上是不同的。为了更好地了解柯克斯体双相发育的生理相关性，有必要对SCV和LCV的形态发生进行进一步的分子和生化分析。然而，柯克斯体的细胞内生长施加了相当大的实验限制。因此，我们研究了我们的新的无宿主细胞生长培养基--酸性半胱氨酸柠檬酸盐培养基(ACCM)的开发情况。在ACCM中SCV向LCV的转变与在体内传播的柯克斯体没有什么区别。ACCM中柯克斯体形态发生的保真性现在为生化研究、超微结构分析和表型鉴定提供了充足的纯细胞群。