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: 10014100
• 负责人: robert a heinzen
• 金额: $ 110.05万
• 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/10014100
• 关键词: Acids; Adaptor Signaling Protein; Amino Acids; Antibiotics; Autophagocytosis; Autophagosome; Bacteria; Binding; Biochemical; Biogenesis; Biological; Biology; Carbohydrates; Cell Line; Cell Membrane Permeability; Cell Wall; Cell physiology; Cells; Cellular biology; Cholesterol; Clathrin-Coated Vesicles; Complex; Coxiella; Coxiella burnetii; Cytoskeleton; Cytosol; Data; Defect; Development; Developmental Biology; Disease; EIF4EBP1 gene; Endocytic Vesicle; Event; Exhibits; FRAP1 gene; Gene Silencing; Generations; Genes; Glycine; Goals; Growth; Hela Cells; Human; Hydrolase; Impairment; Individual; Infection; Laboratories; Link; Lipids; Lipoproteins; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Biology; N-terminal; Natural History; Nutrient; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Peptidoglycan; Peptidyltransferase; Phagolysosome; Phase; Phenotype; Phosphorylation; Physiology; Play; Process; Production; Property; Proteins; Proteome; Proteomics; Q Fever; Regulation; Resistance; Role; Shotguns; Structure; System; Technology; Testing; VDAC1 gene; Vacuole; Variant; Vesicle; Virulence; Waste Products; base; cell envelope; cohort; deprivation; extracellular; genetic technology; innovation; insight; mTOR inhibition; macrophage; mutant; pathogen; pathogenic bacteria; physical property; recruit; residence; response; small molecule; trafficking; uptake

Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, in a large and spacious phagolysosome-like Coxiella-containing vacuole (CCV). Similar to a phagolysosome, the CCV has an acidic pH and contains lysosomal hydrolases obtained via fusion with late endocytic vesicles. Lysosomal hydrolases break down various lipids, carbohydrates, and proteins; thus, it is assumed Coxiella derives nutrients for growth from these degradation products. To investigate this possibility, we utilized a GNPTAB-/- HeLa cell line that lacks lysosomal hydrolases in endocytic compartments. Unexpectedly, examination of Coxiella growth in GNPTAB-/- HeLa cells revealed replication and viability are not impaired, indicating Coxiella does not require by products of hydrolase degradation to survive and grow in the CCV. However, although bacterial growth was normal, CCVs were abnormal, appearing dark and condensed rather than clear and spacious. Lack of degradation within CCVs allowed waste products to accumulate, including intraluminal vesicles, autophagy protein-LC3, and cholesterol. The build-up of waste products coincided with an altered CCV membrane, where LAMP1 was decreased, and CD63 and LAMP1 redistributed from a punctate to uniform localization. This disruption of CCV membrane organization may account for the decreased CCV size due to impaired fusion with late endocytic vesicles. Collectively, these results demonstrate lysosomal hydrolases are not required for Coxiella survival and growth but are needed for normal CCV development. These data provide insight into mechanisms of CCV biogenesis while raising the important question of how Coxiellai obtains essential nutrients from its host. Recruitment of membrane during CCV 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. By using new gene inactivation technologies developed in our laboratory, we have confirmed that a functional T4BSS is required for productive infection of human macrophages by Coxiella. Furthermore, we have verified Dot/Icm-dependent secretion of 40 proteins (among the roughly 120 identified) that are intact in all Coxiella strains. These are likely core effectors needed for successful infection, regardless of strain virulence potential. A critical cohort of effectors is predicted to co-opt vesicular trafficking pathways to promote CCV development. We are currently elucidating the activities of five effector proteins that traffic to the CCV membrane termed CvpA (Coxiella vacuolar protein A), CvpB, CvpC, CvpD, and CvpE that may 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 gained by showing the protein subverts clathrin-coated vesicle trafficking. CCV formation involves interactions with autophagosomes. We examined the activity of the autophagy regulator mTOR in response to Coxiella infection to better understand how the pathogen regulates lysosomal physiology to promote CCV biogenesis. Infected THP-1 cells and primary human macrophages exhibited reduced phosphorylation of the mTOR substrate 4E-BP1. Infected cells also displayed impaired mTORC1 reactivation and lysosomal relocalization when transitioned from amino acid-deprived to nutrient-rich conditions. Inhibition of mTOR was T4BSS-dependent, and cells infected with Coxiella and cultured under mTOR-inhibiting conditions supported larger and more fusogenic CCVs. Hyperactivation of mTOR inhibited Coxiella growth. Infected cells did not exhibit altered autophagic flux under any condition tested. However, during prolonged amino acid deprivation, infected cells accumulated LC3 and p62. Based on these data, inhibition of mTOR is predicted to alter normal lysosomal physiology to generate the expansive Coxiella CCV. 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. The Coxiella icmS mutant 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 displays hyper-secretion by the Coxiella icmS mutant, suggesting IcmS may also suppress secretion of some Dot/Icm substrates. Thus, regulation by IcmS appears complex, with the growth defect of the Coxiella icmS mutant potentially explained by both deficient and aberrant secretion of effector proteins. Coxiella undergoes 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. 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. Using an innovative and sensitive shotgun proteomics approach, we found that SCVs employ a new mechanism of outer membrane (OM) stabilization involving covalent linkage of peptidoglycan (PG ) to OM porins. PG muropeptides are linked to the N-terminal glycine residue of Coxiella OmpA-like porins CBU0307 and CBU0311. Deletion of Coxiella ldt2, encoding L,D transpeptidase 2, abolishes glycine linkages. Striking phenotypes of the deltaldt2 mutant are pronounced membrane blebbing and production of outer membrane vesicles. This hitherto unrecognized mechanism of PG-OM anchoring dramatically expands our understanding of OM stabilization and the function of L,D transpeptidases. These findings also have important implications for understanding how OM permeability is controlled to allow entry of small molecules, such as antibiotics. Moreover, it invokes a new model of OM stabilization in bacteria lacking PG-linked Brauns lipoprotein.

Q热发病机制的核心是病原体伯氏柯克斯体在一个大而宽敞的吞噬溶酶体样含有柯克斯体的液泡（CCV）中的复制。与吞噬溶酶体类似，CCV 具有酸性 pH 值，并含有通过与晚期内吞囊泡融合获得的溶酶体水解酶。溶酶体水解酶分解各种脂质、碳水化合物和蛋白质；因此，推测柯克斯体从这些降解产物中获取生长所需的营养。为了研究这种可能性，我们利用了在内吞区室中缺乏溶酶体水解酶的 GNPTAB-/- HeLa 细胞系。 出乎意料的是，对 GNPTAB-/- HeLa 细胞中柯克斯体生长的检查显示，复制和活力并未受损，这表明柯克斯体不需要水解酶降解的副产物即可在 CCV 中生存和生长。然而，尽管细菌生长正常，CCV 却异常，显得黑暗而浓缩，而不是清晰宽敞。 CCV 内缺乏降解导致废物积聚，包括腔内囊泡、自噬蛋白-LC3 和胆固醇。废物的积累与 CCV 膜的改变同时发生，其中 LAMP1 减少，CD63 和 LAMP1 从点状定位重新分布到均匀定位。 CCV 膜组织的破坏可能是由于与晚期内吞囊泡融合受损而导致 CCV 尺寸减小的原因。总的来说，这些结果表明溶酶体水解酶不是柯克斯体存活和生长所必需的，而是正常 CCV 发育所需的。这些数据提供了对 CCV 生物发生机制的深入了解，同时提出了柯克斯氏菌如何从其宿主获取必需营养物质的重要问题。 CCV 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。通过使用我们实验室开发的新基因灭活技术，我们已证实功能性 T4BSS 是柯克斯体对人类巨噬细胞的有效感染所必需的。此外，我们还验证了 40 种蛋白质（在大约 120 种已鉴定的蛋白质中）的 Dot/Icm 依赖性分泌，这些蛋白质在所有柯克斯体菌株中都是完整的。无论菌株毒力如何，这些都可能是成功感染所需的核心效应器。预计一组关键的效应器将选择囊泡运输途径来促进 CCV 的发展。我们目前正在阐明传输至 CCV 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，它们可能调节膜融合事件。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 的功能有了特别深入的了解。 CCV 的形成涉及与自噬体的相互作用。我们检查了自噬调节剂 mTOR 对柯克斯体感染的反应活性，以更好地了解病原体如何调节溶酶体生理学以促进 CCV 生物合成。受感染的 THP-1 细胞和原代人巨噬细胞表现出 mTOR 底物 4E-BP1 磷酸化降低。当从缺乏氨基酸的条件转变为营养丰富的条件时，受感染的细胞还表现出 mTORC1 重新激活和溶酶体重新定位受损。 mTOR 的抑制是 T4BSS 依赖性的，感染 Coxiella 并在 mTOR 抑制条件下培养的细胞支持更大且更具融合性的 CCV。 mTOR 的过度激活抑制柯克斯体的生长。在任何测试条件下，受感染的细胞都没有表现出自噬通量的改变。然而，在长期缺乏氨基酸的过程中，受感染的细胞积累了 LC3 和 p62。根据这些数据，预测 mTOR 的抑制会改变正常的溶酶体生理学，从而产生膨胀的柯克斯体 CCV。 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 icmS 突变体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 icmS 突变体的一部分底物显示出过度分泌，表明 IcmS 也可能抑制某些 Dot/Icm 底物的分泌。因此，IcmS 的调节显得很复杂，柯克斯体 icmS 突变体的生长缺陷可能由效应蛋白分泌不足和异常来解释。 柯克斯体经历双相发育周期，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。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。 使用创新且灵敏的鸟枪蛋白质组学方法，我们发现 SCV 采用了一种新的外膜 (OM) 稳定机制，涉及肽聚糖 (PG) 与 OM 孔蛋白的共价连接。 PG 胞肽与 Coxiella OmpA 样孔蛋白 CBU0307 和 CBU0311 的 N 末端甘氨酸残基连接。编码 L,D 转肽酶 2 的 Coxiella ldt2 的缺失会消除甘氨酸键。 deltaldt2 突变体的显着表型是明显的膜起泡和外膜囊泡的产生。这种迄今为止未被认识的 PG-OM 锚定机制极大地扩展了我们对 OM 稳定性和 L,D 转肽酶功能的理解。 这些发现对于理解如何控制 OM 渗透性以允许抗生素等小分子进入也具有重要意义。此外，它在缺乏 PG 连接布劳恩脂蛋白的细菌中调用了 OM 稳定的新模型。