Vitox

Wild-type (WT) H. suis strain HS5cLP was grown for 48 h as described previously [29 (link)]. HS5cLPΔggt bacteria were grown under the same conditions as strain HS5cLP, except that the cultivation plates were supplemented with chloramphenicol (30 μg/mL) as described previously [20 (link)].
WT H. pylori strains SS1 and PMSS1 were grown on Columbia agar plates containing 5% (v/v) defibrinated sheep blood for 48–72 h at 37 °C under microaerobic conditions as described previously [29 (link)]. Subsequently, colonies were picked up and cultured in Brucella broth supplemented with Vitox (Oxoid) and 5% fetal calf serum (HyClone) on a rotational shaker under microaerobic conditions (16 h, 125 rpm). SS1Δggt and PMSS1Δggt strains were cultured under the same conditions as the corresponding WT strains on plates supplemented with kanamycin (25 μg/mL).

Escherichia coli JM109 (Table S1) was used for the expression of 6 × histidine‐tagged rFBA and derivatives. E. coli XL10‐Gold ultracompetent cells were used as a host strain for the construction of mutagenic plasmids. E. coli strains were grown at 37°C in Lysogeny Broth (LB) broth or on LB agar supplemented, where appropriate, with ampicillin (100 μg mL⁻¹), kanamycin (30 μg mL⁻¹) or erythromycin (200 μg mL⁻¹). Strains of Neisseria (Table S1) were grown at 37°C in air plus 5% CO₂ on chocolated horse blood agar (Oxoid), Brain Heart Infusion (BHI) agar or BHI broth supplemented with 1% Vitox (Oxoid) and kanamycin (50 μg mL⁻¹) or erythromycin (5 μg mL⁻¹) where appropriate.

Bacterial strains used are listed in supplementary Table S2. H. pylori was grown microaerophilic at 37 °C on GC agar or in Brucella broth supplemented with 1% haemoglobin, 10% horse blood, and 1% Vitox (OXOID). E. coli BL21(DE3) was grown at 37 °C in LB or on LB-agar. Liquid cultures were grown at 200 rpm. Where applicable, 50 μg/ml kanamycin or 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added.

The bacterial strains used in this study are listed in Table 1. Stock cultures were stored at − 80 °C in DMEM/F12 supplemented with 1% fetal calf serum (FCS), 5 mg mL⁻¹ insulin and 15% glycerol. For infection experiments, the stocks were plated on Chocolate Agar with Vitox (Oxoid, Wesel, Germany) and grown at 37 °C in 5% CO₂ atmosphere overnight. Single colonies of the overnight culture were picked and were dissolved and washed in phenol-red free DMEM/F12 with 1% FCS and 5 mg mL⁻¹ insulin and adjusted to an optical density of 0.1 at 600 nm (OD₆₀₀) which corresponded to approximately 1 × 10⁸ colony forming units (CFU) per mL. During the infection experiments, a multiplicity of infection (MOI) of 10 or 100 was used as described for the individual experiments. Bacterial growth was monitored throughout all experiments and in the presence and absence of inhibitors.Table 1

Bacterial strains

N. meningitidis	Serogroup		References
MC58	B	Wild type	[23 (link)]
MC58siaD−	Capsule-deficient mutant	Isogenic siaD mutant	[24 (link)]
WUE2120	C	Wild type	[25 ]
WUE2120siaD−	Capsule-deficient mutant	Isogenic siaD mutant	[24 (link)]

Gonococcal isolates were recovered from cryopreservation on chocolate agar plates (Laborclin, Parana, Brazil) and subcultured on the same medium. To induce resistance to cefixime, we subcultured a single colony on GC agar (Difco™, New Jersey, USA) supplemented with 1% Vitox (Oxoid™, Hampshire, UK) containing different concentrations of cefixime, starting from 0.002 mg/L. The colonies were subcultured on two GC plates: one containing the same concentration of cefixime and another with a higher concentration (1.5- to 2-fold each test). When the isolate grew on the higher-concentration plate, it was challenged to an even higher concentration of cefixime. All plates were incubated at 35 ± 1°C in 5% CO₂ for 18 to 24 h. In vitro induction was considered complete when all isolates achieved stable growth at a cefixime concentration above the resistance breakpoint, according to guidelines of the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) (>0.125 mg/L, http://brcast.org.br/, accessed in March 2021). After resistance induction, all isolates were confirmed as N. gonorrhoeae using a Vitek2 Compact instrument and stored at −80°C in casein-peptone soymeal-peptone broth containing 20% glycerol.

Neisseria meningitidis clinical isolates (electronic supplementary material, table S1) were grown on chocolate horse blood (Oxoid) at 37°C, in an atmosphere of 5% CO₂. Mutagenesis of N. meningitidis MC58 pilQ and porA was described previously [7 (link)]. To mutate pilE, chromosomal DNA extracted from N. meningitidis C311ΔpilE (kindly provided by Prof. C. Tang, University of Oxford, UK) was used to mutate MC58 by natural transformation and allelic exchange as described previously [64 (link)]. The MC58ΔlgtF strain used in this study was described previously [65 (link)]. For selection of mutants, meningococcal cells were cultured on Mueller–Hinton agar plates supplemented with 1% Vitox (Oxoid) and, where appropriate, with streptomycin and spectinomycin (100 μg ml⁻¹) or kanamycin (50 μg ml⁻¹). Invasion assays were performed as previously described [66 (link)].