Fractionation of the Coxiella burnetii Parasitophorous Vacuole.

Fractionation of the Coxiella burnetii Parasitophorous Vacuole. Research Abstract Details 

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Fractionation of the Coxiella burnetii Parasitophorous Vacuole. Abstract Text:

Coxiella burnetii is a bacterial obligate intracellular pathogen that replicates within a spacious parasitophorous vacuole (PV) with lysosomal characteristics. The pathogen actively participates in the biogenesis of its PV by synthesizing proteins that mediate vesicular interactions. Both C. burnetii and host factors that regulate PV formation are likely localized to the PV membrane, and their identification would be aided by an efficient method for isolating the C. burnetii vacuole. To this end, we developed a method to separate intact PV from host cell material that relies on fusion of the vacuole with latex bead-containing phagosomes (LBP). Sequestration of latex beads by the C. burnetii PV increases the vacuole's buoyant density and facilitates its fractionation on a sucrose step gradient. Transmission electron microscopy confirms the isolation of intact PV-containing latex beads from infected MH-S murine alveolar macrophage-like cells. Immunoblotting demonstrates that C. burnetii PV lysates are dramatically enriched for the late endosome/lysosome markers LAMP-1 and LAMP-2 when compared to total host cell lysates. Conversely, PV preparations are devoid of p62 and GM130, markers of the nucleus and Golgi apparatus, respectively, indicating effective separation of the vacuole from these host cell compartments. Two-dimensional gel electrophoresis and immunoblotting reveal distinct protein differences between C. burnetii PV and LBP. Identification of proteins unique to the PV membrane will yield important insight into C. burnetii-host interactions.

Fractionation of the Coxiella burnetii Parasitophorous Vacuole. Publishing Authors By Initials

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Fractionation of the Coxiella burnetii Parasitophorous Vacuole. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Methods in molecular biology (Clifton, N.J.)

VOLUME: 445

Page Numbers: 389-406

Journal Abbreviation: Methods Mol. Biol.

ISSN: 1064-3745

DAY: 21

MONTH: 04

YEAR: 2008

Fractionation of the Coxiella burnetii Parasitophorous Vacuole. Information

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LANGUAGE: eng

NlmUniqueID: 9214969

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Grant and Affiliation Information for Fractionation of the Coxiella burnetii Parasitophorous Vacuole.

AFFILIATION: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT.

Country: United States

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MEDLINETA: Methods Mol Biol

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