The periplasmic disulfide oxidoreductase DsbA contributes to Haemophilus influenzae pathogenesis

Abstract

Haemophilus influenzae is an obligate human pathogen that persistently colonizes the nasopharynx and causes disease when it invades the bloodstream, lungs, or middle ear. Proteins that mediate critical interactions with the host during invasive disease are likely to be secreted. Many secreted proteins require addition of disulfide bonds by the DsbA disulfide oxidoreductase for activity or stability. In this study, we evaluated the role in H. influenzae pathogenesis of DsbA, as well as HbpA, a substrate of DsbA. Mutants of H. influenzae Rd and type b strain Eagan having nonpolar deletions of dsbA were attenuated for bacteremia in animal models, and complemented strains exhibited virulence equivalent to that of the parental strains. Comparison of predicted secreted proteins in H. influenzae to known DsbA substrates in other species revealed several proteins that could contribute to the role of dsbA in virulence. One candidate, the heme transport protein, HbpA, was examined because of the importance of exogenous heme for aerobic growth of H. influenzae. The presence of a dsbA-dependent disulfide bond in HbpA was verified by an alkylation protection assay, and HbpA was less abundant in a dsbA mutant. The hbpA mutant exhibited reduced bacteremia in the mouse model, and complementation restored its in vivo phenotype to that of the parental strain. These results indicate that dsbA is required in vivo and that HbpA and additional DsbA-dependent factors are likely to participate in H. influenzae pathogenesis.

FIG. 1.

Effect of dsbA mutation on survival of H. influenzae in the mouse model of bacteremia. Strains were inoculated i.p. into mice, and bacteremia was assessed after 24 h. The symbols indicate data for individual animals, and the dashed lines indicate the averages. The lower limit of detection (LLD) was 500 CFU/ml. Values less than the lower limit of detection indicate that bacteremia was not detected. (A) Single-strain infection. The asterisk indicates that the P value is 0.039 (t test). (B) Coinfections with the experimental strains indicated and reference strain RdlacZ. The competitive index is the ratio of the LacZ⁻ experimental strain to LacZ⁺ reference strain RdlacZ. The asterisk indicates that the P value is <0.001 (ANOVA with Bonferroni's multiple-comparison test).

FIG. 2.

Effects of dsbA mutation on HbpA protein levels and thiol redox state. (A) Detection of HbpA levels in DsbA⁺ and DsbA⁻ strains. Whole-cell lysates of duplicate cultures of RhbpAC (complemented hbpA deletion mutant carrying hbpA-HA in the xyl locus) (lanes 1 and 2) and RhbpACΔdsbA (RhbpAC with dsbA deletion) (lanes 3 and 4) were resolved by 8% SDS-PAGE under reducing conditions and detected by anti-HA Western blotting (left panel). Equal sample concentrations were verified by Coomassie blue staining (right panel). (B) Differential modification of thiols on HbpA in the dsbA mutant compared to the wild type. Spheroplasts were prepared from log-phase cultures, resolved by 8% SDS-PAGE under nonreducing conditions, and detected by anti-HA Western blotting. The strains were the same as those used for panel A and were treated (+) or not treated (−) with 5 mM MPB as indicated. The arrows indicate the oxidized form of HbpA (ox), the reduced form of HbpA (red), and the reduced form of HbpA with thiols modified with MPB (red + MPB).

FIG. 3.

Effect of hbpA mutation on survival of H. influenzae in the mouse model of bacteremia. Strains were inoculated i.p. into mice, and bacteremia was assessed after 24 h. The symbols indicate data for individual animals, and the dashed lines indicate the averages. The lower limit of detection (LLD) was 500 CFU/ml. Values less than the lower limit of detection indicate that bacteremia was not detected. (A) Single-strain infection. (B) Coinfections with the experimental strains indicated and reference strain RdlacZ. The competitive index is the ratio of the LacZ⁻ experimental strain to LacZ⁺ reference strain RdlacZ. The asterisk indicates that the P value is <0.001.

FIG. 4.

Effect of dsbA mutation on the virulence of H. influenzae type b in infant rats. Strains were inoculated i.p. into 5-day-old infant rats. The symbols indicate data for individual animals, and the dashed lines indicate the averages. An asterisks indicates that the P value is <0.001. The lower limit of detection (LLD) was 500 CFU/ml. Values less than the lower limit of detection indicate that bacteremia was not detected.