The molecular determinants of human alpha-defensins' biological functions

Human alpha-defensins are cationic peptides with potent antitoxic, antimicrobial and chemotactic activities in the innate and adaptive immunity. Bacillus anthracis produces a lethal factor [LF], which is a potent toxin, capable of cell lysis. The molecular mechanism of the inhibition of LF by human alpha-defensins and the bacterial strain selectivity are not fully understood. Also, the pore-formation model that relates the cationic property to the antibacterial activity of human alpha-defensins cannot fully explain the bacterial strain selectivity. The aims of this dissertation were: (a) to elucidate the molecular determinants and the nature of the inhibition of LF by two well-studied alpha-defensins, human neutrophil peptide 1 [HNP1] and human defensin 5 [HD5], and (b) to explore the mechanism of bactericidal activities of HNP1 and HD5 against Staphylococcus aureus [S. aureus]. We hypothesized that: (a) the nature of the contact residues within these peptides influences the inhibitory activity against LF and the antibacterial effect against S. aureus, and (b) the quaternary structures of HNP1 and HD5 are crucial to the inhibition of LF and their antibacterial effect against S. aureus. We used alanine-scan mutagenesis of HNP1 and HD5 followed by functional assays to identify residues that contributed to LF inhibition and bactericidal activity against S. aureus. The results identified that tryptophane 26 [Trp26] in HNP1and leucine 29 [Leu29] in HD5 were the essential residues for the inhibition of LF and the bactericidal activity against S. aureus. Also, the results demonstrated that the hydrophobicity of HNP1 and HD5 as well as the dimerization and/or oligomerization were prerequisite to the LF inhibition and bactericidal activity against S. aureus. We concluded that Trp26 and Leu29 in HNP1 and HD5, respectively, as well as the hydrophobicity and self-association were the molecular determinants for LF inhibition and the bactericidal activity against S. aureus. The HNP1 and HD5 analogs are potentially useful tools for future studies towards the development of new anti-toxins. Finally, the data can potentially lead researchers to the discovery of unknown biological activities of alpha-defensins.