Finally, vapX was amplified from 86-028NP genomic DNA and ligated to the SacI/XhoI-cut pET24b expression vector, resulting in VapX with a C-terminal polyhistidine tag in pDD902. To overexpress each protein for purification, pDD689, pDD791, and pDD902 were grown to logarithmic phase in BL21(DE3) and induced for 3 hours with 1 mM IPTG. Protein isolation Fludarabine from induced cells was performed with the MagneHis protein purification system (Promega, Madison, WI USA) according to the manufacturer’s instructions. NTHi growth dynamics

To compare growth dynamics, the 86-028NP wild type strain and the ΔvapBC-1, ΔvapXD, and ΔvapBC-1 ΔvapXD mutants were re-suspended from chocolate agar plates grown for 18 hours at 37°C in 5% CO2 into fresh sBHI broth to

an OD600 of ~0.1, then 200 microliters of each re-suspension was placed in triplicate into a sterile nontreated flat-bottomed 96 well plate (#351172, BD Biosciences, Bedford, MA, USA). Empty wells were filled with 200 microliters of sterile water to decrease evaporation, and the plate was covered with sterile gas permeable sealing film (#9123-6100, USA Scientific, Ocala, FL, USA). The plate was incubated for 11 hours with shaking at 35°C in a Multiskan FC spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), and the OD595 was read hourly. Two biological replicates and three technical replicates were performed and analyzed by one-way analysis of variance Everolimus in vivo (ANOVA) for independent

samples. Transmission electron microscopy (TEM) of NTHi strains co-cultured with EpiAirway Selleck Crenigacestat tissues Primary human Dehydratase respiratory epithelial tissues grown at the ALI, the EpiAirway model (MatTek #AIR-100-ABF, Ashland, MA USA) was used for co-culture with NTHi [32]. Each 0.6 cm2 tissue was fed basally by 1 ml of the proprietary antibiotic-free maintenance medium, AIR-100-MM-ABF (MM) and cultured at 37°C with 5% CO2. Each insert was washed daily with 200 μl of pre-warmed Dulbecco’s PBS (D-PBS) with calcium and magnesium and the basal MM was renewed daily. NTHi strains were grown overnight on chocolate agar plates at 37°C with 5% CO2. Bacteria were then suspended in D-PBS to an OD600 of ~0.2, and diluted to the desired inoculum. The tissues were inoculated apically with ~1.0 × 107 colony forming units (CFU) in ~25 microliters per insert with the 86-028NP parent strain or the ΔvapBC-1, ΔvapXD, and ΔvapBC-1 ΔvapXD mutants. On day 5 after infection, the tissues were fixed with 1.25% glutaraldehyde and 2.0% paraformaldehyde in 100 mM sodium cacodylate buffer (pH 7.2) for 24 hours.