Bacterial membrane activity of α-peptide/β-peptoid chimeras: Influence of amino acid composition and chain length on the activity against different bacterial strains

<p>Abstract</p> <p>Background</p> <p>Characterization and use of antimicrobial peptides (AMPs) requires that their mode of action is determined. The interaction of membrane-active peptides with their target is often established using model membranes, however, the actual permeabilization of live bacterial cells and subsequent killing is usually not tested. In this report, six α-peptide/β-peptoid chimeras were examined for the effect of amino acid/peptoid substitutions and chain length on the membrane perturbation and subsequent killing of food-borne and clinical bacterial isolates.</p> <p>Results</p> <p>All six AMP analogues inhibited growth of twelve food-borne and clinical bacterial strains including Extended Spectrum Beta-Lactamase-producing <it>Escherichia coli</it>. In general, the Minimum Inhibitory Concentrations (MIC) against Gram-positive and -negative bacteria were similar, ranging from 1 to 5 μM. The type of cationic amino acid only had a minor effect on MIC values, whereas chain length had a profound influence on activity. All chimeras were less active against <it>Serratia marcescens </it>(MICs above 46 μM). The chimeras were bactericidal and induced leakage of ATP from <it>Staphylococcus aureus </it>and <it>S. marcescens </it>with similar time of onset and reduction in the number of viable cells. EDTA pre-treatment of <it>S. marcescens </it>and <it>E. coli </it>followed by treatment with chimeras resulted in pronounced killing indicating that disintegration of the Gram-negative outer membrane eliminated innate differences in susceptibility. Chimera chain length did not influence the degree of ATP leakage, but the amount of intracellular ATP remaining in the cell after treatment was influenced by chimera length with the longest analogue causing complete depletion of intracellular ATP. Hence some chimeras caused a complete disruption of the membrane, and this was parallel by the largest reduction in number of viable bacteria.</p> <p>Conclusion</p> <p>We found that chain length but not type of cationic amino acid influenced the antibacterial activity of a series of synthetic α-peptide/β-peptoid chimeras. The synthetic chimeras exert their killing effect by permeabilization of the bacterial cell envelope, and the outer membrane may act as a barrier in Gram-negative bacteria. The tolerance of <it>S. marcescens </it>to chimeras may be due to differences in the composition of the lipopolysaccharide layer also responsible for its resistance to polymyxin B.</p

Water-membrane partition and the mutant selection window of antimicrobial peptides: insights from liposome studies

The mutant selection window (MSW) is a range of antimicrobial concentrations, where some bacteria are killed, while others survive. Within this interval resistance may develop. Antimicrobial peptides (AMPs) are a promising class of antimicrobials that generally act by perturbing the integrity of bacterial membranes. Their MSW is typically narrower than that of traditional antibiotics, but it still encompasses about one order of magnitude of peptide concentrations. Phenotypic or genetic differences between individual cells may cause this heterogeneous bacterial response to AMPs. Therefore, we minimized the system complexity by investigating pore formation in liposomes with homogeneous size and composition. Surprisingly, the AMPs novicidin, P9-4, and Sub3 formed pores only in a fraction of vesicles, over a wide range of total peptide concentrations. By characterizing the water/membrane partition equilibrium of these three AMPs, we were able to report the vesicle-perturbing activity as a function of the membrane-bound peptide concentration. In this case, the curves became essentially step functions with well-defined (bound) concentration thresholds at which pores were formed in all liposomes. Therefore, the apparent heterogeneous effects of AMPs on vesicles were actually determined by variations in the fraction of membrane-bound peptides under different conditions, due to water-membrane partition. Unexpectedly, the thresholds coincided for all peptides in terms of bound amino acids per lipid (∼0.4), suggesting that the mechanism of pore formation primarily depends on the surface coverage by the AMPs

Simultaneous quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems

Robust, sensitive, and versatile analytical methods are essential for quantification of RNA drug cargos loaded into nanoparticle-based delivery systems. However, simultaneous quantification of multiple RNA cargos co-loaded into nanoparticles remains a challenge. Here, we developed and validated the use of ion-pair reversed-phase high-performance liquid chromatography combined with UV detection (IP-RP-HPLC-UV) for simultaneous quantification of single- and double-stranded RNA cargos. Complete extraction of RNA cargo from the nanoparticle carrier was achieved using a phenol:chloroform:isoamyl alcohol mixture. Separations were performed using either a C18 or a PLRP-S column, eluted with 0.1 M triethylammonium acetate (TEAA) solution as ion-pairing reagent (eluent A), and 0.1 M TEAA containing 25 % (v/v) CH3CN as eluent B. These methods were applied to quantify mRNA and polyinosinic:polycytidylic acid co-loaded into lipid-polymer hybrid nanoparticles, and single-stranded oligodeoxynucleotide donors and Alt-R CRISPR single guide RNAs co-loaded into lipid nanoparticles. The developed methods were sensitive (limit of RNA quantification 0.997), and accurate (≈ 100 % recovery of RNA spiked in nanoparticles). Hence, the present study may facilitate convenient quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems

Quantification of pharmaceutical peptides using selenium as an elemental detection label

Se-labelling of pharmaceutical biomolecules provides detailed quantitative and qualitative information on the fate of the biomolecule in cell uptake studies.</p

Novel Cyclic Lipopeptide Antibiotics:Effects of Acyl Cain Length and Position

Multidrug-resistant bacteria are a global health problem. One of the last-resort antibiotics against Gram-negative bacteria is the cyclic lipopeptide colistin, displaying a flexible linker with a fatty acid moiety. The aim of the present project was to investigate the effect on antimicrobial activity of introducing fatty acid moieties of different lengths and in different positions in a cyclic peptide, S3(B), containing a flexible linker. The lipidated analogues of S3(B) were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis. Following assembly of the linear peptide by Fmoc solid-phase peptide synthesis, on-resin head-to-tail cyclization and fatty acid acylation were performed. The antimicrobial activity was determined against the ESKAPE pathogens, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, hemolytic activity was determined against human erythrocytes. A total of 18 cyclic lipopeptides were synthesized and characterized. It was found that introduction of fatty acids in positions next to the flexible linker was more strongly linked to antimicrobial activity. The fatty acid length altered the overall hydrophobicity, which was the driving force for both high antimicrobial and hemolytic activity. Peptides became highly hemolytic when carbon-chain length exceeded 10 (i.e., C10), overlapping with the optimum for antimicrobial activity (i.e., C8&ndash;C12). The most promising candidate (C8)5 showed antimicrobial activity corresponding to that of S3(B), but with an improved hemolytic profile. Finally, (C8)5 was further investigated in a time-kill experiment