, Australia) and Griffith University (Gold

Coast, Qld., Australia) culture collections. All C. jejuni strains were subcultured no more than once to avoid the influence of passaging. Strains were grown on blood agar, composed of Columbia agar containing 5% (v/v) defibrinated horse blood and Skirrow’s antibiotic supplement (Oxoid), under microaerobic conditions (5% O2, 10% CO2 and 85% N2) at 37°C for 48 h and 42°C for 24 h. LOS preparations For gel electrophoresis Blood agar-grown bacteria were harvested in 1 mL of sterile water, washed once in 1 mL of sterile water, and lysed Selleckchem mTOR inhibitor by heating. Prior to lysis, samples were adjusted for numbers of bacteria using the OD600 measurements of bacterial suspensions. Mini-preparations of LOS were prepared by treating the whole-cell extracts with proteinase K as described previously [33]. The LOS mini-preparations from single colonies were prepared by collecting Epigenetics activator and washing cells in 40 μL of sterile water and then lysing by heating. Purified C. jejuni LOS was prepared by subjecting the biomass to hot phenol-water treatment using 90% (v/v) aqueous phenol at 65°C for 10 min [34]. Extracted LOS was purified by enzymatic treatment as described previously [19]. The LOS preparations were made up to 15 μg/μL in distilled water prior to gel electrophoresis. For NMR analysis C. jejuni 11168 was grown for 24 hr

as described above and bacterial biomass was harvested and washed twice using phosphate-buffered saline pH 7.4 (PBS; Sigma) and centrifugation (5000 × g, 4°C, 15 min). Biomass was lyophilised and 21 g and 20 g dry-cell mass was PFKL collected from cultures grown at 37°C and 42°C, respectively. Dried biomass was pretreated using pronase-E [35]. Extraction of LOS was carried out using hot-phenol water technique [34]. Water-soluble LOS was purified using RNaseA, DNase II and proteinase K (Sigma) and ultra-centrifugation, as previously described [19]. The LOS were treated with 0.1 M HCl at 100°C for 2 hours to cleave the acid-labile ketosidic linkage between the core OS and lipid A [19].

The lipid A precipitate was removed by centrifugation (5000 × g, 4°C, 30 mins), washed and both this and supernatant were lyophilised. The supernatant was fractionated using gel-permeation chromatography on a column of Bio-Gel P4 (1 m × 2 cm) with 0.05 M pyridinium acetate (pH 4.5) as the eluent. The resultant fractions were monitored by capillary-tube spotting on silica gel 60 TLC plates (Merck), followed by charring with 20% H2SO4 in EtOH at 150°C. The water-soluble carbohydrate-containing fractions of core OS were flash-frozen in dry-ice/acetone bath and lyophilized. CPS and whole-cell protein preparations For assessing CPS production, proteinase K-treated whole cell extracts were prepared as described above. Whole-cell protein samples were prepared by incubating SDS-PAGE loading buffer with C. jejuni biomass at 100°C for 5 min to facilitate bacterial lysis and binding of the SDS to the denatured proteins.