10-¬µl wells were bored in a 9-cm2 plate of agarose, buffered with 10¬mM PIPES, pH 7.4,¬containing 5¬mM glucose, and seeded with 1¬ó6 mid-log phase cells. Five-¬µl aliquots of each peptide, dissolved in 0.01% HOAc at 10-300 ¬µg/ml, were added to each well. After incubation at 37¬¬C for 2¬h, the seeded agar was overlaid with molten agarose containing 6% trypticase soy broth (for bacteria) or Sabouraud dextrose broth (for fungi). Plates were incubated at 37¬¬C for 18-24 h, and antimicrobial activity was determined by measuring the diameter of clearing around each well. The microbicidal activities of each peptide were determined by incubating 2¬ó6 colony-forming units/ml with peptides (0.5-12 ¬µg/ml) in 50¬¬µl of low salt diluent, 10¬mM PIPES buffer containing 5¬mM glucose, pH 7.4,¬or the same diluent supplemented with 25-150 mM NaCl. After 2¬h of incubation at 37¬¬C, the cell suspensions were diluted 1:50 with 10¬mM sodium phosphate buffer, pH 7.4,¬and exponentially spread with an Autoplate 400¬(Spiral Biotech) onto trypticase soy agar (bacteria) or Sabouraud dextrose agar (fungi). After incubation at 37¬¬C for 18-48 h, colonies were counted and cell survival was expressed as colony-forming units/ml.
The antimicrobial activities of the three theta-defensins were equivalent against S.¬aureus, C.¬albicans, and C.¬neoformans. RTD-2 was 2-3-fold less active than RTD-1 and RTD-3 against E.¬coli. The bactericidal and fungicidal activities of the three peptides were similar. However, approximately twice as much peptide was required to kill fungal suspensions as was needed to kill the same number of bacterial cells. RTD-2-mediated killing of E. coli showed a steep dose dependence similar to that of RTD-1 and -3, but approximately twice as much RTD-2 was required to achieve the microbicidal activities of RTD-1 and -3. However, at concentrations of 2 ug/ml or higher, all three theta-defensins reduced the viability of E. coli ML35 by at least 99.9%.