Engineering of Porin P2 loop 7 from Haemophilus influenzae: structural requirements for proinflammatory activity

Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children, characterised by inflammation mainly mediated by cytokines and chemokines. One of the most abundant components of the Hib outer membrane is the P2 porin, which has been shown to induce the release of several inflammatory cytokines. Synthetic peptides corresponding to loops L5, L6 and L7 activate JNK and p38 mitogen-activated protein kinase (MAPK) pathways, L7 being the most active peptide. The virulence of microrganisms is the result of complex interactions between the organism and its host. Signals leading to cellular activation are often generated at the cell surface after binding of receptors to their appropriate ligands. This study is the first to identify those amino acid sequences of porins that are likely to be involved in cytokine synthesis. Sequence-activity relationships were explored both by a point-mutation approach, based on Ala-scanning of the whole L7 sequence, and by elongating L7 sequence to different extents either at N-terminus only (N1 and N2), or at C-terminus only (C1), or at both termini (CKR). In addition, a cyclic form, closed by a disulfide bridge between two Cys residues introduced at the ends of an elongated L7 sequence, was also tested (CCC). The ability of mutant peptides to induce activation of signal transduction pathways and release of TNF-α and IL-6 has been determined, and, in conjunction with CD spectra, bioinformatics analysis and molecular dynamics data, showed that 6 out of 8 amino acids contribute significantly to the overall activity. Molecular dynamics showed that L7 modifications increased loop rigidity and helicity after Gly6 mutation, thus providing a possible structural explanation for observed loss of bioactivity. These results clearly show that the octapeptide L7 is the most active derivative among loops of protein P2. This work provides insights into essential molecular details of P2 that may impact on the pathogenesis of Hib infections where interruption of the signalling cascade could represent an attractive therapeutic strategy.