Polytron homogeniser

An overnight culture of a single X-33_VirB9-1 or XX-33_VirB10 colony in 2 mL of Buffered glycerol-complex medium (BMGY) was used to inoculate 25 mL cultures of BMGY (Life Technologies).These cultures were grown at 28 °C in a shaking incubator (260 rpm) to an optical density at 600 nm (OD₆₀₀) of 2–6. The yeast cell pellets were collected by centrifugation at 2000× g, at 20 °C for 5 min. The supernatant was decanted and discarded. The cell pellet was re-suspended in Buffered Methanol-complex Medium (BMMY) containing 0.5% methanol to an OD₆₀₀ of 1.0 to induce expression. Methanol (100%) was added to a final concentration of 0.5% methanol every 24 h to maintain induction. The supernatants and cell pellets were collected and stored at −80 °C for protein purification after 72 h of methanol induction. The cell pellets were homogenised by Polytron^® homogeniser (Kinematica AG, Luzern, Switzerland) in lysis buffer (100 mM NaH₂PO₄, 10 mM Tris-Cl, 8 M Urea, pH 8.0). The lysates were incubated for 30 min with gentle shaking. The resultant solution was centrifuged at 15,000× g at 4 °C for 30 min. The supernatants were stored at −80 °C until required. Purification of 6-His-tagged proteins were performed with Ni-NTA Agarose (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.

H. pylori infection levels within mouse gastric tissues were quantified by colony-forming assay. Briefly, stomachs were opened along the inner curvature and bisected longitudinally. One half was placed in BHI and homogenized (GmbH Polytron homogeniser, Kinematica, Switzerland) and the other half rapidly frozen for RNA extraction. Ten-fold serial dilutions were prepared in BHI broth and aliquots spread over GSSA selective agar plates [Blood Agar Base No. 2 with 5% horse blood, vancomycin (12 mg/mL), polymyxin B (0.40 mg/mL), bacitracin (24 mg/mL), nalidixic acid (1.3 mg/mL), and amphotericin B (3.75 mg/mL), all from Sigma]. After 5 days culture as above, colonies were counted, and the number of colony-forming units was calculated per stomach (McGuckin et al., 2007 (link)). Colonies were confirmed to be H. pylori by the rapid urease test as previously described (Sutton et al., 2000 (link)).

Frozen mouse tissues were rapidly defrosted in the ice-cold lysis buffer [50 mM Tris–HCl, pH 7.5, 1% (v/v) Triton X-100, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM NaF, 10 mM 2-glycerophosphate, 5 mM sodium pyrophosphate, 0.1 µg/ml microcystin-LR (Enzo Life Sciences), 270 mM sucrose, and complete EDTA-free protease inhibitor cocktail (Sigma–Aldrich Cat # 11836170001)] and homogenised using a POLYTRON homogeniser (KINEMATICA) on ice (5 s homogenisation, 10 s interval, and 5 s homogenisation). Lysates were clarified by centrifugation at 20 800 g for 30 min at 4°C and supernatants were quantified by Bradford assay and used for subsequent immunoblot analysis.