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Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development.

ABSTRACT: The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome- and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.

SUBMITTER: Martinez E

PROVIDER: S-EPMC4988616 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development.

Martinez Eric E Allombert Julie J Cantet Franck F Lakhani Anissa A Lakhani Anissa A Yandrapalli Naresh N Neyret Aymeric A Norville Isobel H IH Favard Cyril C Muriaux Delphine D Bonazzi Matteo M

Proceedings of the National Academy of Sciences of the United States of America 20160525 23

The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxie ...[more]

PMID: 27226300

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Project description:The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a β-lactamase enzyme (BlaM) fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into host cells after bacteria establish residency in a lysosome-derived organelle.

Project description:The Q fever agent Coxiella burnetii is a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify the Coxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report that C. burnetii inhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However, C. burnetii did not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 by C. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulates C. burnetii intracellular growth.IMPORTANCECoxiella burnetii is an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of the Coxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report that C. burnetii growth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefit C. burnetii growth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate that C. burnetii inhibition of mTORC1 without accelerated autophagy promotes bacterial growth.

Project description:Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small noncoding RNAs (sRNAs) of C. burnetii One of them, CbsR12 (Coxiella burnetiismall RNA 12), is highly transcribed during axenic growth and becomes more prominent during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in stem loops with consensus AGGA/ANGGA motifs. We subsequently determined that CbsR12 binds to recombinant C. burnetii CsrA-2, but not CsrA-1, proteins in vitro Moreover, through a combination of in vitro and cell culture assays, we identified several in trans mRNA targets of CbsR12. Of these, we determined that CbsR12 binds and upregulates translation of carA transcripts coding for carbamoyl phosphate synthetase A, an enzyme that catalyzes the first step of pyrimidine biosynthesis. In addition, CbsR12 binds and downregulates translation of metK transcripts coding for S-adenosylmethionine synthetase, a component of the methionine cycle. Furthermore, we found that CbsR12 binds to and downregulates the quantity of cvpD transcripts, coding for a type IVB effector protein, in mammalian cell culture. Finally, we found that CbsR12 is necessary for expansion of Coxiella-containing vacuoles and affects growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first characterization of a trans-acting sRNA of C. burnetii and the first example of a bacterial sRNA that regulates both CarA and MetK synthesis. CbsR12 is one of only a few identified trans-acting sRNAs that interacts with CsrA.IMPORTANCE Regulation of metabolism and virulence in C. burnetii is not well understood. Here, we show that C. burnetii small RNA 12 (CbsR12) is highly transcribed in the metabolically active large-cell variant compared to the nonreplicative small-cell variant. We show that CbsR12 directly regulates several genes involved in metabolism, along with a type IV effector gene, in trans In addition, we demonstrate that CbsR12 binds to CsrA-2 in vitro and induces autoaggregation and biofilm formation when transcribed ectopically in Escherichia coli, consistent with other CsrA-sequestering sRNAs. These results implicate CbsR12 in the indirect regulation of a number of genes via CsrA-mediated regulatory activities. The results also support CbsR12 as a crucial regulatory component early on in a mammalian cell infection.

Dataset Information

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development.

Publications

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development.

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