Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4(red)) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4(ox)) and Crp4(red) can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4(red), the bactericidal activities of Crp4(ox) and Crp4(red), Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4(red) and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4(ox) formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4(red) targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4(red) is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4(ox) and Crp4(red).

Automated summary

This study reveals that the potent bactericidal activity of Crp4(red) is derived from its hydrophobicity, and its bactericidal mechanism involves the disruption of bacterial membrane. Additionally, Crp4(ox) is found to form electrostatic interactions with the bacterial membrane surface, although it lacks bactericidal activity.