Michael L. Vasil

Rational Design of α-Helical Antimicrobial Peptides with Enhanced Activities and Specificity/Therapeutic Index

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 °C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the d-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with l-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected d-/l-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.

Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa

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.

GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes

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 ΔpvdS 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.

The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence

During the past decade significant progress has been made towards identifying some of the schemes that Pseudomonas aeruginosa uses to obtain iron and towards cataloguing and characterizing many of the genes and gene products that are likely to play a role in these processes. This review will largely recount what we have learned in the past few years about how P. aeruginosa regulates its acquisition, intake and, to some extent, trafficking of iron, and the role of iron acquisition systems in the virulence of this remarkable opportunistic pathogen. More specifically, the genetics, biochemistry and biology of an essential regulator (Ferric uptake regulator — Fur) and a Fur‐regulated alternative sigma factor (PvdS), which are central to these processes, will be discussed. These regulatory proteins directly or indirectly regulate a substantial number of other genes encoding proteins with remarkably diverse functions. These genes include: (i) other regulatory genes, (ii) genes involved in basic metabolic processes (e.g. Krebs cycle), (iii) genes required to survive oxidative stress (e.g. superoxide dismutase), (iv) genes necessary for scavenging iron (e.g. siderophores and their cognate receptors) or genes that contribute to the virulence (e.g. exotoxin A) of this opportunistic pathogen. Despite this recent expansion of knowledge about the response of P. aeruginosa to iron, many significant biological issues surrounding iron acquisition still need to be addressed. Virtually nothing is known about which of the distinct iron acquisition mechanisms P. aeruginosa brings to bear on these questions outside the laboratory, whether it be in soil, in a pipeline, on plants or in the lungs of cystic fibrosis patients.

Role of Peptide Hydrophobicity in the Mechanism of Action of α-Helical Antimicrobial Peptides

ABSTRACT In the present study, the 26-residue amphipathic α-helical antimicrobial peptide V13KL (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 reversed-phase 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.

Architecture of a protein central to iron homeostasis : crystal structure and spectroscopic analysis of the ferric uptake regulator.

Iron is an essential element for almost all organisms, although an overload of this element results in toxicity because of the formation of hydroxyl radicals. Consequently, most living entities have developed sophisticated mechanisms to control their intracellular iron concentration. In many bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, this task is performed by the ferric uptake regulator (Fur). Fur controls a wide variety of basic physiological processes including iron uptake systems and the expression of exotoxin A. Here, we present the first crystal structure of Fur from P. aeruginosa in complex with Zn2+ determined at a resolution of 1.8 Å. Furthermore, X‐ray absorption spectroscopic measurements and microPIXE analysis were performed in order to characterize the distinct zinc and iron binding sites in solution. The combination of these complementary techniques enables us to present a model for the activation and DNA binding of the Fur protein.

Involvement of the twin-arginine translocation system in protein secretion via the type II pathway.

The general secretory pathway (GSP) is a two‐step process for the secretion of proteins by Gram‐negative bacteria. The translocation across the outer membrane is carried out by the type II system, which involves machinery called the secreton. This step is considered to be an extension of the general export pathway, i.e. the export of proteins across the inner membrane by the Sec machinery. Here, we demonstrate that two substrates for the Pseudomonas aeruginosa secreton, both phospholipases, use the twin‐arginine translocation (Tat) system, instead of the Sec system, for the first step of translocation across the inner membrane. These results challenge the previous vision of the GSP and suggest for the first time a mosaic model in which both the Sec and the Tat systems feed substrates into the secreton. Moreover, since P.aeruginosa phospholipases are secreted virulence factors, the Tat system appears to be a novel determinant of bacterial virulence.

Enhanced Pseudomonas aeruginosa Biofilm Development Mediated by Human Neutrophils

ABSTRACT Cystic fibrosis (CF) lung disease features persistent neutrophil accumulation to the airways from the time of infancy. CF children are frequently exposed to Pseudomonas aeruginosa, and by adulthood, 80% of CF patients are chronically infected. The formation of biofilms is a particularly important phenotypic characteristic of P. aeruginosa that allows for bacterial survival despite aggressive antibiotic therapy and an exuberant immune response. Here, we show that the presence of neutrophils enhances initial P. aeruginosa biofilm development over a period of 72 h through the formation of polymers comprised of actin and DNA. F-actin was found to be a site of attachment for P. aeruginosa. These actin and DNA polymers are present in CF sputum, and disruption of the polymers dispersed the associated P. aeruginosa cells and reduced biofilm development. These findings demonstrate a potential maladaptation of the primary innate response. When the host fails to eradicate the infection, cellular components from necrotic neutrophils can serve as a biological matrix to facilitate P. aeruginosa biofilm formation.

Effects of net charge and the number of positively charged residues on the biological activity of amphipathic α-helical cationic antimicrobial peptides

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).

Role of the Pseudomonas aeruginosa oxyR-recG Operon in Oxidative Stress Defense and DNA Repair: OxyR-Dependent Regulation of katB-ankB, ahpB, and ahpC-ahpF

Pseudomonas aeruginosa possesses an extensive armament of genes involved in oxidative stress defense, including katB-ankB, ahpB, and ahpC-ahpF. Transcription of these genes was regulated in response to H(2)O(2), paraquat, or organic peroxides. Expression of katB-lacZ and the observed KatB catalase levels in P. aeruginosa PAO1 were induced up to 250-fold after exposure to oxidative stress-generating compounds. Also, ahpB-lacZ and ahpC-lacZ expression was 90- and 3-fold higher, respectively, upon exposure to paraquat. The dose- and time-response curves revealed that 1 microM paraquat was sufficient for half-maximal activation of each reporter fusion within 5 min of exposure. Expression of these genes was not observed in a DeltaoxyR mutant, indicating that OxyR was essential for this response. The transcriptional start sites of katB-ankB, ahpB, and ahpC-ahpF were mapped, putative OxyR-binding sites were identified upstream of the -35 promoter elements, and direct binding of purified OxyR protein to these target promoters was demonstrated. The oxyR mutant was hypersusceptible to oxidative stress-generating agents, including H(2)O(2) and paraquat, in spite of total KatA catalase activity being comparable to that of the wild type. The oxyR phenotype was fully complemented by a plasmid containing the oxyR gene, while any of the katB, ahpB, or ahpCF genes alone resulted in only marginal complementation. Increased katB-lacZ expression and higher KatB catalase levels were detected in a DeltaahpCF background compared to wild-type bacteria, suggesting a compensatory function for KatB in the absence of AhpCF. In P. aeruginosa, oxyR is located upstream of recG, encoding a putative DNA repair enzyme. oxyR-lacZ and recG-lacZ reporter activities and oxyR-recG mRNA analysis showed that oxyR and recG are organized in an operon and expressed constitutively with regard to oxidative stress from a single promoter upstream of oxyR. Mutants affected in recG but not oxyR were dramatically impaired in DNA damage repair as measured by sensitivity to UV irradiation. In conclusion, we present evidence that the oxyR-recG locus is essential for oxidative stress defense and for DNA repair.