Dataset Information

Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole.

ABSTRACT: The gram-negative bacterium Coxiella burnetii causes human Q fever, a disease characterized by a debilitating flu-like illness in acute cases and endocarditis in chronic patients. An obligate intracellular pathogen, Coxiella burnetii survives within a large, lysosome-like vacuole inside the host cell. A unique feature of the Coxiella parasitophorous vacuole (PV) is high levels of fusion with the host endocytic pathway, with PV-endosome fusion critical for Coxiella survival within the host cell. This unit describes quantitating PV-endosome fusion by measuring delivery of the fluid phase endosome marker dextran to the PV using live cell imaging. To study the effect of host cell proteins involved in PV-endosome fusion, details are provided for using siRNA knockdown host cells. This method is a powerful tool for understanding mechanisms underlying Coxiella's ability to manipulate host cell trafficking pathways. © 2017 by John Wiley & Sons, Inc.

SUBMITTER: Winfree S

PROVIDER: S-EPMC5558434 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole.

Winfree Seth S Gilk Stacey D SD

Current protocols in microbiology 20170811

The gram-negative bacterium Coxiella burnetii causes human Q fever, a disease characterized by a debilitating flu-like illness in acute cases and endocarditis in chronic patients. An obligate intracellular pathogen, Coxiella burnetii survives within a large, lysosome-like vacuole inside the host cell. A unique feature of the Coxiella parasitophorous vacuole (PV) is high levels of fusion with the host endocytic pathway, with PV-endosome fusion critical for Coxiella survival within the host cell. ...[more]

PMID: 28800156

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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 intracellular bacterium that causes the human disease Q fever. C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a type 4B secretion system to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport and metabolism is critical for successful infection. The mammalian lipid transport protein ORP1L (oxysterol binding protein-like protein 1 Long) localizes to the CCV membrane and mediates CCV-endoplasmic reticulum (ER) membrane contact sites. ORP1L functions in lipid sensing and transport, including cholesterol efflux from late endosomes and lysosomes (LELs), and the ER. Its sister isoform, ORP1S (oxysterol binding protein-like protein 1 Short) also binds cholesterol but has cytoplasmic and nuclear localization. In ORP1-null cells, we found that CCVs were smaller than in wild-type cells, highlighting the importance of ORP1 in CCV development. This effect was consistent between HeLa cells and murine alveolar macrophages (MH-S cells). CCVs in ORP1-null cells had higher cholesterol content than CCVs in wild-type cells at 4 days of infection, suggesting ORP1 functions in cholesterol efflux from the CCV. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrated that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol. IMPORTANCE Coxiella burnetii is an emerging zoonotic pathogen and bioterrorism threat. No licensed vaccine exists in the United States, and the chronic form of the disease is difficult to treat and potentially lethal. Postinfectious sequelae of C. burnetii infection, including debilitating fatigue, place a significant burden on individuals and communities recovering from an outbreak. C. burnetii must manipulate host cell processes in order to promote infection. Our results establish a link between host cell lipid transport processes and C. burnetii's avoidance of cholesterol toxicity during infection of alveolar macrophages. Elucidating the mechanisms behind bacterial manipulation of the host will yield insight for new strategies to combat this intracellular pathogen.

Dataset Information

Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole.

Publications

Quantitative Dextran Trafficking to the Coxiella burnetii Parasitophorous Vacuole.

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