Cellular and Developmental Biology of Coxiella burnetii

伯内氏柯克斯体的细胞和发育生物学

基本信息

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
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/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

项目摘要

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内氏立克次体在一个大而宽敞的吞噬溶酶体样寄生液泡（PV）内的复制。 PV生物发生所需的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，并且药物抑制宿主胆固醇代谢会对PV的产生和病原体复制产生负面影响。胆固醇是哺乳动物膜的重要组成部分，它提供结构稳定性、称为脂筏的信号平台，并作为第二信使分子的前体。为了更好地了解胆固醇在柯克斯体发病机制中的作用，并避免胆固醇代谢抑制剂潜在的多效性作用，我们使用 DHCR24-/- 小鼠胚胎成纤维细胞 (MEF) 开发了一种无胆固醇细胞系统，该系统缺乏产生胆固醇所需的哺乳动物 Δ24 甾醇还原酶。胆固醇生物合成的最后一个酶促步骤。这些细胞的膜积聚去莫甾醇（一种无法形成脂筏的甾醇）而不是胆固醇。研究了柯克斯体定植 DHCR24-/- MEF 的能力以及作为对照生物体的沙眼衣原体和鼠伤寒沙门氏菌的定植能力。 DHCR24-/- MEF 中沙门氏菌和衣原体的摄取没有改变。此外，宿主入侵所必需的沙门氏菌 III 型效应物的分泌在没有胆固醇的情况下不会受到影响。相比之下，柯克斯体在 DHCR24-/- MEF 中内化的效率较低，这表明富含胆固醇的脂筏在柯克斯体进入宿主细胞中发挥着作用。一旦内化，所有三种病原体都会建立各自的液泡生态位并正常复制。然而，在 DHCR24-/- MEF 中，柯克斯体 PV 缺乏野生型细胞中发现的典型多层膜，表明胆固醇在囊泡运输至 PV 中发挥作用。这些数据表明胆固醇对于沙门氏菌和衣原体的入侵和细胞内复制并不是必需的，但可能在柯克斯体-宿主细胞相互作用中发挥作用。我们已经鉴定出 40 种 Coxiella 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目标将提供有关柯克斯体/宿主关系的所需信息。最初使用军团菌作为替代宿主鉴定了 Coxiella Dot/Icm 底物。然而，通过使用我们实验室开发的新的柯克斯体遗传转化方法，我们已经证实柯克斯体具有Dot/Icm依赖性效应子分泌。六个效应子的一个有趣的子集是由 Coxiella 神秘的 QpH1 质粒编码的。当在 HeLa 细胞中异位表达时，质粒效应子会运输到不同的亚细胞位点，包括自噬体、富含泛素的区室和内质网。总的来说，这些结果表明柯克斯体质粒编码的 T4SS 底物在颠覆宿主细胞功能中发挥着重要作用，从而提供对于这种病原体绝对维持质粒基因的合理解释。与荧光蛋白融合的染色体编码效应子在哺乳动物细胞中的异位表达也揭示了各种亚细胞定位，包括微管和柯克斯体PV膜。酵母双杂交分析鉴定了六种 Coxiella Dot/Icm 底物的潜在真核结合伴侣。这些初步结果现在为定义效应器功能奠定了基础。事实上，我们目前拥有表达 T4SS 效应子的 Coxiella 转化体，该效应子与表位标签融合，以便 Coxiella 过度表达。标记效应子的亚细胞运输将提供有关功能的重要线索。我们还生成了 icmD 基因被 Himar1 转座子 (Tn) 灭活的柯克斯体菌株。该菌株最近被用来确定人类巨噬细胞在柯克斯体感染过程中 IV 型分泌的要求。细胞内双相发育周期被认为是柯克斯体毒力的基础，其中从大细胞变体（LCV）形态形式产生高抗性小细胞变体（SCV）形态形式。我们实验室之前的工作表明，LCV 是柯克斯体的复制形式，并且 SCV 和 LCV 在成分和抗原性上都不同。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 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，我们研究了新型宿主无细胞生长培养基——酸化半胱氨酸柠檬酸盐培养基（ACCM）的开发。 ACCM 中的 SCV 到 LCV 转变与体内繁殖的柯克斯体无法区分。 ACCM 中柯克斯体形态发生的保真度现在为生化研究、超微结构分析和表型分析提供了充足的纯细胞群。