Non-typeable Haemophilus influenzae P5 binds C4b-binding protein promoting serum resistance

Abstract

Abstract Haemophilus influenzae is a Gram-negative bacterium considered as a commensal in pre-school children, mainly giving rise to infections locally in the upper and lower respiratory tract. Historically invasive disease has been caused by encapsulated strains, mainly H. influenzae serotype b (Hib). However, since introduction of the Hib vaccine, there has been an increased incidence of invasive disease with unencapsulated non-typeable H. influenzae (NTHi). Since NTHi lacks a capsule they must rely on other mechanisms for serum resistance, including binding of complement regulators. We have previously demonstrated that binding of C4b-binding protein (C4BP) is important for NTHi-dependent complement evasion. Here we identified outer membrane protein 5 (P5) as the C4BP-binding protein in NTHi based on an outer membrane proteomic pull down-assay. NTHi P5 has previously been found to bind factor H, an inhibitor of the alternative complement pathway. We found that NTHi grown in nasopharyngeal-simulated conditions has a lower surface expression of P5. This was also corresponding to decreased serum resistance. Importantly, we found that P5-deficient NTHi mutants showed significantly decreased C4BP binding and serum resistance. Additionally, surface expression of P5 on Escherichia coli conferred increased serum resistance and C4BP binding. Our results further highlights P5 as an important protein for protecting NTHi against complement-mediated killing.