Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Antibiotic-resistant bacterial pathogens threaten public health. Because many antibiotics target specific bacterial enzymes or reactions, corresponding genes may mutate under selection and lead to antibiotic resistance. Accordingly, antimicrobials that selectively target overall microbial cell integrity may offer alternative approaches to therapeutic design. Naturally occurring mammalian α- and θ-defensins are potent, non-toxic microbicides that may be useful for treating infections by antibiotic-resistant pathogens because certain defensin peptides disrupt bacterial, but not mammalian, cell membranes. To test this concept, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including vancomycin heteroresistant strains, and ciprofloxacin-resistant Pseudomonas aeruginosa (Cip(R)-PA) were tested for sensitivity to α-defensins Crp-4, RMAD-4 and HNPs 1-3, and to RTD-1, macaque θ-defensin-1. In vitro, 3 µM Crp-4, RMAD-4 and RTD-1 reduced MRSA cell survival by 99%, regardless of vancomycin susceptibility. For PA clinical isolates that differ in fluoroquinolone resistance and virulence phenotype, peptide efficacy was independent of strain ciprofloxacin resistance, site of isolation or virulence factor expression. Thus, Crp-4, RMAD-4 and RTD-1 are effective in vitro antimicrobials against clinical isolates of MRSA and Cip(R)-PA, perhaps providing templates for development of α- and θ-defensin-based microbicides against antibiotic resistant or virulent infectious agents.

Hydrophobic determinants of α-defensin bactericidal activity.

Mammalian α-defensins are approximately 4- to 5-kDa broad-spectrum antimicrobial peptides and abundant granule constituents of neutrophils and small intestinal Paneth cells. The bactericidal activities of amphipathic α-defensins depend in part on electropositive charge and on hydrophobic amino acids that enable membrane disruption by interactions with phospholipid acyl chains. Alignment of α-defensin primary structures identified conserved hydrophobic residues in the loop formed by the Cys(III)-Cys(V) disulfide bond, and we have studied their role by testing the effects of mutagenesis on bactericidal activities. Mouse α-defensin 4 (Crp-4) and rhesus myeloid α-defensin 4 (RMAD-4) were selected for these studies, because they are highly bactericidal in vitro and have the same overall electropositive charge. Elimination of hydrophobicity by site-directed mutagenesis at those positions in Crp-4 attenuated bactericidal activity markedly. In contrast to native Crp-4, the (I23/F25/L26/G)-Crp-4 variant lacked bactericidal activity against Salmonella enterica serovar Typhimurium and did not permeabilize Escherichia coli ML35 cells as a result of removing aliphatic side chains by Gly substitutions. Ala replacements in (I23/F25/L26/A)-Crp-4 restored activity, evidence that hydrophobicity contributed by Ala methyl R-groups was sufficient for activity. In macaques, neutrophil α-defensin RMAD-6 is identical to RMAD-4, except for a F28S difference, and (F28S)-RMAD-4 mutagenesis attenuated RMAD-4 bactericidal activity and E. coli permeabilization. Interestingly, (R31/32D)-Crp-4 lacks activity in these assays despite the presence of the Ile23, Phe25, and Leu26 hydrophobic patch. We infer that electrostatic interactions between cationic α-defensin residues and negative charge on bacteria precede interactions between critical hydrophobic residue positions that mediate membrane disruption and bacterial cell killing.