Gc agar

All bacterial strains used in this study are listed in Table 1. All Neisseria strains and Streptococcus pyogenes strains were grown on GC agar (Acumedia) containing Kellogg's supplement (Kellogg et al., 1963 (link)). Pseudomonas aeruginosa, Staphylococcus aureus, all Salmonella strains and the E. coli strains were grown on Luria agar (Acumedia). The Lactobacillus strains were grown on Rogosa agar (Oxoid). All aforementioned bacteria were cultured at 37°C and 5% CO₂ for 16–18 h before experimentation. The Helicobacter pylori strains were grown on Colombia blood agar (Acumedia) supplemented with 5% defibrinated horse blood and 5% inactivated horse serum (Håtunalab) for 3 days at 37°C under microaerophilic conditions (5% O₂, 10% CO₂). Before each experiment, the bacteria were washed once and resuspended in cell culture medium without serum that was specific to the cell line that was used.

The Neisseria meningitidis FAM20 serogroup C strain and mutant deficient in PilC1 have been described previously [24 (link), 51 (link)]. The Neisseria meningitidis JB515 serogroup W strain and Neisseria gonorrhoeae MS11 strain have been described previously [24 (link), 52 (link)]. The strains were grown on GC agar (Acumedia) supplemented with 1% Kelloggs’ for 18 h at 37°C in a 5% CO₂ environment. Antibiotics for selection of FAM20 mutant strains were used in following concentrations: tetracycline 1 μg/ml, kanamycin 50 μg/ml and spectinomycin 40 μg/ml.

Neisseria meningitidis serogroup C strain FAM20 and its pilE mutant (ΔpilE) have been previously described (Rahman et al., 1997 (link); Jones et al., 2009 (link); Engman et al., 2016 (link)). The strains were grown on GC agar (Acumedia) with 1% Kellogg’s supplements (Kellogg et al., 1968 (link)) at 37°C and 5% CO₂ for 16-18 h before experiments. Lactobacillus reuteri ATCC PTA5289 (de Klerk et al., 2016 (link)) was grown at 37°C 5% CO₂ on Rogosa (Oxoid) agar for 30-72 h and then in MRS broth for 16-18 h. Escherichia coli DH5α was grown on LA-plates and then in LB broth (Acumedia) at 37°C 5% CO₂ at 200 rpm shaking.
The human pharyngeal epithelial cell line FaDu (ATCC-HBT43) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM) and supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) at 37°C and 5% CO₂ in a humidified environment.