Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH(2) resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called "carpet-like" mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MIC(tethered)/MIC(free). In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.
This study compares the effect of cyclic R-, W-rich peptides with variations in amino acid sequences and sizes from 5 to 12 residues upon Gram negative and Gram positive bacteria as well as outer membrane-deficient and LPS mutant Escherichia coli (E. coli) strains to analyze the structural determinants of peptide activity. Cyclo-RRRWFW (c-WFW) was the most active and E. coli-selective sequence and bactericidal at the minimal inhibitory concentration (MIC). Removal of the outer membrane distinctly reduced peptide activity and the complete smooth LPS was required for maximal activity. c-WFW efficiently permeabilised the outer membrane of E. coli and promoted outer membrane substrate transport. Isothermal titration calorimetric studies with lipid A-, rough-LPS (r-LPS)- and smooth-LPS (s-LPS)-doped POPC liposomes demonstrated the decisive role of O-antigen and outer core polysaccharides for peptide binding and partitioning. Peptide activity against the inner E. coli membrane (IM) was very low. Even at a peptide to lipid ratio of 8/1, c-WFW was not able to permeabilise a phosphatidylglycerol/phosphatidylethanolamine (POPG/POPE) bilayer. Low influx of propidium iodide (PI) into bacteria confirmed a low permeabilising ability of c-WFW against PE-rich membranes at the MIC. Whilst the peptide effect upon eukaryotic cells correlated with the amphipathicity and permeabilisation of neutral phosphatidylcholine bilayers, suggesting a membrane disturbing mode of action, membrane permeabilisation does not seem to be the dominating antimicrobial mechanism of c-WFW. Peptide interactions with the LPS sugar moieties certainly modulate the transport across the outer membrane and are the basis of the E. coli selectivity of this type of peptides.
Antimicrobial peptides (AMPs) are at the focus of attention due to their therapeutic importance and developing computational tools for the identification of efficient antibiotics from the primary structure. Here, we utilized the 13CNMR spectral of amino acids and clustered them into various groups. These clusters were used to build feature vectors for the AMP sequences based on the composition, transition, and distribution of cluster members. These features, along with the physicochemical properties of AMPs were exploited to learn computational models to predict active AMPs solely from their sequences. Naïve Bayes (NB), k-nearest neighbors (KNN), support-vector machine (SVM), random forest (RF), and eXtreme Gradient Boosting (XGBoost) were employed to build the classification system using the collected AMP datasets from the CAMP, LAMP, ADAM, and AntiBP databases. Our results were validated and compared with the CAMP and ADAM prediction systems and indicated that the synergistic combination of the 13CNMR features with the physicochemical descriptors enables the proposed ensemble mechanism to improve the prediction performance of active AMP sequences. Our web-based AMP prediction platform, IAMPE, is available at .
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