Antibacterial and Hemolytic Activities of Single Tryptophan Analogs of Indolicidin

An Antibody as Surrogate Receptor Reveals Determinants of Activity of an Innate Immune Peptide Antibiotic

“…The fact that IN-411 displays an unequal change in minimum inhibitory concentration versus mean hemolytic concentration indicates that membrane interaction and antibacterial activity may not be perfectly correlated, in agreement with previous studies (20,33). Also, although mAb V2D2 may display membrane-like features in its binding to indolicidin, it does not implicate the lipid bilayer as the sole target of the peptide.…”

“…Interaction curves were simultaneously fit using bivalent model in BIAevaluation program to determine Radial diffusion assay using double-layered agarose (19) involved incubating ϳ2 ϫ 10 7 mid-log phase bacterial cells with 10 mM peptide solution in nutrient-poor agarose at 37°C for 3 h. Overlayering with 3% tryptic soy broth agarose revealed circular bacteria-free zones. Minimum inhibitory concentration was determined by broth microdilution (20) in which ϳ10 4 bacterial cells in phosphate buffer were incubated with serially diluted peptide solution in a 96-well microtiter plate. Addition of LB medium subsequently was used to ascertain the minimum peptide concentration sufficient to prevent bacterial growth, observable as A 600 .…”

“…Sheep erythrocytes were used to ascertain mean hemolytic concentration (MHC) (20). Briefly, a 0.02 dilution of 200-l packed erythrocyte volume in PBS was incubated with serially diluted peptides in round-bottom 96-well microtiter plates at 37°C for 90 min.…”

“…Comparable concentrations of both indolicidin and IN-411 were able to completely inhibit microbial growth across the spectrum of species tested (Table 2), reinforcing the notion of IN-411 as the focal antibiotic motif. Because a number of reports in the past have blamed Trp residues for the undesirable cytotoxicity characteristic of indolicidin (20,33), we also compared the effects of IN-411 and indolicidin on mammalian erythrocytes. Interestingly, the IN-411 motif, despite containing all five Trp residues, exhibited a significant reduction in its mean hemolytic concentration.…”

Length Effects in Antimicrobial Peptides of the (RW)nSeries

“…Other studies suggest that the antimicrobial activity of peptides containing R is higher than those of peptides containing K (34,41), while peptides containing W are more potent than those with either F or Y (8,47,49). The guanidinium group of R has a more dispersed positive charge than the single amine of K, possibly enhancing electrostatic interactions between peptides and the negatively charged bacterial membrane surface (39,53).…”