Hepes

The PDCoV strain-HNZK-04 (GenBank accession number MH708124) was isolated and identified by the laboratory and propagated in porcine kidney proximal tubular epithelial cell line (LLC-PK1, ATCC CL-101). LLC-PK1 cells were cultured at 37°C in 5% CO₂ minimum essential medium (MEM, Solarbio) containing 8% fetal bovine serum (FBS, Gibco), 1% HEPES (Solarbio) and 1% MEM-nonessential amino acids (NEAA, Solarbio). Passage 34 (P34) of PDCoV HNZK-04 was cultured in MEM supplemented with 1% HEPES and 3 μg/mL trypsin (Sigma).

INS-1 cells were obtained from Bioleaf Biotech (Shanghai, China). Cells were cultured in RPMI 1640 (Gibco, CA, USA) containing 11.1 mM glucose, 10% (v/v) fetal bovine serum (FBS) (10099141, Gibco, Australia), 50 µM β-mercaptoethanol (M3148, Sigma), 10 mM HEPES (H1090, Solarbio), 2 mM L-glutamine (G0200, Solarbio), 1 mM sodium pyruvate (SP0100, Solarbio), 100 U/ml penicillin (P1400, Solarbio), and 100 µg/ml streptomycin(P1400, Solarbio), as described previously (Jiao et al., 2018 (link)). Human embryonic kidney 293 (HEK-293) cells were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Dulbecco’s modified essential media (10564029, DMEM) (Gibco) supplemented with 10% (v/v) FBS (10099141, Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin (P1400, Solarbio) (Zhang et al., 2018 (link)). The cells were maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO₂.

Bovine mammary epithelial cells (BMECs) were provided by Animal Clinical Laboratory of Hebei Agricultural University (Baoding, China) and grown in Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (DMEM/F12, Gibco, Grand Island, USA) supplemented with 15% FBS (Gibco), 0.1% hydrocortisone (Sigma-Aldrich, MO, USA), 100 U/mL penicillin-streptomycin (Solarbio, Beijing, China) and 0.025 M HEPES (Solarbio) at 37℃ with 5% CO₂. When cells were 70–80% confluence, they were treated as follows in six-well plates: the CON group (DMEM/F12), the ECOL group (10⁷ CFU/mL E. coli), pretreated with L. casei 03 (10⁴, 10⁵ and 10⁶ CFU/mL) for 3 h before the addition of E. coli, the LC group (10⁶ CFU/mL L. casei 03). Eight hours after E. coli (10⁷ CFU/mL) infection, cells were washed with PBS and collected for subsequent assays.

Splenocytes were harvested from the BALB/c mice. Primary CD3⁺ T cells, which included CD4⁺ T cells and CD8⁺ T cells, were purified from the splenocytes using the mouse CD3 T cell isolation kit (BioLegend) and were cultured at 10⁶/mL in RPMI-1640 medium (HyClone) supplemented with 10% FBS (Gibco), HEPES (10 mM, Solarbio), sodium pyruvate (1 mM, GENOM), 1× non-essential amino acids (Gibco), β-mercaptoethanol (50 μM, Sigma) and 100 IU/mL penicillin and streptomycin (10 mg/mL, Gibco). T cells were stimulated under the condition of anti-mouse CD3 antibody (1 μg/mL, BioLegend) and anti-mouse CD28 antibody (2 μg/mL, BioLegend). 48 h after T cells stimulation, T cells were transduced with retroviral supernatants from the Plat-E cell line in the presence of polybrene (6 μg/mL, Yeasen) by centrifugal infection. T cells were analyzed by flow cytometry 2 days after transduction and were used for further experiments.

The permeability of OaBac5mini to E. coli ATCC 25922 cell membranes was determined by N-Phenyl-1-naphthylamine (NPN, sigma, India), which is a fluorescent dye that only can pass through the damaged cell membrane and exhibit an increase of fluorescence. The NPN uptake assay was conducted following the instructions of Helander and Mattila (25 (link)) with minor modifications. In brief, E. coli. ATCC 25922 in logarithm growth period was centrifuged and resuspended with N′-a-hydroxythylpiperazine-N′-ethanesulfanic acid (HEPES, Solarbio, China, 5 mM, pH 7.2–7.4). NPN (10 μM final concentration, diluted with 5 mM HEPES) containing OaBac5mini (25, 50, 100, and 200 μg/ml final concentrations), PMB (positive control, 50 μg/ml final concentrations) or PBS (negative control) were mixed with an equal volume of prepared bacteria solution and were added into a 96-well plate. Fluorescence units were monitored immediately using INFINITE 200 PRO (Infinite^® 2000, TECAN, Austria) at the excitation wavelength of 350 nm and emission wavelength of 420 nm for 100 min at 5 min intervals. Experiments were performed in biological triplicate.