title: Hydrophobic determinants of α-defensin bactericidal activity.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Southern California Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California.
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Tai
authors.forename: Valerie V
authors.initials: VV
authors.lastname: Le
authors.forename: Michael E
authors.initials: ME
authors.lastname: Selsted
authors.forename: André J
authors.initials: AJ
authors.lastname: Ouellette
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001419:Bacteria / Q000187:drug effects*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D057927:Hydrophobic and Hydrophilic Interactions; D008253:Macaca mulatta; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000494:pharmacology; D023084:alpha-Defensins / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D011994:Recombinant Proteins; D023084:alpha-Defensins