Bacitracin

The H. pylori strains used in this study were PMSS1 (20 (link)), 7.13 (48 (link)), the G27 (49 (link)) derivatives NSH57 (23 (link)) and LSH100 (37 (link)), and four representative isolates from the J99 culture collection (25 (link), 27 (link), 29 (link)): J99, SC4, D1 and C2. All H. pylori isolates were grown on solid media, horse blood agar (HB agar). HB agar plates contain 4% Columbia agar base (Oxoid), 5% defibrinated horse blood (Hemostat Labs), 10 mg/mL vancomycin (Thermo Fisher), 2.5 U/mL polymyxin B (Sigma-Aldrich), 8 mg/mL amphotericin B (Sigma-Aldrich), and 0.2% β-cylodextrin (Thermo Fisher). For HB agar plates used to grow H. pylori from homogenized mouse stomach, 5 mg/l cefsulodin (Thermo Fisher), 5 mg/l trimethoprim (Sigma) and 0.2 mg/mL of bacitracin (Acros Organics, Fisher) were added to prevent outgrowth of mouse microflora. For competition experiments, 15 μg/mL chloramphenicol was added to distinguish between mutant (chloramphenicol-resistant) and wild-type (chloramphenicol-sensitive) bacteria. Shaking liquid cultures were grown in BB10, Brucella broth (Thermo Fisher) supplemented with 10% heat-inactivated FBS (Gemini BioProducts). H. pylori on plates and in liquid culture were grown at 37°C in a microaerobic conditions (10% CO₂, 10% O₂, and 80% N₂) using a tri-gas incubator.

Antibiotic susceptibility test of the Bacillus species strains was performed using standardized Single-Disc Method, as described previously [20 (link)]. All antibiotics, except for bacitracin B10, streptomycin S10, and kanamycin K30, which were purchased from BioMaxima S.A. (Lublin, Polska), used in this study were obtained from Emapol Species z o.o. (Gdansk, Poland). Sources of antibiotics were as follows: kanamycin K30 ug, ampicillin AM10, ampicillin & sulfbactam SAM20, streptomycin S10, rifampicin RA5, oxacillin OX5, bacitracin B10, and chloramphenicol C30. The overnight bacterial cultures were rejuvenated and adjusted to OD = 0.3 (at 600 nm), then were subsequently rubbed onto the surface of TYM agar plates. On 60 mL of the solid medium, 250 µL of the culture of the appropriate probiotic bacteria Bacillus species was rubbed. A commercial dispenser was used to apply antibiotic discs. All antibiotic discs were the standard high-concentration discs from 5 to 30 µg/µL. The incubation time was from 20 to 24 h at 37 °C. Zone diameters were measured against a black background illuminated with a high-intensity lamp to provide transmitted light. Inhibition of approximately 80% or more of the growth around a disc was considered to be a zone of inhibition. Some strains exhibited an inner area of light growth immediately around the antibiotic disc with an obvious area of inhibition outside this hazy growth. In these instances, the outer zone of inhibition was measured. The most obvious zone of inhibition was measured in those instances when several concentric zones were observed around an antibiotic disc. Zone diameters of 7 mm did not correspond to any measurable zone of inhibition since the antibiotic discs were between 6 and 7 mm in diameter.

Endogenous CALCRL-expressing BON-1 cells (DSMZ, Braunschweig, Germany) were seeded onto poly-L-lysine-coated 60-mm dishes and grown to 80% confluence. Cells were either left untreated or treated with chemically synthesised, double-stranded CALCRL siRNA duplexes (Santa Cruz Biotechnology, Dallas, TX, USA) in accordance with the manufacturer’s instructions. A scrambled siRNA was used as the negative control (Santa Cruz Biotechnology). Subsequently, the cells were lysed in detergent buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES, pH 7.4], 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulphate, 0.2 mM phenylmethylsulfonylfluoride, 10 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mg/mL aprotinin, and 10 mg/mL bacitracin). CALCRL enrichment was conducted using wheat germ lectin agarose beads (J-OIL MILLS, Inc., Tokyo, Japan), as previously described [130 (link)]. Subsequently, the protein content of the samples was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, and the samples (20 µg of protein per lane) were subjected to 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotted onto polyvinylidene fluoride membranes. Blots were incubated with the rabbit monoclonal anti-CALCRL antibody 8H9L8 (1:500 dilution) overnight at 4 °C, then incubated with peroxidase-conjugated secondary anti-rabbit antibody (1:5000 dilution; Santa Cruz Biotechnology) for 2 h at room temperature and visualised by enhanced chemiluminescence (Amersham, Braunschweig, Germany).
For adsorption controls, the anti-CALCRL antibody was preincubated for 2 h at room temperature with either 10 µg/mL of the immunising peptide (peptide 2) or 10 µg/mL of a control peptide that corresponded to a different region of the receptor (peptide 1; residues 23–40; sequence: ELEESPEDSIQLGVTRNK).