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发烧发病机理的中心是在宽敞而宽敞的吞噬体样寄生虫液泡（PV）中复制Coxiella burnetii的复制。 PV生物发生过程中膜的募集是一个复杂的过程，均由宿主和细菌因子调节。 Coxiella编码一个专门的DOT/ICM类型IVB分泌系统（T4BS），该系统将蛋白质直接函数直接分泌到宿主细胞细胞质中。预计效应子蛋白会调节促进病原体生长的一系列宿主细胞过程，例如囊泡运输。最初使用肺炎军团菌作为替代宿主研究了Coxiella dot/ICM功能。但是，通过使用在我们的实验室中开发的新基因灭活技术，我们最近确认，Coxiella对人类巨噬细胞的生产性感染需要功能性T4BS。此外，我们已经验证了30多个蛋白质的Coxiella依赖DOT/ICM依赖性分泌。 Coxiella必须选择囊泡贩运途径以促进PV的发展。当前，当在被称为CVPA的感染细胞（Coxiella acciurolar蛋白A），CVPB，CVPC和CVPD中异位表达时，我们目前正在阐明四种蛋白质的活性，这些蛋白的活性被调节膜融合事件。特别了解CVPA功能已被播种。 Coxiella 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DERQXXXLL，I和酪氨酸（YXXφ）基于基于网状蛋白适配器蛋白（AP）配合物AP1，AP2和AP3的内吞分选基序。异位表达的MCHERRY-CVPA定位于Rab11和转铁蛋白受体呈阳性的周细胞体回收的内体的管状和囊泡结构域，而在内吞作用基序突变的突变后，CVPA膜相互作用消失。在下拉测定中，含有CVPA排序基序和全长CVPA的肽与AP2亚基相互作用和网格蛋白重链。此外，通过siRNA处理对AP2或网格蛋白的耗竭会显着抑制Coxiella的复制。因此，我们的结果揭示了网格蛋白涂层的囊泡运输在Coxiella感染中的重要性，并确定了CVPA在颠覆这些转运机制中的新作用。 尽管Coxiella的生产性感染显然需要T4BSS将蛋白质递送到宿主细胞细胞质中，但其他分泌系统可能负责修饰促进病原体复制的PV Lumen微环境。 为了评估考克斯氏菌将蛋白质分泌到PV中的潜力，我们通过质谱法分析了轴突生长培养基的蛋白质含量，以便在病原体蛋白的存在中分析。 从55种鉴定蛋白的候选列表中，通过在考克斯氏菌中表达旗帜标记的蛋白质，然后对培养上清液进行免疫印迹证实了27个分泌。 在感染的Vero细胞的可溶性部分中还发现了由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包含一个信号序列，并且从选定的蛋白质中删除了该序列，以取消分泌。 这些数据表明，蛋白质的分泌最初需要通过SEC转运酶的活性在整个内膜跨膜转移到周期。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降解。我们提出，大多数依赖SEC的分泌组是由于外膜囊泡的释放（OMV）导致的。 EM在培养基和哺乳动物宿主细胞中表现出明显的膜出现和OMV的产生来支持这一想法。 细胞内的双相发育周期，其中抗性小细胞变异（SCV）形态形式是由大细胞变异（LCV）形态形式产生的，被认为是coxiella毒力的基础。然而，谷烷发育的分子生物学知识很少。由于考克斯菌的细胞内生长施加了相当大的实验约束，因此我们试图确定在第一和第二代酸化的柠檬酸酸性半胱氨酸培养基（ACCM-1和ACCM-2）中，宿主细胞中的coxiella发育过渡是否在宿主细胞无宿主细胞（ACCM-1和ACCM-2）中概括。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到LCV过渡，但在延长孵育后，LCV对SCV转换并未发生（21天）。取而代之的是，考克斯菌（Coxiella）表现出类似幽灵的外观，含有浓缩染色质的细菌，但没有细胞质含量。该表型与培养14至21天之间的生存能力几乎总损失相关。在孵育14天后，考克斯菌的转录分析表明，ACCM-1培养细菌中氧化应激基因的表达升高。 ACCM-1和ACCM-2之间的唯一区别是胎牛血清替代甲基-Beta-Cyclodextrin。接种后7天，将甲基-Beta-Cyclodextrin添加到ACCM-1中，从而挽救了Coxiella活力，并降低了氧化应激基因的表达。因此，甲基β-环糊精似乎可以减轻ACCM-2中的氧化应激，从而导致Coxiella发育过渡和生存能力模仿宿主细胞培养的生物。 Coxiella在ACCM-2中的轴承培养，以及用于基因操纵的新方法，现在提供了研究Coxiella Biphasic发育的分子基础和生物学相关性的强大工具。 实际上，转录微阵列，整个细菌细胞蛋白质组学和轴承培养的Coxiella的脂质分析已经揭示了对发育形式的新决定性的。