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: 10927789
• 负责人: robert a heinzen
• 金额: $ 17.28万
• 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/10927789
• 关键词: Antibiotics; Autophagocytosis; Bacteria; Biochemical; Biogenesis; Biological; Biological Assay; Biology; Bulla; CRISPR interference; Carbohydrates; Cell Line; Cell Membrane Permeability; Cell Wall; Cell physiology; Cells; Cellular biology; Cholesterol; Clathrin-Coated Vesicles; Complex; Coxiella; Coxiella burnetii; Cytosol; Darkness; Data; Defect; Development; Developmental Biology; Disease; Endocytic Vesicle; Event; Family; Gene Silencing; Generations; Genes; Genetic Transcription; Genome; Glycine; Goals; Growth; Hela Cells; Human; Hydrolase; Impairment; Individual; Infection; Invaded; Laboratories; Link; Lipids; Lipoproteins; Macrophage; Mammalian Cell; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Biology; N-terminal; Natural History; Nutrient; Pathogenesis; Pathway interactions; Peptidoglycan; Peptidyltransferase; Phagolysosome; Phase; Phenotype; Process; Production; Productivity; Property; Protein Secretion; Protein translocation; Proteins; Proteome; Proteomics; Q Fever; Reporter; Reporting; Resistance; Role; Shotguns; System; Techniques; Technology; Testing; VDAC1 gene; Vacuole; Variant; Vero Cells; Vesicle; Virulence; Waste Products; cell envelope; cohort; extracellular; genetic technology; innovation; insight; late endosome; mutant; parasitism; pathogen; pathogenic bacteria; permissiveness; physical property; recruit; reference genome; residence; 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 Coxiella 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 and silencing 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. Over 150 C. burnetii proteins are reported exported in a T4BSS-dependent manner, but relatively few have assigned effector functions. Cross genome comparisons of T4BSS substrate-encoding genes shows many genes are psuedogenized or absent in reference genomes and thus may be dispensable for mammalian cell infection. To identify T4BSS substrates important for C. burnetii parasitism, 37 substrate proteins conserved among diverse C. burnetii genomes were selected for phenotypic analysis. Using either CyaA or BlaM translocation reporter assays, T4BSS-dependent translocation by C. burnetii was confirmed for 14 of the T4BSS substrates tested. Interference CRISPR (iCRISPR) was used to silence transcription of genes encoding validated C. burnetii T4BSS substrates. Transcriptional silencing of cbu0122, cbu1566, cbu1752, cbu1819, cbu1825, or cbu2007 decreased C. burnetii replication in THP-1 cells and impaired CCV biogenesis in Vero cells. Ectopically expressed CBU0122 localized to the late endosomal compartment and the CCV membrane indicating it belongs to the family of Coxiella vacuolar proteins (Cvp) involved in CCV biogenesis. Collectively, these data further define the functional repertoire of C. burnetii T4BSS 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，同时提出了柯克斯体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失活和沉默技术，我们已经证实，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 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。 据报道，超过 150 种 C. burnetii 蛋白以 T4BSS 依赖性方式输出，但相对较少具有指定的效应子功能。 T4BSS 底物编码基因的跨基因组比较显示，许多基因在参考基因组中被假源化或缺失，因此对于哺乳动物细胞感染来说可能是可有可无的。为了鉴定对伯内氏衣藻寄生重要的 T4BSS 底物，选择了不同伯内氏衣藻基因组中保守的 37 个底物蛋白进行表型分析。使用 CyaA 或 BlaM 易位报告基因测定，对 14 种测试的 T4BSS 底物证实了 C.burnetii 的 T4BSS 依赖性易位。干扰 CRISPR (iCRISPR) 用于沉默编码经过验证的 C.burnetii T4BSS 底物的基因的转录。 cbu0122、cbu1566、cbu1752、cbu1819、cbu1825 或 cbu2007 的转录沉默会减少 THP-1 细胞中伯氏念珠菌的复制，并损害 Vero 细胞中的 CCV 生物合成。异位表达的 CBU0122 定位于晚期内体区室和 CCV 膜，表明它属于参与 CCV 生物发生的柯克斯体液泡蛋白 (Cvp) 家族。总的来说，这些数据进一步定义了 C.burnetii T4BSS 效应蛋白的功能。 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，但对该过程的分子生物学知之甚少。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 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。

项目成果

Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic.

• DOI: 10.1128/mbio.02313-16
• 发表时间: 2017-02-28
• 期刊: mBio
• 影响因子: 6.4
• 作者: Mulye M;Samanta D;Winfree S;Heinzen RA;Gilk SD
• 通讯作者: Gilk SD

Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.

• DOI: 10.1371/journal.ppat.1007005
• 发表时间: 2018-04
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Miller HE;Larson CL;Heinzen RA
• 通讯作者: Heinzen RA

High-Content Imaging Reveals Expansion of the Endosomal Compartment during Coxiella burnetii Parasitophorous Vacuole Maturation.

高内涵成像揭示伯内氏立克次体寄生液泡成熟过程中内体室的扩张。

• DOI: 10.3389/fcimb.2017.00048
• 发表时间: 2017
• 期刊: Frontiers in cellular and infection microbiology
• 影响因子: 5.7
• 作者: Larson CL;Heinzen RA
• 通讯作者: Heinzen RA

Characterization of Coxiella burnetii Dugway Strain Host-Pathogen Interactions In Vivo.

• DOI: 10.3390/microorganisms10112261
• 发表时间: 2022-11-15
• 期刊: Microorganisms
• 影响因子: 4.5
• 作者: Tesfamariam M;Binette P;Cockrell D;Beare PA;Heinzen RA;Shaia C;Long CM
• 通讯作者: Long CM

Morphological remodeling of Coxiella burnetii during its biphasic developmental cycle revealed by cryo-electron tomography.

• DOI: 10.1016/j.isci.2023.107210
• 发表时间: 2023-07-21
• 期刊: ISCIENCE
• 影响因子: 5.8
• 作者: Kaplan, Doulin C.;Kaplan, Mohammed;Vankadari, Naveen;Kim, Ki Woo;Larson, Charles L.;Dutak, Przemysla;Beare, Paul A.;Krzymowski, Edward;Heinzen, Robert A.;Jensen, Grant J.;Ghosal, Debnath
• 通讯作者: Ghosal, Debnath