Results of ureC were normalized with gyrA, a gene that is constitutively expressed . Transcription of ureC in media plus sputum was 3.32 ± 0.066 (mean ± standard deviation) fold greater than transcription of ureC in media alone (1.0 ± 0.223). We conclude that transcription of ureC is up regulated when H. influenzae grows in media with added human sputum compared to growth in laboratory media alone. Human antibody responses To determine whether urease was expressed by H. influenzae selleck kinase inhibitor during infection
of the human respiratory tract, 18 serum pairs from patients who experienced exacerbations due to H. influenzae were assayed for the development of antibody to purified recombinant urease following exacerbation. The cutoff value for a significant percentage change between pre-exacerbation
this website and post-exacerbation serum IgG levels was determined as previously described [41–44]. Eight control pairs of serum samples obtained 2 months apart (the same time interval for the experimental samples) from adults with COPD who were clinically stable and who had negative sputum cultures for H. influenzae were subjected Fedratinib concentration to ELISA with the purified recombinant urease. The % change in OD450 values between the paired control samples was calculated. These paired control serum samples demonstrated a 3.36% ± 6.01 (mean ± SD) change when tested with urease. A change in OD of 9.37% represented the upper limit of the 99% confidence interval isometheptene for the control samples. Therefore, any increase in value from pre to post exacerbation serum pairs of ≤ 9.37% was regarded as a significant change. A significant increase of serum IgG antibodies to urease was seen in 7 of 18 serum pairs (Figure 9).
We conclude that H. influenzae expresses urease during infection of the human respiratory tract and is a target of human serum antibodies in adults with COPD. Figure 9 Human antibody response to urease. Results of ELISAs measuring serum IgG to purified recombinant urease C in serum samples from adults with COPD who experienced exacerbations due to H. influenzae. Patient numbers (N = 18) are noted on the X-axis. The per cent changes from pre exacerbation to post exacerbation are shown on the Y-axis. The cutoff value (dotted line) for a significant increase in antibody level was determined by averaging the difference between 8 control pairs of sera from patients who had negative sputum cultures and were clinically stable (see text). Susceptibility of H. influenzae to acid conditions The ability of wild type and urease mutant to survive exposure to acid was investigated in the presence and absence of urea. Incubation of H. influenzae at pH 4 in the absence of urea, resulted in ~35% survival of wild type and mutant strains. However, in the presence of either 50 mM or 100 mM urea, survival of the wild type strain increased whereas no change in survival was observed in the urease C mutant or the urease operon mutant (Figure 10).