Role of complement in defense of the middle ear revealed by restoring the virulence of nontypeable Haemophilus influenzae siaB mutants

Abstract

Nontypeable (NT) Haemophilus influenzae is an important cause of otitis media in children. We have shown previously that NT H. influenzae mutants defective in their ability to sialylate lipopolysaccharide (LPS), called siaB mutants, show attenuated virulence in a chinchilla model of experimental otitis media (EOM). We show that complement is a key arm of host innate immunity against NT H. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulent siaB mutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused by siaB mutants in CoVF-treated animals coincided with reappearance of C3. Wild-type strains were more resistant to direct complement-mediated killing than their siaB mutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NT H. influenzae strain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding to siaB mutants. LPS sialylation had a more profound impact on the amount of alternative-pathway-mediated C3 binding ( approximately 5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an approximately 1.5-fold decrease in fluorescence intensity of C3 binding was seen with the serum-sensitive strain, where surface proteins predominantly bound C3. Differences in binding sites for C3 and C4 may account for variations in serum resistance between NT H. influenzae strains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NT H. influenzae infections and specifically EOM.

FIG. 1.

Detection of C3 α-chain in chinchillas following treatment with CoVF (CoVF+) to assess the extent and duration of complement depletion. pre, samples collected 24 h prior to CoVF administration; 375siaB CoVF−, control animal that did not receive CoVF.

FIG. 2.

Natural history of EOM due to 486 WT and 486siaB in control animals and chinchillas treated with CoVF. The proportion of animals with culture-positive MEF at the time points indicated in each group is represented by the percentage. Symbols: ▴, 486siaB with CoVF (n = 10); ▪, 486siaB without CoVF (n = 5); ⧫, 486 WT without CoVF (n = 2); •, 486 WT with CoVF (n = 2).

FIG. 3.

Natural history of EOM due to 375 WT and 375siaB in control animals and chinchillas treated with CoVF. The proportion of animals with culture-positive MEF at the time points indicated in each group is represented by the percentage.

FIG. 4.

Bactericidal activities of NHS against 375 WT and 375siaB and 486 WT and 486siaB. Percent survival with 95% confidence interval around the mean percentage is shown in the figures. (1), P = 0.0002 (percent survival of 375 WT compared to that of 375siaB at a 5% NHS concentration); (2), P = 0.0007 (percent survival of 486 WT compared to that of 486siaB at 3.5% NHS).

FIG. 5.

Factor H binding to 375 and 375siaB by flow cytometry. The shaded histogram represents binding to 375 WT, the solid line binding to 375siaB, and the broken line binding to the positive-control strain (N. gonorrhoeae strain 15253). The isotype control for strain 375 (no factor H in the reaction mixture) was identical to the histograms observed for that strain with factor H and has been omitted to simplify the illustration. One experiment representative of two separate experiments is shown. The x axis represents fluorescence on a log₁₀ scale, and the y axis the number of events (counts).

FIG. 6.

C4 and C3 binding to strains 375 WT and 486 WT (shaded histograms) and their siaB mutants (solid lines). Bacteria were incubated with NHS (20%, vol/vol, for strains 375 and 375siaB and 10%, vol/vol, for strains 486 and 486siaB) or with NHS chelated with 10 mM MgEGTA (20%, vol/vol, for all strains). Numbers beside each histogram represent geometric mean fluorescence. Values for the wild-type strains are indicated in shaded boxes. Controls (either no serum or heat-inactivated serum in the reaction mixture) yielded fluorescence values that lay within the first decade (<10) in every instance and have been omitted for simplicity. One experiment representative of two separate and comparable experiments is shown. Axes are described in the legend for Fig. 5.

FIG. 7.

C4b binding to strains 375 and 486 and their siaB mutants. Bacteria were incubated either with NHS (20%, vol/vol, for strains 375 and 375siaB and 10%, vol/vol, for strains 486 and 486siaB) or with NHS chelated with 10 mM MgEGTA (20%, vol/vol, for all strains). The upper blot shows samples not treated with methylamine (both amide and ester linkages intact), while the lower blot shows methylamine-treated samples, where ester linkages are disrupted by nucleophilic attack while amide linkages are unaffected. Coomassie-stained gels, where 10 μl of each sample was loaded per lane, were used as loading controls and are shown below each blot. The asterisks indicate binding of C4b α′-chain to an ∼5-kDa target. The arrow indicates the C4b α′-chain bound to an ∼55-kDa target. The bracket indicates the location of C4b-containing heterodimers (C5 convertases). The arrowhead in the lower blot indicates the 87-kDa C4b α′-chain that was ester linked to its targets and has been released by nucleophilic attack by methylamine. The C4 α-chain (95 kDa) and C4 β-chain (75 kDa) are indicated in the lane marked NHS alone (serum with no bacteria). The lanes marked HI serum represent bacteria incubated with heat-inactivated serum. The far left lane indicates the molecular mass marker.

FIG. 8.

iC3b binding to strains 375 WT and 486 WT and their siaB mutants. Bacteria were incubated either with NHS or with MgEGTA-NHS as described in the legend for Fig. 7. The upper blot shows samples not treated with methylamine (both amide and ester linkages intact), while the lower blot shows methylamine-treated samples, where ester linkages are disrupted by nucleophilic attack while amide linkages are unaffected. The asterisk indicates the iC3b α₁′-chain bound to an ∼5-kDa target. The arrow at ∼125 kDa indicates the iC3b α₁′-chain bound to an ∼57-kDa target. The arrowhead in the lower blot indicates the ∼68-kDa iC3b α₁′-chain that was ester linked to its targets and has been released by nucleophilic attack by methylamine. The lanes marked HI serum are controls using bacteria incubated with heat-inactivated serum. The same samples used for Fig. 7 were used in these experiments, and loading controls for these samples are illustrated in Fig. 7. Pure iC3b, iC3b made from fluid-phase C3b.