Sandra Jonsson

Background: Regulation of transcription is essential for the pathoadaptation of non-typeable Haemophilus influenzae (NTHi). We identified fatty acid biosynthesis repressor (FabR) as a previously unrecognized transcriptional regulator in NTHi through its binding to the promoter region of ompP5 and investigated its contribution to bacterial virulence and physiology. Methods: A fabR deletion mutant (ΔfabR) was generated in NTHi strain 3655. The mutant was compared with the wild-type strain in assays of host cell adherence, serum resistance, immunoglobulin G (IgG) deposition, vitronectin binding, murine nasopharyngeal colonization, protein expression, membrane fatty acid composition, and metabolic activity. Metabolic activity was also evaluated in ΔfabR mutants generated in additional NTHi strains. Results: Deletion of fabR reduced host cell adherence, increased serum sensitivity, enhanced IgG deposition, and decreased vitronectin binding compared with the wild-type strain. The ΔfabR mutant also exhibited impaired colonization of the murine nasopharynx. Proteomic analyses demonstrated altered expression of metabolic proteins, accompanied by an increased unsaturated-to-saturated fatty acid ratio. Furthermore, ΔfabR mutants from multiple NTHi strains showed increased metabolic activity, indicating a conserved role for FabR in metabolic regulation. Conclusions: FabR is a novel transcriptional regulator that may contribute to NTHi virulence. By modulating bacterial metabolism and membrane lipid composition, FabR may influence outer membrane architecture, thereby affecting host interaction, immune evasion, and colonization.

Acquisition of complement regulators is a virulence strategy used by nontypeable Haemophilus influenzae (NTHi) to evade complement-mediated killing by the host. The major outer membrane protein of NTHi, P5, binds C4b-binding protein and factor H to promote bacterial serum resistance. We show that P5 also binds vitronectin, which inhibits the formation of the membrane attack complex at the terminal stage of the complement pathway. Heterologous surface expression of P5 variants from NTHi strains 3655, KR271, KR317, and P652 promoted vitronectin binding to the P5-expressing Escherichia coli. In contrast, deletion of P5 from the NTHi strains reduced vitronectin binding. Vitronectin acquisition conferred serum resistance to P5-expressing E. coli, but not to NTHi ΔompP5 mutants. Using site-directed mutagenesis, extracellular loop 2 of the P5 variants was identified as the binding site for vitronectin. In conclusion, our findings highlight P5 as a receptor for vitronectin that promotes NTHi serum resistance.

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative human pathogen that causes infections mainly in the upper and lower respiratory tract. The bacterium is associated with bronchitis and exacerbations in patients suffering from chronic obstructive pulmonary disease and frequently causes acute otitis media in preschool children. We have previously demonstrated that the binding of C4b binding protein (C4BP) is important for NTHi complement evasion. In this study, we identified outer membrane protein 5 (P5) of NTHi as a novel ligand of C4BP. Importantly, we observed significantly lower C4BP binding and decreased serum resistance in P5-deficient NTHi mutants. Surface expression of recombinant P5 on Escherichia coli conferred C4BP binding and consequently increased serum resistance. Moreover, P5 expression was positively correlated with C4BP binding in a series of clinical isolates. We revealed higher levels of P5 surface expression and consequently more C4BP binding in isolates from the lower respiratory tract of chronic obstructive pulmonary disease patients and tonsil specimens compared with isolates from the upper respiratory tract and the bloodstream (invasive strains). Our results highlight P5 as an important protein for protecting NTHi against complement-mediated killing. Key Points Exposure of P5 at the surface of NTHi positively correlates with C4BP binding. C4BP bound to the bacterial surface retains its complement inhibitory capacity. C4BP binding to P5 is important for NTHi serum resistance.