Structural requirements for proinflammatory activity of porin P2 Loop 7 from Haemophilus influenzae.
Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children, characterized 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. Therefore, sequence-activity relationships and key residues were identified by elongating sequence to different extents, designing cyclic peptides, and performing an alanine scan of L7. The ability of mutant peptides to induce activation of signal transduction pathways and release of TNF-alpha 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. This work provides insights into essential molecular details of P2 that may impact on the pathogenesis of Hib infections where interruption of the signaling cascade could represent an attractive therapeutic strategy.
Department of Experimental Medicine, II University of Naples, Via De Crecchio 7, 80138 Naples, Italy.
Stefania
S
Galdiero
Mariateresa
M
Vitiello
Pietro
P
Amodeo
Marina
M
D'Isanto
Marco
M
Cantisani
Carlo
C
Pedone
Massimiliano
M
Galdiero
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D003198:Computer Simulation; D006193:Haemophilus influenzae / Q000737:chemistry*; D006801:Humans; D018836:Inflammation Mediators / Q000494:pharmacology*; D015850:Interleukin-6 / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020298:U937 Cells
D001426:Bacterial Proteins; D018836:Inflammation Mediators; D015850:Interleukin-6; D018272:Porins; D014409:Tumor Necrosis Factor-alpha; C451502:ompP2 protein, Haemophilus influenzae; D048369:MAP Kinase Kinase 1