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: 8946368
• 负责人: robert a heinzen
• 金额: $ 66.42万
• 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/8946368
• 关键词: Appearance; Arginine; Bacteria; Biochemical; Biogenesis; Biological; Biological Assay; Bulla; Cell physiology; Cells; Cellular biology; Cereals; Chromatin; Citrates; Clathrin; Clathrin Adaptor Protein Complexes; Clathrin Heavy Chains; Clathrin-Coated Vesicles; Complex; Coxiella; Coxiella burnetii; Culture Media; Cysteine; Cytoplasm; Cytosol; Data; Defect; Development; Developmental Biology; Drug Metabolic Detoxication; Endosomes; Event; Exhibits; Gene Expression Profile; Gene Silencing; Generations; Genes; Goals; Growth; Human; Immunoblotting; Infection; Laboratories; Legionella pneumophila; Length; Lipids; Mass Spectrum Analysis; Mediating; Membrane; Membrane Fusion; Methods; Modification; Molecular; Molecular Biology; Mutation; Natural History; Nutrient; Organism; Oxidative Stress; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Peptides; Phagolysosome; Phagosomes; Phase; Phenotype; Process; Production; Property; Protein Biosynthesis; Protein Secretion; Proteins; Proteome; Proteomics; Q Fever; Reactive Oxygen Species; Recycling; Resistance; Role; Sequence Deletion; Small Interfering RNA; Sorting - Cell Movement; System; Technology; Transcription Factor AP-1; Transcription Factor AP-2 Alpha; Transferrin Receptor; Tubular formation; Tyrosine; Vacuole; Variant; Vero Cells; Vesicle; Virulence; base; cell envelope; cohort; extracellular; fetal bovine serum; genetic manipulation; genetic technology; genome annotation; in vivo; insight; macrophage; methyl-beta-cyclodextrin; mutant; novel; pathogen; periplasm; protein degradation; protein transport; residence; tool; trafficking; translocase; 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 that is 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 four proteins that traffic to the PV membrane when ectopically expressed in infected cells termed CvpA (Coxiella vacuolar protein A), CvpB, CvpC, and CvpD that are speculated to modulate membrane fusion events. Particular insight into the function of CvpA has been grained. A Coxiella cvpA mutant exhibits significant defects in replication and PV development. CvpA contains multiple dileucine DERQXXXLL,I and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. Ectopically expressed mCherry-CvpA localizes to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions are lost upon mutation of endocytic sorting motifs. In pull-down assays, peptides containing CvpA sorting motifs and full-length CvpA interact with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibits Coxiella replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in Coxiella infection and define a novel role for CvpA in subverting these transport mechanisms. Although T4BSS delivery of proteins into the host cell cytoplasm is clearly required for productive infection by Coxiella, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promotes pathogen replication. To assess the potential of Coxiella to secrete proteins into the PV, we analyzed by mass spectrometry the protein content of axenic growth media for the presence of pathogen proteins. From a candidate list of 55 identified proteins, secretion of 27 was confirmed by expressing FLAG-tagged proteins in Coxiella followed by immunoblotting of culture supernatants. Tagged proteins expressed by Coxiella transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs in vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase. Possible roles for secreted proteins based on genome annotation include detoxification of reactive oxygen species, transport of arginine, and degradation of protein. We propose that the majority of the sec-dependent secretome results from release of outer membrane vesicles (OMV). This idea is supported by EM showing obvious membrane blebbing and OMV production during growth of Coxiella in media and within mammalian host cells. An intracellular biphasic developmental cycle where resistant small cell variant (SCV) morphological forms are generated from large cell variant (LCV) morphological forms is considered fundamental to Coxiella virulence. However, the molecular biology of Coxiella development is poorly understood. Because intracellular growth of Coxiella imposes considerable experimental constraints, we sought to establish whether Coxiella developmental transitions in host cells are recapitulated during host cell-free (axenic) growth in first and second generation acidified citrate cysteine media (ACCM-1 and ACCM-2, respectively). We show that ACCM-2 supports developmental transitions and viability. Although ACCM-1 also supported SCV to LCV transition, LCV to SCV transition did not occur after extended incubation (21 days). Instead, Coxiella exhibited a ghost-like appearance with bacteria containing condensed chromatin but otherwise devoid of cytoplasmic content. This phenotype correlated with a near total loss in viability between 14 and 21 days of cultivation. Transcriptional profiling of Coxiella following 14 days of incubation revealed elevated expression of oxidative stress genes in ACCM-1 cultivated bacteria. The only difference between ACCM-1 and ACCM-2 is the substitution of fetal bovine serum for methyl-beta-cyclodextrin. Addition of methyl-beta-cyclodextrin to ACCM-1 at 7 days post-inoculation rescued Coxiella viability and lowered expression of oxidative stress genes. Thus, methyl-beta-cyclodextrin appears to alleviate oxidative stress in ACCM-2 to result in Coxiella developmental transitions and viability that 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. Indeed, transcriptional microarrays, whole bacterial cell proteomics and lipid analyses of axenically cultured Coxiella have revealed novel determinates of developmental forms.

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）中的复制。 PV 生物发生过程中膜的募集是一个复杂的过程，受宿主和细菌因素的调节。柯克斯体编码一种特殊的 Dot/Icm 型 IVB 分泌系统 (T4BSS)，该系统将具有效应功能的蛋白质直接分泌到宿主细胞胞质中。预计效应蛋白会调节一系列宿主细胞过程，例如促进病原体生长的囊泡运输。最初使用嗜肺军团菌作为替代宿主研究了 Coxiella Dot/Icm 功能。然而，通过使用我们实验室开发的新基因失活技术，我们最近证实，柯克斯体对人类巨噬细胞的有效感染需要功能性 T4BSS。此外，我们还验证了柯克斯体对 30 多种蛋白质的 Dot/Icm 依赖性分泌。柯克斯体必须选择囊泡运输途径来促进 PV 的发展。我们目前正在阐明四种蛋白的活性，当在感染细胞中异位表达时，这些蛋白运输到 PV 膜，称为 CvpA（柯克斯体液泡蛋白 A）、CvpB、CvpC 和 CvpD，推测它们可以调节膜融合事件。对 CvpA 功能的特别深入的了解已经深入人心。 柯克斯体 cvpA 突变体在复制和 PV 发育方面表现出明显的缺陷。 CvpA 包含多个基于二亮氨酸 DERQXXXLL,I 和酪氨酸 (YXXΦ) 的内吞分选基序，如网格蛋白接头蛋白 (AP) 复合物 AP1、AP2 和 AP3 识别的基序。异位表达的 mCherry-CvpA 定位于 Rab11 和转铁蛋白受体阳性的中心体周围回收内体的管状和囊泡结构域，并且 CvpA 膜相互作用因内吞分选基序的突变而丢失。在 Pull-down 测定中，含有 CvpA 分选基序和全长 CvpA 的肽与 AP2 亚基和网格蛋白重链相互作用。此外，通过 siRNA 处理消除 AP2 或网格蛋白可显着抑制柯克斯体复制。因此，我们的结果揭示了网格蛋白包被的囊泡运输在柯克斯体感染中的重要性，并确定了 CvpA 在颠覆这些运输机制中的新作用。 尽管 T4BSS 将蛋白质递送到宿主细胞的细胞质中显然是柯克斯体有效感染所必需的，但额外的分泌系统可能负责改变 PV 腔微环境，从而促进病原体复制。 为了评估柯克斯体将蛋白质分泌到PV中的潜力，我们通过质谱分析了无菌生长培养基的蛋白质含量，以确定病原体蛋白质的存在。 从 55 种已鉴定蛋白质的候选列表中，通过在柯克斯体中表达 FLAG 标记的蛋白质，然后对培养物上清液进行免疫印迹，确认了 27 种蛋白质的分泌。 在受感染的 Vero 细胞的可溶部分中也发现了由柯克斯体转化体表达的标记蛋白，表明分泌发生在体内。 所有分泌的蛋白质都含有信号序列，从选定的蛋白质中删除该序列就会消除分泌。 这些数据表明蛋白质分泌最初需要通过 Sec 易位酶的活性跨内膜易位到周质。基于基因组注释的分泌蛋白的可能作用包括活性氧的解毒、精氨酸的运输和蛋白质的降解。我们认为大多数 sec 依赖性分泌蛋白组是由外膜囊泡 (OMV) 释放引起的。 这一想法得到了电镜的支持，电镜显示柯克斯体在培养基中和哺乳动物宿主细胞内生长期间出现明显的膜起泡和 OMV 产生。 细胞内双相发育周期，其中抗性小细胞变体（SCV）形态形式是从大细胞变体（LCV）形态形式产生的，被认为是柯克斯体毒力的基础。然而，人们对柯克斯体发育的分子生物学知之甚少。由于柯克斯体的细胞内生长施加了相当大的实验限制，我们试图确定在第一代和第二代酸化柠檬酸盐半胱氨酸培养基（ACCM-1和ACCM-2，ACCM-1和ACCM-2，分别）。我们证明 ACCM-2 支持发育转变和生存能力。尽管 ACCM-1 也支持 SCV 到 LCV 的转变，但在延长孵育（21 天）后并未发生 LCV 到 SCV 的转变。相反，柯克斯体表现出幽灵般的外观，细菌含有浓缩的染色质，但缺乏细胞质内容物。这种表型与培养 14 至 21 天期间活力几乎完全丧失相关。孵育 14 天后，Coxiella 的转录谱显示 ACCM-1 培养细菌中氧化应激基因的表达升高。 ACCM-1 和 ACCM-2 之间的唯一区别是用胎牛血清替代甲基-β-环糊精。接种后 7 天将甲基-β-环糊精添加到 ACCM-1 中可挽救柯克斯体的活力并降低氧化应激基因的表达。因此，甲基-β-环糊精似乎可以减轻 ACCM-2 中的氧化应激，从而导致柯克斯体模仿宿主细胞培养的生物体的发育转变和活力。 ACCM-2 中柯克斯体的无菌培养以及遗传操作的新方法现在为研究柯克斯体双相发育的分子基础和生物学相关性提供了强大的工具。 事实上，转录微阵列、全细菌细胞蛋白质组学和无菌培养的柯克斯体的脂质分析揭示了发育形式的新决定因素。