Synergy Pattern of Short Cationic Antimicrobial Peptides Against Multidrug-Resistant Pseudomonas aeruginosa.

With the rise of various multidrug-resistant (MDR) pathogenic bacteria, worldwide health care is under pressure to respond. Conventional antibiotics are failing and the development of novel classes and alternative strategies is a major priority. Antimicrobial peptides (AMPs) cannot only kill MDR bacteria, but also can be used synergistically with conventional antibiotics. We selected 30 short AMPs from different origins and measured their synergy in combination with polymyxin B, piperacillin, ceftazidime, cefepime, meropenem, imipenem, tetracycline, erythromycin, kanamycin, tobramycin, amikacin, gentamycin, and ciprofloxacin. In total, 403 unique combinations were tested against an MDR Pseudomonas aeruginosa isolate (PA910). As a measure of the synergistic effects, fractional inhibitory concentrations (FICs) were determined using microdilution assays with FICs ranges between 0.25 and 2. A high number of combinations between peptides and polymyxin B, erythromycin, and tetracycline were found to be synergistic. Novel variants of indolicidin also showed a high frequency in synergist interaction. Single amino acid substitutions within the peptides can have a very strong effect on the ability to synergize, making it possible to optimize future drugs toward synergistic interaction.

Optimization of oncocin for antibacterial activity using a SPOT synthesis approach: extending the pathogen spectrum to Staphylococcus aureus.

The identification of lead molecules against multidrug-resistant bacteria ensuing the development of novel antimicrobial drugs is an urgent task. Proline-rich antimicrobial peptides are highly active in vitro and in vivo, but only against a few Gram-negative human pathogens, with rather weak activities against Pseudomonas aeruginosa and Staphylococcus aureus. This reduced level of efficacy could be related to inadequate uptake mechanisms or structural differences of the intracellular target proteins, i.e., the 70S ribosome or chaperone DnaK. Here we synthesized peptide arrays on cellulose membranes using cleavable linkers to release the free individual peptides for further antimicrobial tests. Thus, a library of singly substituted oncocin analogs was produced by replacing each residue by all other 19 canonical amino acids yielding a set of 361 individual peptides to be evaluated against a luminescent P. aeruginosa strain. Thirteen substitutions appeared promising and their improved antibacterial activities were confirmed for different bacteria after larger scale synthesis of these analogs. By combining two favorable substitutions into one peptide, we finally obtained an oncocin analog that was ten times more active against P. aeruginosa and even 100-fold more active against S. aureus than the original oncocin, providing minimal inhibitory concentrations of 4-8 and 0.5 µg/mL, respectively.

Improving short antimicrobial peptides despite elusive rules for activity.

Antimicrobial peptides (AMPs) can effectively kill a broad range of life threatening multidrug-resistant bacteria, a serious threat to public health worldwide. However, despite great hopes novel drugs based on AMPs are still rare. To accelerate drug development we studied different approaches to improve the antibacterial activity of short antimicrobial peptides. Short antimicrobial peptides seem to be ideal drug candidates since they can be synthesized quickly and easily, modified and optimized. In addition, manufacturing a short peptide drug will be more cost efficient than long and structured ones. In contrast to longer and structured peptides short AMPs seem hard to design and predict. Here, we designed, synthesized and screened five different peptide libraries, each consisting of 600 9-mer peptides, against Pseudomonas aeruginosa. Each library is presenting a different approach to investigate effectiveness of an optimization strategy. The data for the 3000 peptides were analyzed using models based on fuzzy logic bioinformatics and plausible descriptors. The rate of active or superior active peptides was improved from 31.0% in a semi-random library from a previous study to 97.8% in the best new designed library. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Targeting Mycobacterium tuberculosis and other microbial pathogens using improved synthetic antibacterial peptides.

The lack of effective therapies for treating tuberculosis (TB) is a global health problem. While Mycobacterium tuberculosis is notoriously resistant to most available antibiotics, we identified synthetic short cationic antimicrobial peptides that were active at low micromolar concentrations (less than 10 µM). These small peptides (averaging 10 amino acids) had remarkably broad spectra of antimicrobial activities against both bacterial and fungal pathogens and an indication of low cytotoxicity. In addition, their antimicrobial activities displayed various degrees of species specificity that were not related to taxonomy. For example, Candida albicans and Staphylococcus aureus were the best surrogates to predict peptide activity against M. tuberculosis, while Mycobacterium smegmatis was a poor surrogate. Principle component analysis of activity spectrum profiles identified unique features associated with activity against M. tuberculosis that reflect their distinctive amino acid composition; active peptides were more hydrophobic and cationic, reflecting increased tryptophan with compensating decreases in valine and other uncharged amino acids and increased lysine. These studies provide foundations for development of cationic antimicrobial peptides as potential new therapeutic agents for TB treatment.

Screening for antifungal peptides and their modes of action in Aspergillus nidulans.

Many short cationic peptides have been identified as potent antimicrobial agents, but their modes of action are not well understood. Peptide synthesis on cellulose membranes has resulted in the generation of peptide libraries, while high-throughput assays have been developed to test their antibacterial activities. In this paper a microtiter plate-based screening method for fungi has been developed and used to test nine antibacterial peptides against the model fungus Aspergillus nidulans. Microscopical studies using sublethal peptide concentrations caused defects in polarized growth, including increased branch formation and depolarized hyphae. We characterized the mode of action for one of our target peptides, Sub5 (12 amino acids), which has already been shown to possess pharmacological potential as an antibacterial agent and is able to interact with ATP and ATP-dependent enzymes. The MIC for A. nidulans is 2 µg/ml, which is in the same range as the MICs reported for bacteria. Fluorescein isothiocyanate (FITC)-labeled Sub5 targeted the cytoplasmic membrane, particularly hyphal tips, and entered the cytoplasm after prolonged exposure, independent of endocytosis. Interestingly, Sub5 peptide treatment disturbed sterol-rich membrane domains, important for tip growth, at hyphal tips. A very similar peptide, FITC-P7, also accumulated on the cell membrane but did not have antibacterial or antifungal activity, suggesting that the cytoplasmic membrane is a first target for the Sub5 peptide; however, the antifungal activity seems to be correlated with the ability to enter the cytoplasm, where the peptides might act on other targets.

Short cationic antimicrobial peptides interact with ATP.

The mode of action of short, nonhelical antimicrobial peptides is still not well understood. Here we show that these peptides interact with ATP and directly inhibit the actions of certain ATP-dependent enzymes, such as firefly luciferase, DnaK, and DNA polymerase. α-Helical and planar or circular antimicrobial peptides did not show such interaction with ATP.

Synergistic interaction between silver nanoparticles and membrane-permeabilizing antimicrobial peptides.

Silver nanoparticles, as well as antimicrobial peptides (AMPs), can be used to fight infectious diseases. Since AMPs are known to permeabilize bacterial membranes and might therefore help silver nanoparticles to access internal target sites, we investigated their combined activities and showed synergistic effects between polymyxin B and silver nanoparticles for gram-negative bacteria.