Role of Complement in Defense of the Middle Ear Revealed by Restoring the Virulence of NontypeableHaemophilus influenzae siaBMutants

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Abstract
Nontypeable (NT)Haemophilus influenzaeis an important cause of otitis media in children. We have shown previously that NTH. influenzaemutants defective in their ability to sialylate lipopolysaccharide (LPS), calledsiaBmutants, 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 NTH. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulentsiaBmutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused bysiaBmutants in CoVF-treated animals coincided with reappearance of C3. Wild-type strains were more resistant to direct complement-mediated killing than theirsiaBmutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NTH. influenzaestrain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding tosiaBmutants. LPS sialylation had a more profound impact on the amount of alternative-pathway-mediated C3 binding (∼5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an ∼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 NTH. influenzaestrains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NTH. influenzaeinfections and specifically EOM.