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贝氏柯克斯体在一个大而宽敞的吞噬溶酶体样寄生虫空泡（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。Coxiella编码一种专门的Dot/Icm型IVB分泌系统（T4 BSS），其将具有效应子功能的蛋白质直接分泌到宿主细胞胞质溶胶中。预期效应蛋白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。Coxiella Dot/Icm功能最初使用嗜肺军团菌作为替代宿主进行研究。然而，通过使用我们实验室开发的新的基因失活技术，我们最近证实了一个功能性的T4 BSS是生产性感染人巨噬细胞所需的柯克斯体。此外，我们已经验证了超过30种蛋白质的Coxiella的Dot/Icm依赖性分泌。柯克斯体必须增选囊泡运输途径，以促进PV的发展。我们目前正在阐明的活动，交通PV膜异位表达时，被称为CvpA（柯克斯体空泡蛋白A），CvpB，CvpC，和CvpD被推测为调节膜融合事件的感染细胞的蛋白质。对CvpA功能的特别了解已经得到了细化。 Coxiella cvpA突变体在复制和PV发育中表现出显著缺陷。CvpA含有多个基于双亮氨酸DERQXXXLL、I和酪氨酸（YXX）的内吞分选基序，如由网格蛋白衔接蛋白（AP）复合物AP 1、AP 2和AP 3识别的那些。异位表达的mCherry-CvpA定位于Rab 11和转铁蛋白受体阳性的核内体周围再循环内体的管状和囊泡结构域，并且CvpA膜相互作用在内吞分选基序突变后丢失。在下拉测定中，含有CvpA分选基序和全长CvpA的肽与AP 2亚基和网格蛋白重链相互作用。此外，通过siRNA处理消耗AP 2或网格蛋白显著抑制柯克斯体复制。因此，我们的研究结果揭示了在柯克斯体感染网格蛋白包被的囊泡贩运的重要性，并定义了一个新的作用，CvpA在颠覆这些运输机制。 尽管T4 BSS将蛋白质递送到宿主细胞质中显然是柯克斯体属的生产性感染所需的，但额外的分泌系统可能负责PV腔微环境的修饰，从而促进病原体复制。 为了评估柯克斯体分泌蛋白到PV中的潜力，我们通过质谱分析了无菌生长培养基中是否存在病原体蛋白的蛋白含量。 从55个鉴定的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，然后通过培养上清液的免疫印迹来确认27个的分泌。 在感染的Vero细胞的可溶性部分中也发现了Coxiella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一个信号序列，从选定的蛋白质中删除该序列可消除分泌。 这些数据表明蛋白质分泌最初需要通过Sec易位酶的活性穿过内膜易位到周质中。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、精氨酸的转运和蛋白质的降解。我们提出，大部分的sec依赖的分泌组的结果从外膜囊泡（OMV）的释放。 这一观点得到了EM的支持，EM显示在培养基中和哺乳动物宿主细胞内的柯克斯体生长期间明显的膜起泡和OMV产生。 细胞内的双相发育周期，其中抗性小细胞变体（SCV）的形态形式产生的大细胞变体（LCV）的形态形式被认为是根本的柯克斯体毒力。然而，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，我们试图建立柯克斯体在宿主细胞中的发育转变是否在第一代和第二代酸化柠檬酸半胱氨酸培养基（分别为ACCM-1和ACCM-2）中的宿主无细胞（无菌）生长期间重现。我们表明，ACCM-2支持发展过渡和生存能力。尽管ACCM-1也支持SCV向LCV的转变，但在延长孵育（21天）后未发生LCV向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，但没有细胞质内容物。这种表型与培养14至21天之间的活力几乎完全丧失相关。在孵育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取代甲基-β-环糊精。在接种后7天向ACCM-1中添加甲基-β-环糊精拯救了柯克斯体的存活力并降低了氧化应激基因的表达。因此，甲基-β-环糊精似乎减轻ACCM-2中的氧化应激，从而导致模拟宿主细胞培养的生物体的柯克斯体发育转变和活力。在ACCM-2中进行柯克斯体的无菌培养，沿着新的遗传操作方法，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有力的工具。 事实上，转录微阵列，全细菌细胞蛋白质组学和脂质分析的无菌培养柯克斯体揭示了新的决定因素的发展形式。