Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus

Abstract

Human α-defensin 5 (HD5, HD5ox to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and –positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys3—Cys31, Cys5—Cys20, Cys10—Cys30) were mutated to Ser or Ala residues were overexpressed in E. coli, purified and characterized. A hexa mutant peptide, HD5[Serhexa], where all six native Cys residues are replaced by Ser residues was also evaluated. Removal of a single native S—S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low-micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low-micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5ox against this Gram-positive bacterial strain. This observation supports the notion that the HD5ox mechanism of antibacterial action differs for Gram-negative and Gram-positive species (Wei, G.; de Leeuw, E., Pazgier, M., Yuan, W., Zou, G., Wang, J., Ericksen, B., Lu, W.-Y.; Lehrer, R. I.; Lu, W. (2009) J. Biol. Chem. 284, 29180-29192), and that the native disulfide array is a requirement for its activity against S. aureus.

Intracellular Targeting Mechanisms by Antimicrobial Peptides

“…The human ␣-defensin 5 (HD5) is able to traverse the outer membrane and impairs the inner membrane of E. coli (122,123). Peptide entering the cytosol will then induce multiple cellular damages, as observed with blebbing and clumping of cells.…”

Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs

“…Analogs where cysteines were replaced with serine [32] or α-amino butyric acid (Abu) [18] showed activity comparable to HD5 against E. coli but were inactive against S. aureus. The effect of deleting all the six cysteine residues (D5R) was examined.…”

“…In a recent report, an analog of HD5 with increased positive charge that does not form a dimer shows enhanced antibacterial activity [36]. Also, the linear forms of HNP-1 [37] as well as HD5 [32] do show activity against E. coli. Contacts between the amino acids in the dimers would not be present in the less structured forms.…”

“…Analogs where cysteine residues were replaced by serine or α-amino butyric acid were active against Escherichia coli but not Staphylococcus aureus [18,32]. Analogs of HD5 with less than three disulfide bridges and non-native connectivities exhibited antibacterial activity [32].…”

“…Analogs of HD5 with less than three disulfide bridges and non-native connectivities exhibited antibacterial activity [32].…”