Introduction: The Porcine Myeloid Antibacterial Peptide (PMAP)-23 is a porcine host defence peptide with strong antibacterial activity against Gram-positive and Gram-negative bacteria, and fungi.Objective: PMAP-23 and truncated/mutated derivatives were tested for antibacterial and immunomodulatory activities to determine core elements of the peptide required for functionality. Methods: PMAP-23 and truncated and/or mutated derivatives were synthesized. Antibacterial activity against Gram positive and negative bacteria was determined using colony counting assays. Cytotoxicity was measured against red blood cells and epithelial cells. Peptide induced cytokine production of epithelial cells was determined by ELISA. LPS neutralization was measured using isothermal titration calorimetry and inhibition of LPS induced cytokine production by macrophages. The effect of peptides on phagocytosis was performed by measuring uptake of fluorescently labelled beads by porcine macrophages. Results: Truncation of the peptide did not lead to a strong reduction in antibacterial activity, but interestingly, all C-terminal truncated forms were strongly inhibited by salt addition, unlike the full length peptide or the two N-terminally truncated peptides. None of the peptides were hemolytic or toxic in concentrations up to 40 μM. Full length PMAP-23 induced IL-8 production in porcine epithelial cells, however, this activity was lost in all truncated peptides. None of the peptides bound LPS and subsequently did not inhibit LPS-induced cytokine production of monocytes. Finally, all PMAP-23 derived peptides reduced the uptake of beads by freshly isolated monocytes. Conclusion: PMAP-23 is mainly antibacterial with only limited immunomodulating capacity; the full length peptide is required for the full spectrum of activities.
Keywords: Antimicrobial peptide, innate immunity, defence, immunomodulation, LPS, alternative to antibiotics.