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: 9161549
• 负责人: robert a heinzen
• 金额: $ 72.55万
• 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/9161549
• 关键词: Appearance; Arginine; Bacteria; Binding; Biochemical; Biogenesis; Biological; Biology; Cardiolipins; Cell Wall; Cell physiology; Cells; Cellular biology; Cereals; Clathrin-Coated Vesicles; Complex; Coxiella; Coxiella burnetii; Cryoelectron Microscopy; Cytosol; Defect; Development; Developmental Biology; Employee Strikes; Epithelial Cells; Escherichia coli; Ethanolamines; Event; Exhibits; Gene Expression Profile; Gene Silencing; Generations; Genes; Genetic Determinism; Goals; Growth; Homologous Gene; Human; Individual; Infection; Laboratories; Lactams; Legionella pneumophila; Lipids; Mass Spectrum Analysis; Membrane; Membrane Fusion; Metabolism; Methods; Molecular; Molecular Biology; Natural History; Nonesterified Fatty Acids; Nutrient; Organism; Oxidative Stress; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Peptides; Peptidoglycan; Peptidyltransferase; Phagolysosome; Phagosomes; Phase; Phosphatidylethanolamine; Phosphatidylglycerols; Phospholipases A; Plasmalogens; Process; Property; Protein Biosynthesis; Proteins; Proteome; Q Fever; Regulation; Relative (related person); Resistance; Role; System; Technology; Thick; Thin Layer Chromatography; Up-Regulation; Vacuole; Variant; Vesicle; Virulence; adapter protein; base; biological adaptation to stress; cell envelope; cohort; crosslink; extracellular; genetic manipulation; genetic technology; insight; macrophage; mutant; neurotensin mimic 2; pathogen; periplasm; physical property; protein transport; residence; tool; trafficking; 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 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 five effector proteins that traffic to the PV membrane termed CvpA (Coxiella vacuolar protein A), CvpB, CvpC, CvpD, and CvpE that are speculated to modulate membrane fusion events. Mutants in individual cvp genes all display significant defects in replication and PV development. Particular insight into the function of CvpA has been grained by showing the protein subverts clathrin-coated vesicle trafficking. Regulation of the Coxiella T4BSS is poorly defined. IcmS is a predicted cytoplasmic adapter protein that facilitates translocation of certain T4BSS effectors by binding an internal signal sequence(s). We examined the function of Coxiella IcmS by generating an icmS deletion mutant. Coxiella ΔicmS grows normally in axenic media while having a pronounced growth defect in host cells that is rescued with a single chromosomal copy of icmS. Optimal secretion of individual substrates is either IcmS-dependent or independent. Additionally, a subset of substrates display hyper-secretion in Coxiella ΔicmS, suggesting IcmS may also suppress secretion of some Dot/Icm substrates. Thus, regulation by IcmS appears complex with the growth defect of Coxiella ΔicmS potentially explained by both deficient and aberrant secretion of effector proteins. A hallmark of Coxiella is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCV are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. Although the developmental cycle is considered fundamental to Coxiella virulence, the molecular biology of this process is poorly understood. Recently, we discovered that Coxiella developmental transitions and viability in the synthetic medium ACCM-2 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. Ultrastructural studies show marked differences in the cell envelope between cell variants, but little is known about biochemical differences between SCV and LCV that confer their distinct biological and physical properties. We analyzed the lipid composition of Coxiella after 4 (LCV), 7 (intermediate forms) and 14 (SCV) days of growth in synthetic medium, using thin layer chromatography and mass spectrometry. Similar to Escherichia coli, Coxiella contains cardiolipin, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), with some PE in an unusual plasmalogen form. PE and PG are present in lower quantities in the SCV relative to the LCV. However, three additional major lipid species are present in higher quantities in the SCV: lyso-phosphatidylethanolamine, a breakdown product of PE; glycerophospho-N-acyl-ethanolamine, a lipid previously not found in bacteria; and free fatty acids, which are normally toxic for bacteria. Mutational analysis indicates that these three lipids are generated via the activity of a Coxiella outer membrane phospholipase A homolog (CBU0489). A cbu0489 mutant exhibits a significant growth defect in THP-1 macrophage-like cells, suggesting developmentally regulated lipid synthesis is required for optimal intracellular growth and could contribute to the distinct properties of LCV and SCV. To further identify genetic determinants of LCV to SCV transition, we profiled the Coxiella transcriptome by microarray at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV) and 21 (late SCV) days post-infection (dpi) of Vero epithelial cells. Transcriptional signatures of SCV are up-regulation of genes involved in oxidative stress responses, arginine metabolism, and cell wall remodeling. Genes down-regulated in SCV are primarily associated with intermediary metabolism. A striking transcriptional signature of the SCV is induction (10-fold) of five genes encoding predicted L,D transpeptidases that catalyze β-lactam resistant 3-3 peptide crosslinks typically found in the peptidoglycan (PG) of stationary phase bacteria. Cryo-electron microscopy reveals an unusually thick and dense periplasmic layer specific to SCV, suggestive of PG, whereas the periplasm and inner and outer membranes of LCV exhibits a more typical gram-negative appearance. Muropeptide analysis of Coxiella PG shows an increasing percentage of 3-3 cross-links as LCV transition to SCV raising the possibility that the Coxiella L,D transpeptidase homologs up-regulated by microarray may be important in cross-linking the PG of SCV. Collectively, these results indicate the SCV produces a unique transcriptome with a major subset of these genes directed towards remodeling a PG layer that may contribute to Coxiellas environmental resistance.

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受宿主和细菌因素调节的复杂过程。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。我们目前正在阐明转运至 PV 膜的五种效应蛋白的活性，称为 CvpA（柯克斯体液泡蛋白 A）、CvpB、CvpC、CvpD 和 CvpE，推测它们可调节膜融合事件。单个 cvp 基因的突变体在复制和 PV 发育中都表现出显着的缺陷。通过显示蛋白质破坏网格蛋白包被的囊泡运输，人们对 CvpA 功能的了解更加深入。 Coxiella T4BSS 的调节尚不清楚。 IcmS 是一种预测的细胞质接头蛋白，可通过结合内部信号序列促进某些 T4BSS 效应子的易位。我们通过生成 icmS 缺失突变体来检查柯克斯体 IcmS 的功能。柯克斯体 ΔicmS 在无菌培养基中正常生长，但在宿主细胞中具有明显的生长缺陷，可通过 icmS 的单个染色体拷贝来挽救。各个底物的最佳分泌要么依赖于 IcmS，要么独立于 IcmS。 此外，Coxiella ΔicmS 中的一部分底物表现出过度分泌，表明 IcmS 也可能抑制某些 Dot/Icm 底物的分泌。因此，IcmS 的调节似乎与 Coxiella ΔicmS 的生长缺陷很复杂，这可能是由效应蛋白分泌不足和异常引起的。 柯克斯体的一个标志是双相发育周期，产生生物学、超微结构和组成上不同的大细胞变体 (LCV) 和小细胞变体 (SCV) 形式。 LCV 是复制型、指数期形式，而 SCV 是非复制型、稳定期形式。 SCV 具有多种特性，例如浓缩的核仁和不寻常的细胞包膜，怀疑可增强环境稳定性。尽管发育周期被认为是柯克斯体毒力的基础，但这一过程的分子生物学却知之甚少。最近，我们发现合成培养基 ACCM-2 中柯克斯体的发育转变和活力模仿了宿主细胞培养的生物体。 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。 超微结构研究显示不同细胞变体之间的细胞包膜存在显着差异，但人们对 SCV 和 LCV 之间的生化差异知之甚少，正是这些差异赋予了它们独特的生物学和物理特性。我们使用薄层色谱和质谱分析了在合成培养基中生长 4 天（LCV）、7 天（中间形式）和 14 天（SCV）后柯克斯体的脂质组成。 与大肠杆菌类似，柯克斯体含有心磷脂、磷脂酰甘油 (PG) 和磷脂酰乙醇胺 (PE)，其中一些 PE 以不寻常的缩醛磷脂形式存在。 相对于 LCV，SCV 中 PE 和 PG 的含量较低。 然而，另外三种主要脂质种类在 SCV 中的含量较高：溶血磷脂酰乙醇胺，PE 的分解产物；甘油磷酸-N-酰基乙醇胺，一种以前在细菌中未发现的脂质；和游离脂肪酸，通常对细菌有毒。 突变分析表明，这三种脂质是通过柯克斯体外膜磷脂酶 A 同源物 (CBU0489) 的活性产生的。 cbu0489 突变体在 THP-1 巨噬细胞样细胞中表现出显着的生长缺陷，表明发育调节的脂质合成是最佳细胞内生长所必需的，并且可能有助于 LCV 和 SCV 的独特特性。 为了进一步确定 LCV 向 SCV 转变的遗传决定因素，我们在术后 3 天（早期 LCV）、5 天（晚期 LCV）、7 天（中间形式）、14 天（早期 SCV）和 21 天（晚期 SCV）通过微阵列分析了柯克斯体转录组。 -Vero上皮细胞的感染(dpi)。 SCV 的转录特征是参与氧化应激反应、精氨酸代谢和细胞壁重塑的基因上调。 SCV 中下调的基因主要与中间代谢相关。 SCV 的一个引人注目的转录特征是诱导（10 倍）编码预测的 L,D 转肽酶的 5 个基因，这些基因催化通常在稳定期细菌的肽聚糖 (PG) 中发现的 β-内酰胺抗性 3-3 肽交联。冷冻电镜显示 SCV 特有的异常厚且致密的周质层，提示 PG，而 LCV 的周质和内膜和外膜表现出更典型的革兰氏阴性外观。 柯克斯体 PG 的胞肽分析显示，随着 LCV 过渡到 SCV，3-3 个交联的百分比不断增加，这提高了微阵列上调的柯克斯体 L,D 转肽酶同系物可能在 SCV 的 PG 交联中起重要作用的可能性。 总的来说，这些结果表明 SCV 产生了一个独特的转录组，其中这些基因的主要子集直接用于重塑可能有助于柯克斯体环境抵抗力的 PG 层。