Type 3 porcine gastric mucin

A. baumannii ATCC 19606^T was purchased from the American Type Culture Collection (ATCC) and porcine gastric mucin type III was obtained from Sigma-Aldrich. A. baumannii ATCC 19606^T was stored as Luria-Bertani (LB) broth/glycerol stocks at -80°C [31 ]. Escherichia coli MG1655 was obtained from F. Blattner, University of Wisconsin, USA. Cultures on LB agar were used to inoculate either LB broth [31 ] or swimming broth (SB, 1% tryptone, 0.5% NaCl), which were incubated overnight (12 to 14 h) at 37°C with shaking at 200 rpm. Swimming agar (SA) plates were made by adding 0.3% agarose to SB. Solutions containing mucin were sterilized by autoclaving as described previously [15 (link), 19 (link)–21 (link)]. A549 human alveolar epithelial cells, which were obtained from Dr. E. Lafontaine, College of Veterinary Medicine, University of Georgia, USA, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, ampicillin and streptomycin at 37°C in the presence of 5% CO₂, as previously described [32 (link)].

We generated droplets from an aqueous solution containing NaCl (9 g l⁻¹, physiological concentration), mucin (3 g l⁻¹ porcine gastric mucin type III, Sigma-Aldrich^®), and 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC, 0.5 g l⁻¹, Avanti Inc.). DPPC is among the most abundant phospholipid lung surfactants that reduces surface tension during breathing [31 (link)]. We investigated phase transition and evaporation for droplets with (4C) and without (3C) surfactant. One of the authors donated the saliva samples. In this study, we measured gross changes only and not specific reactive sites on the mucin; we, therefore, used porcine gastric mucin type III as a surrogate for human mucin, which has similar mechanical properties [32 (link)].

Human milk and blood antigen oligosaccharides used in this study are described in Table S1. N-acetylneuraminic acid (Neu5Ac), α-d-galactose-1-phosphate (Gal1P) and α-l-fucose (Fuc) were form Carbosynth and xylotetraose was from Megazyme. Galactose (Gal), Glucose (Glc), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc) and porcine gastric mucin type III, (PGM) were from Sigma Aldrich. Bovine submaxillary mucin (BSM) was from VWR. 2-aminoanthranilic acid (2-AA) was from Nacalai Tesque and pooled human milk samples were purchased from Hvidøvre hospital (Hvidøvre, Denmark). All chemicals were of analytical grade unless otherwise stated.

A. muciniphila AH39 was cultured in BHI broth supplemented with 0.25% porcine gastric mucin (type III, 81 Sigma-Aldrich) for 72 h. C. butyricum FHLJZD47T10 was cultured in BHI broth for 24 h. B.bifidum JSNJJNM2 and another 85 B. bifidum strains were cultured in MRS broth for 24–48 h. All bacteria were grown in an anaerobic chamber (Electrotek, Shipley, UK) with the following gaseous characteristics: 5% hydrogen, 5% 85 carbon dioxide, and 90% nitrogen. For in vitro MLN model, freshly cultured bacterial cells were collected and washed with phosphate-buffered saline (PBS). After that, bacterial cells were re-suspended and adjusted to 0.5 of optical density at 600 nm for further experiments. For in vivo MLN experiment, bacterial cells were collected and concentrated to 5 × 10⁹ CFU/mL for oral administration.

The sample (100 μl) would be mixed with 1 ml of 25% modified version of Artificial Salivary Medium (ASM) before inoculation in the flow cell (Fig 2). The ASM includes the following components: 0.50 g/L tryptone (Oxoid, Hampshire, England), 0.50 g/L neutralised bacteriological peptone (Oxoid), 0.625 g/L type III porcine gastric mucin (Sigma-Aldrich, Steinheim, Germany), 0.25 g/L yeast extract (Oxoid), 0.05 g/L KCl, 0.05 g/L CaCl2, 0.088 g/L NaCl, and 1mg/L haemin (Sigma-Aldrich). The solution was supplemented with 2.5mM DTT (Sigma-Aldrich) and the pH was adjusted to 7.0 [42 (link)]. The sample would be injected into the inoculation port (Fig 2F) then be cultured on hydroxyapatite (HA) discs (Clarkson Chromatography Products Inc. PA, USA) in 3D-printed flow cells for a period of 14 days. The flow cell would remain static for 12h to ensure each HA discs would be coated with ASM, as this would facilitate the formation of acquired pellicle for bacterial adhesion. Based on our unpublished preliminary data, each flow cell will produce 16 discs with ~1.15x10⁴ live cells on each. After 14 days, the flow cell would be dissembled and all the 16 discs would be extracted. Half of the discs would be used for microscopic examination and the other half would be stored at -80° C until used for amplicon and shotgun sequencing.

We used a nonredundant transposon mutant library collection constructed in the El Tor C6706 background (83 (link)). Using a 96-well plate replicator, the library was replica plated onto large LB kanamycin (0.05 mg/ml) agar plates and incubated overnight at 37°C. Subsequently, this LB-grown library was replica plated onto minimal MCLMAN medium (84 (link)) plates supplemented with 0.5% type III porcine gastric mucin (Sigma). Transposon insertion mutants that were qualitatively defective for growth compared to neighboring transposon insertion mutants were marked as deficient for mucin utilization for further investigation. The complete list of identified mutants is in Table S1 at https://doi.org/10.5281/zenodo.3966283.

Mucin influence on microbial growth was assessed for each strain by evaluating the growth in presence of Type III porcine gastric mucin (Sigma Aldrich, St Louis, MO, United States). Lb. plantarum cells growth on MRS broth supplemented with 1 mg/ml of Type III porcine gastric mucin, was monitored at OD₆₀₀ using an EnSpire^® multimode plate reader (PerkinElmer, Waltham, MA, United States). The plate reader was run in discontinuous mode, with absorbance readings performed in 60-min intervals and preceded by 30 s shaking at medium speed. Cultures were grown in three biologically independent replicates and the resulting data were expressed as mean ± SEM.