Francisella tularensis live vaccine strain: Proteomic analysis of membrane proteins enriched fraction

Pávková, Ivona; Hubálek, Martin; Zechovská, Jana; Lenčo, Juraj; Stulı́k, Jiřı́

doi:10.1002/pmic.200401213

Cited by 30 publications

(40 citation statements)

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“…Given the identification of KatG from pooled OM fractions (Table 2) and its high PSORTb OM localization score, it is likely that KatG is an OMP. Our results are consistent with the previous identification of KatG as a putative OMP from F. tularensis extracts (33). Antibodies are currently being developed against KatG to verify its membrane localization in sucrose gradient fractions.…”

Section: Discussionsupporting

confidence: 92%

“…2 and 3). While our optimized method was rigorous and subject to some experimental variability (see Results), our results appear to have much greater utility than those garnered from the use of detergents, lithium chloride, or sodium carbonate (2,20,33,41,46,51). We identified 15 bona fide F. tularensis OMPs, which colocalized at densities between 1.17 and 1.20 g/ml.…”

Section: Discussionmentioning

confidence: 82%

“…6), but bioinformatic analyses do not support their OM localization. Previous 2DE studies that used sodium carbonate to extract membrane proteins from F. tularensis also identified FTT1103 and FTT0831c (33,51), and we plan to generate antibodies to confirm their OM localization. The presence of certain cytoplasmic (water-soluble) proteins, including pyruvate dehydrogenase (FTT1485c) and GroEL (FTT1696), was not surprising in that other cytoplasmic proteins were uniformly distributed across the sucrose gradients (data not shown) and thus were difficult to exclude from OM fractions.…”

Section: Discussionmentioning

confidence: 99%

“…However, only three of those identified by mass spectrometry were proposed to be membrane proteins, including TUL4. More recently, two separate studies have used sodium carbonate to putatively enrich for F. tularensis membrane proteins (33,51). Whereas the former study identified roughly 200 proteins by 2DE and mass spectrometry, only 7 were predicted to be OMPs.…”

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Characterization ofFrancisella tularensisOuter Membrane Proteins

et al. 2007

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Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

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Characterization ofFrancisella tularensisOuter Membrane Proteins

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“…As noted above, growth phase of the bacterial cells, careful monitoring during osmotic lysis, and gentle resuspension of membrane pellets are critical steps that appear to correlate with success/failure of this membrane separation procedure. While this optimized method is rigorous and subject to some experimental variability, the resulting OMs appear to be of remarkably enhanced purity as compared to those garnered from the use of detergents, lithium chloride, or sodium carbonate 7,8,9,10,11,12 .…”

Section: Discussionmentioning

confidence: 99%

Method for the Isolation of Francisella tularensis Outer Membranes

Huntley

Robertson

Norgard

2010

JoVE

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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...

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Proteome Analysis of Bacterial Protein Expression after Ingestion of Microbes by Macrophages

Brychta

Pávková

2011

BSL3 and BSL4 Agents

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Francisella tularensis live vaccine strain: Proteomic analysis of membrane proteins enriched fraction

Cited by 30 publications

References 22 publications

Characterization ofFrancisella tularensisOuter Membrane Proteins

Characterization ofFrancisella tularensisOuter Membrane Proteins

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

Proteome Analysis of Bacterial Protein Expression after Ingestion of Microbes by Macrophages

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