In the present study, the 26-residue amphipathic ␣-helical antimicrobial peptide V13K L (Y. Chen et al., J. Biol. Chem. 2005, 280:12316-12329, 2005) was used as the framework to study the effects of peptide hydrophobicity on the mechanism of action of antimicrobial peptides. Hydrophobicity was systematically decreased or increased by replacing leucine residues with less hydrophobic alanine residues or replacing alanine residues with more hydrophobic leucine residues on the nonpolar face of the helix, respectively. Hydrophobicity of the nonpolar face of the amphipathic helix was demonstrated to correlate with peptide helicity (measured by circular dichroism spectroscopy) and self-associating ability (measured by reversedphase high-performance liquid chromatography temperature profiling) in aqueous environments. Higher hydrophobicity was correlated with stronger hemolytic activity. In contrast, there was an optimum hydrophobicity window in which high antimicrobial activity could be obtained. Decreased or increased hydrophobicity beyond this window dramatically decreased antimicrobial activity. The decreased antimicrobial activity at high peptide hydrophobicity can be explained by the strong peptide self-association which prevents the peptide from passing through the cell wall in prokaryotic cells, whereas increased peptide self-association had no effect on peptide access to eukaryotic membranes.Antibiotic resistance, due to the extensive clinical use of classical antibiotics (22, 32), has become a great concern in recent years, prompting an urgent need for a new class of antibiotics. Antimicrobial peptides have been proposed as potent candidates of a new class of antibiotics, with characteristics including an ability to kill target cells rapidly, an unusually broad spectrum of activity, activity against some of the more serious antibiotic-resistant pathogens in clinics, and the relative difficulty in selecting resistant mutants in vitro (13,35). Although the exact mode of action of antimicrobial peptides has not been established, it is generally accepted that the cytoplasmic membrane is the main target of antimicrobial peptides, whereby peptide accumulation in the membrane causes increased permeability and a loss of barrier function, resulting in the leakage of cytoplasmic components and cell death (13,28).Factors believed to be important for antimicrobial activity have been identified, including peptide hydrophobicity, the presence of positively charged residues, an amphipathic nature that segregates basic and hydrophobic residues, and secondary structure. Recently, Hodges and coworkers increased this list to include (i) the importance of a lack of structure in benign medium (nondenaturing conditions; see Materials and Methods) but an inducible structure in the presence of the hydrophobic environment of the membrane, (ii) the presence of a positively charged residue in the center of the nonpolar face of amphipathic cyclic ␤-sheet and ␣-helical peptides as a determinant for locating the peptides at the interface r...

Numerous bacteria secrete low molecular weight compounds termed siderophores that have a high affinity for iron ions. Siderophores have a well-documented role as iron-scavenging chemicals, chelating iron ions in the environment whereupon the ferrisiderophores reenter the bacterial cells by means of specific cell-surface receptors. The iron is then released for incorporation into bacterial proteins. Here we show that in addition to its role as an iron-scavenger, the siderophore pyoverdine that is secreted by Pseudomonas aeruginosa regulates the production of at least three virulence factors (exotoxin A, an endoprotease, and pyoverdine itself), which are major contributors to the ability of this bacterium to cause disease. Regulation occurs through a transmembrane signaling system that includes an outer membrane receptor for ferripyoverdine, a signal-transducing protein that is predicted to extend from the periplasm into the cytoplasm, and a sigma factor. Expression of genes that form part of the regulon is triggered by pyoverdine so that this siderophore acts as a signaling molecule to control the production of secreted products. Recognition that a siderophore acts as a signaling molecule has important implications for the understanding of interactions between bacterial cells.

Upon iron restriction, the opportunistic pathogen Pseudomonas aeruginosa produces various virulence factors, including siderophores, exotoxin, proteases and haemolysin. The ferric uptake regulator (Fur) plays a central role in this response and also controls other regulatory genes, such as pvdS, which encodes an alternative sigma factor. This circuit leads to a hierarchical cascade of direct and indirect iron regulation. We used the GeneChip to analyse the global gene expression profiles in response to iron. In iron-starved cells,the expression of 118 genes was increased at least fivefold compared with that in iron-replete cells, whereas the expression of 87 genes was decreased at least fivefold. The GeneChip data correlated well with results obtained using individual lacZ gene fusions. Strong iron regulation was observed for previously identified genes involved in biosynthesis or uptake of the siderophores pyoverdine and pyochelin, utilization of heterologous siderophores and haem and ferrous iron transport. A low-iron milieu led to increased expression of the genes encoding TonB, alkaline protease,PrpL protease, exotoxin A, as well as fumarase C, Mn-dependent superoxide dismutase SodA, a ferredoxin and ferredoxin reductase and several oxidoreductases and dehydrogenases. Iron-controlled regulatory genes included seven alternative sigma factors and five other transcriptional regulators. Roughly 20% of the iron-regulated genes encoded proteins of unknown function and lacked any conclusive homologies. Under low-iron conditions, expression of 26 genes or operons was reduced in a DeltapvdS mutant compared with wild type, including numerous novel pyoverdine biosynthetic genes. The GeneChip proved to be a very useful tool for rapid gene expression analysis and identification of novel genes controlled by Fur or PvdS.

In our previous study, we utilized a 26‐residue amphipathic α‐helical antimicrobial peptide L‐V13K (Chen et al., Antimicrob Agents Chemother 2007, 51, 1398–1406) as the framework to study the effects of peptide hydrophobicity on the mechanism of its antimicrobial action. In this study, we explored the effects of net charge and the number of positively charged residues on the hydrophilic/polar face of L‐V13K on its biological activity (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity, helicity, and peptide self‐association). The net charge of V13K analogs at pH 7 varied between −5 and +10 and the number of positively charged residues varied from 1 to 10. The minimal inhibitory concentrations (MIC) against six strains of Pseudomonas aeruginosa as well as other gram‐negative and gram‐positive bacteria were determined along with the maximal peptide concentration that produces no hemolysis of human red blood cells (MHC). Our results show that the number of positively charged residues on the polar face and net charge are both important for both antimicrobial activity and hemolytic activity. The most dramatic observation is the sharp transition of hemolytic activity on increasing one positive charge on the polar face of V13K i.e., the change from +8 to +9 resulted in greater than 32‐fold increase in hemolytic activity (250 μg/ml to <7.8 μg/ml, respectively). © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 369–383, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com