Characterization of igaB, a second immunoglobulin A1 protease gene in nontypeable Haemophilus influenzae

Abstract

Nontypeable Haemophilus influenzae is an important respiratory pathogen, causing otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Immunoglobulin A1 (IgA1) protease is a well-described protein and potential virulence factor in this organism as well as other respiratory pathogens. IgA1 proteases cleave human IgA1, are involved in invasion, and display immunomodulatory effects. We have identified a second IgA1 protease gene, igaB, in H. influenzae that is present in addition to the previously described IgA1 protease gene, iga. Reverse transcriptase PCR and IgA1 protease assays indicated that the gene is transcribed, expressed, and enzymatically active in H. influenzae. The product of this gene is a type 2 IgA1 protease with homology to the iga gene of Neisseria species. Mutants that were deficient in iga, igaB, and both genes were constructed in H. influenzae strain 11P6H, a strain isolated from a patient with COPD who was experiencing an exacerbation. Analysis of these mutants indicated that igaB is the primary mediator of IgA1 protease activity in this strain. IgA1 protease activity assays on 20 clinical isolates indicated that the igaB gene is associated with increased levels of IgA1 protease activity. Approximately one-third of 297 strains of H. influenzae of diverse clinical and geographic origin contained igaB. Significant differences in the prevalence of igaB were observed among isolates from different sites of isolation (sputum > middle ear > nasopharynx). These data support the hypothesis that the newly discovered igaB gene is a potential virulence factor in nontypeable H. influenzae.

FIG. 1.

Diagram of the igaB locus. The upstream and downstream genes are labeled based on designations in the sequenced strain KW20 Rd. Shaded boxes of igaB denote regions of high homology to neisserial iga genes. Open boxes labeled “Repeats” indicate the positions of 423-bp unique tandem repeats in the igaB gene. Asterisks indicate positions of four direct repeats 13 bp in length found in the context of the larger tandem repeats. Also shown are the conserved domains of neisserial IgA1 protease proteins as they relate to igaB. S, 25-amino-acid signal sequence; protease, 961-amino-acid secreted IgA1 protease domain; P, 32-amino-acid short peptide conserved among IgA1 proteases; alpha protein, 476-amino-acid region, including repeated sequence, potentially corresponding to the alpha protein of neisserial IgA1 proteases; beta-core, 393-amino-acid β-core (autosecretory domain).

FIG. 2.

Results of RT-PCR of IgA1 protease genes from H. influenzae strain 11P6H. (A) RT-PCR with iga primers; (C) RT-PCR with igaB primers; (B and D) RT-PCR with ompP2 primers for positive control. Lanes 1 and 2, purified 11P6H RNA template; lanes +, H. influenzae strain 11P6H genomic DNA template for positive control; lanes −, water template for negative control. The bottom panels (E to H) depict the same templates and primers corresponding to upper panels amplified with Taq polymerase only, confirming the absence of DNA contamination.

FIG. 3.

Southern blot assays of genomic DNA of H. influenzae strain 11P6H and mutants. Lanes 1, strain 11P6H; lanes 2, Δiga mutant; lanes 3, ΔigaB mutant; lanes 4, Δiga ΔigaB double mutant. (A) iga probe; (B) igaB probe; (C) spectinomycin cassette probe; (D) chloramphenicol cassette probe. Molecular size markers are labeled in kilobases.

FIG. 4.

Results of RT-PCR of IgA1 protease mutants. Lanes 1, Δiga mutant RNA template; lanes 2, ΔigaB mutant RNA template; lanes 3, Δiga ΔigaB double mutant RNA template; lanes +, H. influenzae strain 11P6H genomic DNA template for positive control; lanes −, water template for negative control. (A) RT-PCR with iga primers; (B) RT-PCR with igaB primers; (C) RT-PCR with ompP2 primers for positive control. The bottom panels (D to F) depict the same templates and primers corresponding to upper panels amplified with Taq polymerase only, confirming the absence of DNA contamination.

FIG. 5.

RT-PCR of genes upstream and downstream of igaB. Lanes 1, H. influenzae strain 11P6H RNA template; lanes 2, ΔigaB mutant RNA template; lanes 3, Δiga ΔigaB double mutant RNA template; lanes +, H. influenzae strain 11P6H genomic DNA template for positive control; lanes −, water template for negative control. (A) RT-PCR with HI0185 primers; (B) RT-PCR with HI0184 primers; (C) RT-PCR with HI0164 primers; (D) RT-PCR with HI0166 primers; (E) RT-PCR with ompP2 primers for positive control. The bottom panels (F to J) depict the same templates and primers corresponding to upper panels amplified with Taq polymerase only, confirming the absence of DNA contamination.

FIG. 6.

Coomassie-stained SDS-polyacrylamide gel of concentrated culture supernatants from cultures in chemically defined media. Lane A, H. influenzae strain 11P6H; lane B, Δiga mutant; lane C, ΔigaB mutant; lane D, Δiga ΔigaB double mutant. Molecular mass markers are labeled in kilodaltons. Arrows denote IgaB-secreted protease bands.

FIG. 7.

Western blot assays of IgA digests. (A) Lanes contain purified human IgA1 digested with culture supernatants of H. influenzae or PBS in place of the supernatant, as follows: strain 11P6H (lane 1); Δiga mutant (lane 2); ΔigaB mutant (lane 3); Δiga ΔigaB double mutant (lane 4); type 1 IgA1 protease control, H. influenzae strain KW20 Rd (lane 5); type 2 IgA1 protease control, H. influenzae biogroup aegyptius strain BPF 17 (lane 6); sterile BHI broth (lane 7); and PBS (lane 8). (B) Lanes contain purified human IgA2 digested with culture supernatants of H. influenzae or broth or PBS in place of the supernatant, as follows: strain 11P6H (lane 1); Δiga mutant (lane 2); ΔigaB mutant (lane 3); Δiga ΔigaB double mutant (lane 4); sterile BHI broth (lane 5); PBS (lane 6); and PBS (unincubated) (lane 7). Molecular mass markers are labeled in kilodaltons.

FIG. 8.

IgA1 protease activity assays. Lines indicate LOESS plots of the IgA1 protease activity as a percentage of control of culture supernatants of H. influenzae strain 11P6H and the mutant strains as noted. x axis, the degree of twofold dilution of the original samples; y axis, activity as a percentage of maximal activity based on positive (IgA with sterile BHI broth) and negative (undiluted culture supernatants absent IgA) controls. Each data point is an average of eight replicates. Error bars represent the standard error for each dilution.

FIG. 9.

Agarose gel showing specificity of primers for PCR amplification of IgA1 protease genes. DNA from strain 11P6H and the mutant strains were used as the templates for PCRs as noted. Primers used in each PCR are noted at the top of the gel. Primers used for the amplification of iga were the “P” primers used by Vitovski et al. (62).

FIG. 10.

Percentage of strains containing igaB by site of infection. Chi-square P values are shown.

FIG. 11.

Results of IgA1 protease activity assays on 20 strains of H. influenzae from adults with COPD. y axis, the dilution factor required to produce 50% IgA1 protease activity from culture supernatants as determined by four replicates. The bars are the averages of two groups of 10 strains each with iga and igaB genes as noted. The difference between groups is significant (P = 0.04; t test). Error bars represent the standard errors for each group.