Strategic Single-Residue Substitution in the Antimicrobial Peptide Esc(1-21) Confers Activity against Staphylococcus aureus, Including Drug-Resistant and Biofilm Phenotype.

Staphylococcus aureus, a bacterium resistant to multiple drugs, is a significant cause of illness and death worldwide. Antimicrobial peptides (AMPs) provide an excellent potential strategy to cope with this threat. Recently, we characterized a derivative of the frog-skin AMP esculentin-1a, Esc(1-21) (1) that is endowed with potent activity against Gram-negative bacteria but poor efficacy against Gram-positive strains. In this study, three analogues of peptide 1 were designed by replacing Gly8 with α-aminoisobutyric acid (Aib), Pro, and dPro (2-4, respectively). The single substitution Gly8 → Aib8 in peptide 2 makes it active against the planktonic form of Gram-positive bacterial strains, especially Staphylococcus aureus, including multidrug-resistant clinical isolates, with an improved biostability without resulting in cytotoxicity to mammalian cells. Moreover, peptide 2 showed a higher antibiofilm activity than peptide 1 against both reference and clinical isolates of S. aureus. Peptide 2 was also able to induce rapid bacterial killing, suggesting a membrane-perturbing mechanism of action. Structural analysis of the most active peptide 2 evidenced that the improved biological activity of peptide 2 is the consequence of a combination of higher biostability, higher α helical content, and ability to reduce membrane fluidity and to adopt a distorted helix, bent in correspondence of Aib8. Overall, this study has shown how a strategic single amino acid substitution is sufficient to enlarge the spectrum of activity of the original peptide 1, and improve its biological properties for therapeutic purposes, thus paving the way to optimize AMPs for the development of new broad-spectrum anti-infective agents.

Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.

Effects of antimicrobial peptides on membrane dynamics: A comparison of fluorescence and NMR experiments.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed "sand in the gearbox". Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptide-induced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer.

Isoamphipathic antibacterial molecules regulating activity and toxicity through positional isomerism.

Peptidomimetic antimicrobials exhibit a selective interaction with bacterial cells over mammalian cells once they have achieved an optimum amphiphilic balance (hydrophobicity/hydrophilicity) in the molecular architecture. To date, hydrophobicity and cationic charge have been considered the crucial parameters to attain such amphiphilic balance. However, optimization of these properties is not enough to circumvent unwanted toxicity towards mammalian cells. Hence, herein, we report new isoamphipathic antibacterial molecules (IAMs: 1-3) where positional isomerism was introduced as one of the guiding factors for molecular design. This class of molecules displayed good (MIC = 1-8 µg mL-1 or µM) to moderate [MIC = 32-64 µg mL-1 (32.2-64.4 µM)] antibacterial activity against multiple Gram-positive and Gram-negative bacteria. Positional isomerism showed a strong influence on regulating antibacterial activity and toxicity for ortho [IAM-1: MIC = 1-32 µg mL-1 (1-32.2 µM), HC50 = 650 µg mL-1 (654.6 µM)], meta [IAM-2: MIC = 1-16 µg mL-1 (1-16.1 µM), HC50 = 98 µg mL-1 (98.7 µM)] and para [IAM-3: MIC = 1-16 µg mL-1 (1-16.1 µM), HC50 = 160 µg mL-1 (161.1 µM)] isomers. Co-culture studies and investigation of membrane dynamics indicated that ortho isomer, IAM-1 exerted more selective activity towards bacterial over mammalian membranes, compared to meta and para isomers. Furthermore, the mechanism of action of the lead molecule (IAM-1) has been characterized through detailed molecular dynamics simulations. In addition, the lead molecule displayed substantial efficacy against dormant bacteria and mature biofilms, unlike conventional antibiotics. Importantly, IAM-1 exhibited moderate in vivo activity against MRSA wound infection in a murine model with no detectable dermal toxicity. Altogether, the report explored the design and development of isoamphipathic antibacterial molecules to establish the role of positional isomerism in achieving selective and potential antibacterial agents.

Structural and Functional Characterization of the Newly Designed Antimicrobial Peptide Crabrolin21.

(1) Background: antimicrobial resistance is becoming a dramatic problem for public health, and the design of new antimicrobial agents is an active research area. (2) Methods: based on our previous work, we designed an improved version of the crabrolin peptide and characterized its functional and structural properties with a wide range of techniques. (3) Results: the newly designed peptide, crabrolin21, is much more active than the previous ones and shows specific selectivity towards bacterial cells. (4) Conclusions: crabrolin21 shows interesting properties and deserves further studies.