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发烧发病机理的中心是在大而宽敞的含吞噬体样胶状液泡（CCV）中复制Coxiella burnetii的复制。与吞噬体类似，CCV具有酸性pH值，并包含通过与晚期内吞囊泡融合获得的溶酶体水解酶。溶酶体水解酶分解了各种脂质，碳水化合物和蛋白质；因此，假定Coxiella从这些降解产物中得出了生长的营养。为了研究这种可能性，我们利用了一种Gnptab - / - HeLa细胞系，该细胞系缺乏内吞区室中的溶酶体水解酶。 出乎意料的是，检查GNPTAB - / - HELA细胞中考克斯菌生长的检查发现复制和生存能力不会受到损害，表明水解酶降解产物不需要Coxiella在CCV中生存和生长。然而，尽管细菌生长正常，但CCV是异常的，显得黑暗而凝结，而不是透明而宽敞。 CCV中缺乏降解允许废物积聚，包括腔内囊泡，自噬蛋白LC3和胆固醇。废物的积累与改变的CCV膜相吻合，其中LAMP1的减少，CD63和LAMP1从点状定位重新分布。 CCV膜组织的这种破坏可能是由于与晚期囊泡的融合受损而导致的CCV大小降低。总的来说，这些结果表明，考氏菌的存活和生长不需要溶酶体水解酶，而是正常的CCV发育所必需的。这些数据提供了对CCV生物发生机制的见解，同时提出了Coxiellai如何从其宿主那里获得必需营养素的重要问题。 CCV生物发生过程中膜的募集是由宿主和细菌因子调节的复杂过程。 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。通过使用在我们的实验室中开发的新基因失活技术，我们已经证实，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40种蛋白质（大约120个蛋白质）的DOT/ICM依赖性分泌，这些蛋白质在所有Coxiella菌株中都是完整的。这些可能是成功感染所需的核心效应子，而不论菌株毒力潜力如何。预计效应子的关键队列将是促进CCV发展的囊泡贩运途径。我们目前正在阐明五种效应子蛋白的活性，这些蛋白质流通于CCV膜（称为CVPA）（Coxiella acciell actroul蛋白A），CVPB，CVPC，CVPD和CVPE，可能调节膜融合事件。单个CVP基因中的突变体在复制和PV发育中均显示出明显的缺陷。通过显示蛋白质颠覆网状蛋白包被的囊泡运输，已经获得了对CVPA功能的特别洞察力。 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的活性，以响应Coxiella感染，以更好地了解病原体如何调节溶酶体生理学以促进CCV生物发生。感染的THP-1细胞和原发性人巨噬细胞表现出MTOR底物4E-BP1的磷酸化降低。当从氨基酸剥夺到富含营养的条件的情况下，被感染的细胞还显示出受损的MTORC1重新激活和溶酶体重新定位。 MTOR的抑制作用依赖于T4BSS，并在MTOR抑制条件下感染了Coxiella的细胞，这些细胞支持更大，更大的fusogenic CCV。 MTOR的过度激活抑制了考氏菌生长。在任何测试的条件下，感染的细胞不会显示自噬通量改变。但是，在长时间的氨基酸剥夺期间，感染的细胞积累了LC3和P62。基于这些数据，预计MTOR的抑制作用会改变正常的溶酶体生理，从而产生膨胀的Coxiella CCV。 Coxiella T4BSS的调节定义很差。 ICMS是一种预测的细胞质衔接蛋白，通过结合内部信号序列（S）来促进某些T4BSS效应子的易位。我们通过产生ICMS缺失突变体来检查Coxiella ICM的功能。 Coxiella ICMS突变体通常在轴突培养基中生长，同时在宿主细胞中具有明显的生长缺陷，该缺陷被ICM的单个染色体拷贝救出。单个底物的最佳分泌是ICMS依赖性或独立的。 此外，底物的一个子集显示了Coxiella ICMS突变体的超分子分泌，这表明ICMS也可能抑制某些点/ICM底物的分泌。因此，ICMS的调节似乎是复杂的，而Coxiella ICMS突变体的生长缺陷可能通过效应蛋白的不足和异常分泌可能解释。 Coxiella经历了双相发育循环，该循环在生物学，超微结构和组成上不同的大细胞变体（LCV）和小细胞变体（SCV）形式产生。 LCV正在复制指数相的形式，而SCV则是不复制的，固定相的形式。 SCV具有多种特性，例如凝结的核苷和异常细胞包膜，涉嫌赋予增强的环境稳定性。尽管发育周期被认为是毒力性的基础，但该过程的分子生物学知识很少。超微结构研究表明，细胞变体之间细胞膜的差异很大，但对SCV和LCV之间的生化差异知之甚少，从而赋予其独特的生物学和物理性质。 使用创新且敏感的shot弹枪蛋白质组学方法，我们发现SCVS采用了一种新的外膜（OM）稳定机制，涉及肽聚糖（PG）与OM Porins的共价连接。 PG杂肽与Coxiella oSPA样孔蛋白CBU0307和CBU0311的N末端甘氨酸残基有关。 Coxiella LDT2的缺失，编码L，D转肽酶2，废除甘氨酸链接。 deltaldt2突变体的醒目表型是明显的膜爆炸和外膜囊泡的产生。迄今为止，这种锚定锚定的未识别机制极大地扩展了我们对OM稳定化的理解和L，D转肽酶的功能。 这些发现对于理解如何控制OM渗透性以允许进入小分子（例如抗生素）具有重要意义。此外，它调用了缺乏PG连锁Brauns脂蛋白的细菌中OM稳定的新模型。