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

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:
  • 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:
  • 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:
  • 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.
  • DOI:
    10.3390/microorganisms10112261
  • 发表时间:
    2022-11-15
  • 期刊:
  • 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
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。
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robert a heinzen其他文献

robert a heinzen的其他文献

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{{ truncateString('robert a heinzen', 18)}}的其他基金

Genetics of Coxiella burnetii
伯内氏柯克斯体的遗传学
  • 批准号:
    6987135
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
Genetics of Coxiella burnetii
伯氏柯克斯体的遗传学
  • 批准号:
    8555887
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
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:
    8336171
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
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
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
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:
    9161549
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
Genetics of Coxiella burnetii
伯内氏柯克斯体的遗传学
  • 批准号:
    10272106
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
Genetics of Coxiella burnetii
伯内氏柯克斯体的遗传学
  • 批准号:
    7592301
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
Genetics of Coxiella burnetii
伯内氏柯克斯体的遗传学
  • 批准号:
    7964514
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
Genetics of Coxiella burnetii
伯氏柯克斯体的遗传学
  • 批准号:
    8336184
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
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:
    8946368
  • 财政年份:
  • 资助金额:
    $ 17.28万
  • 项目类别:
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