Method for the Isolation of <em>Francisella tularensis</em> Outer Membranes

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. Video LinkThe Day 2: F. tularensis liquid media inoculation1. Prepare and autoclave 1 liter of cation-adjusted Mueller-Hinton medium (containing 1.23 mM calcium chloride dihydrate and 1.03 mM magnesium chloride hexahydrate). When cooled to 37°C, further supplement medium with 0.1% (wt/vol) glucose, 0.025% (wt/vol) iron pyrophosphate, and 2% (vol/vol) IsoVitaleX. Filter sterilize (0.22 μ) medium into two 500-ml aliquots. 2. Remove 12 ml of supplemented Mueller-Hinton medium and transfer into a sterile 50-ml Falcon tube. 3. Using a sterile 10-μl inoculation loop, scrape a large loopful of F. tularensis growth from agar plates (from Day 1) and transfer bacteria into 12 ml of Mueller-Hinton medium (see Step 2). Pipette solution multiple times to break-up clumps and prepare homogenous bacterial suspension. Do not vortex. 4. Using a sterile pipette, inoculate each 500-ml vessel with 5 ml of bacterial suspension from Step 3. Grow broth cultures at 37°C for 14 to 18 h with gentle shaking (190-200 rpm on a New Brunswick Innova 2300 series shaker).Day 3: Spheroplasting, osmotic lysis, and sucrose density gradient centrifugation1. Obtain F. tularensis cultures from the shaking incubator and remove a 1-ml aliquot from each 500-ml culture to check the respective optical densities. We have found optimum membrane extraction and isolation results when using F. tularensis cells in early logarithmic phase of growth, which correlates with an OD 600 between...

Francisella tularensisSchuS4 and SchuS4 Lipids Inhibit IL-12p40 in Primary Human Dendritic Cells by Inhibition of IRF1 and IRF8

“…Total membrane fraction (MF) from LVS and SchuS4 were isolated as previously described (13–15). Briefly, SchuS4 was grown in modified Mueller-Hinton broth as previously described (10, 12, 13).…”

Vibrio cholerae leuO Transcription Is Positively Regulated by ToxR and Contributes to Bile Resistance

“…The cell pellet was resuspended in 10 mM Tris-HCl (pH 8.0) in 0.75 M sucrose. Spheroplasts were then induced by the addition of 150 g/ml lysozyme, followed by the addition of 2 volumes of buffer A (10 mM Tris-HCl [pH 8.0], 10 mM EDTA) (36,37). The cells were then lysed by passage through a model M-11P Microfluidizer (Microfluidics).…”

IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

“…A previously described osmotic lysis and sucrose density gradient centrifugation procedure (40,48) was used to monitor the subcellular localization of IglE or an IglE ⌬(Asn96-Asp125) variant. Sequential gradient fractions were collected dropwise, and the density (g/ml) of each was determined on the basis of the refractive indices.…”

“…In IglE, the N ϩ 2 amino acid is isoleucine, which should facilitate export to the OM. To verify this directly and compare the membrane distribution of IglE, strain TP569 constitutively expressing wild-type IglE from PrpsL in attTn7 was subjected to osmotic lysis and OM and IM fractionation experiments (40,48). These experiments were initially performed using strain TP569 (LVS ⌬iglE 1 ⌬iglE 2 ::FRT-Tn7-iglE ϩ ) to increase the cellular content of IglE.…”