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纳克希菌在一个大而宽敞的吞噬溶酶体样含克希菌液泡（CCV）中复制。与吞噬溶酶体类似，CCV具有酸性pH值，并含有溶酶体水解酶，通过与晚期内吞囊泡融合获得。溶酶体水解酶分解各种脂质、碳水化合物和蛋白质；因此，假定Coxiella从这些降解产物中获得生长所需的营养物质。为了研究这种可能性，我们使用了一种内吞区缺乏溶酶体水解酶的GNPTAB-/- HeLa细胞系。出乎意料的是，对Coxiella在GNPTAB-/- HeLa细胞中生长的检测显示，Coxiella的复制和活力没有受到损害，这表明Coxiella不需要水解酶降解的副产物就能在CCV中存活和生长。然而，尽管细菌生长正常，但ccv异常，呈现黑暗和浓缩，而不是清晰和宽敞。ccv内缺乏降解使得废物积累，包括腔内囊泡、自噬蛋白lc3和胆固醇。废物的积累与CCV膜的改变相一致，LAMP1减少，CD63和LAMP1从点状重新分布到均匀定位。这种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大小减小的原因。综上所述，这些结果表明溶酶体水解酶不是科希氏菌存活和生长所必需的，但却是正常CCV发育所必需的。这些数据提供了对CCV生物发生机制的深入了解，同时提出了一个重要的问题，即柯西莱如何从宿主那里获得必需的营养物质。