Antibacterial Activity and Specificity of the Six Human α-Defensins

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Abstract
We developed a kinetic, 96-well turbidimetric procedure that is capable of testing the antimicrobial properties of six human α-defensins concurrently on a single microplate. The defensins were prepared by solid-phase peptide synthesis and tested against gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Enterobacter aerogenes and Escherichia coli). Analysis of the growth curves provided virtual lethal doses (vLDs) equivalent to conventional 50% lethal doses (LD50s), LD90s, LD99s, and LD99.9s obtained from colony counts. On the basis of their respective vLD90s and vLD99s, the relative potencies of human myeloid α-defensins against S. aureus were HNP2 > HNP1 > HNP3 > HNP4. In contrast, their relative potencies against E. coli and E. aerogenes were HNP4 > HNP2 > HNP1 = HNP3. HD5 was as effective as HNP2 against S. aureus and as effective as HNP4 against the gram-negative bacteria in our panel. HD6 showed little or no activity against any of the bacteria in our panel, including B. cereus, which was highly susceptible to the other five α-defensins. The assay described provides a quantitative, precise, and economical way to study the antimicrobial activities of host-defense peptides. Its use has clarified the relative potencies of human α-defensins and raised intriguing questions about the in vivo function(s) of HD6.