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Hydrophobic determinants of ?-defensin bactericidal activity.

ABSTRACT: Mammalian ?-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 ?-defensins depend in part on electropositive charge and on hydrophobic amino acids that enable membrane disruption by interactions with phospholipid acyl chains. Alignment of ?-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 ?-defensin 4 (Crp-4) and rhesus myeloid ?-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 ?-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 ?-defensin residues and negative charge on bacteria precede interactions between critical hydrophobic residue positions that mediate membrane disruption and bacterial cell killing.

SUBMITTER: Tai KP

PROVIDER: S-EPMC4019156 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Hydrophobic determinants of α-defensin bactericidal activity.

Tai Kenneth P KP Le Valerie V VV Selsted Michael E ME Ouellette André J AJ

Infection and immunity 20140310 6

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)- ...[more]

PMID: 24614658

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Hydrophobic determinants of ?-defensin bactericidal activity.

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Hydrophobic determinants of α-defensin bactericidal activity.

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