Method for the isolation of Francisella tularensis outer membranes.

Jason F. Huntley, Gregory T. Robertson, Michael V. Norgard

Research output: Contribution to journalArticle

Abstract

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (<10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number40
StatePublished - 2010

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Francisella tularensis
Membranes
Membrane Proteins
Centrifugation
Sucrose
Francisella
Bacteria
Proteins
Tularemia
Immune Evasion
Sonication
Density Gradient Centrifugation
Zoonoses
Sugar (sucrose)
Virulence Factors
Gram-Negative Bacteria
Detergents
Pathogens
Virulence
Lipopolysaccharides

ASJC Scopus subject areas

  • Medicine(all)

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Method for the isolation of Francisella tularensis outer membranes. / Huntley, Jason F.; Robertson, Gregory T.; Norgard, Michael V.

In: Journal of visualized experiments : JoVE, No. 40, 2010.

Research output: Contribution to journalArticle

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