Antibacterial activity and specificity of the six human α-defensins

We developed a kinetic, 96-well turbidimetric procedure that is capable of testing the antimicrobial properties of six human alpha-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 (LD(50)s)l LD(90)s, LD(99)s, and LD(99.9)s obtained from colony counts. On the basis of their respective vLD(90)s and vLD(99)s, the relative potencies of human myeloid alpha-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 alpha-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 alpha-defensins and raised intriguing questions about the in vivo function(s) of HD6.