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Cellular and Developmental Biology of Coxiella burnetii

伯内氏柯克斯体的细胞和发育生物学

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

项目成果

期刊论文数量（29）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

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

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