Interaction of W-Substituted Analogs of Cyclo-RRRWFW with Bacterial Lipopolysaccharides: the Role of the Aromatic Cluster in Antimicrobial Activity

Abstract

The activity of cyclo-RRRWFW (c-WFW) against Escherichia coli has been shown to be modulated by the aromatic motif and the lipopolysaccharides (LPS) in the bacterial outer membrane. To identify interaction sites and to elucidate the mode of c-WFW action, peptides were synthesized by the replacement of tryptophan (W) with analogs having altered hydrophobicity, dipole and quadrupole moments, hydrogen-bonding ability, amphipathicity, and ring size. The peptide activity against Bacillus subtilis and erythrocytes increased with increasing hydrophobicity, whereas the effect on E. coli revealed a more complex pattern. Although they had no effect on the E. coli inner membrane even at concentrations higher than the MIC, peptides permeabilized the outer membrane according to their antimicrobial activity pattern, suggesting a major role of LPS in peptide transport across the wall. For isothermal titration calorimetry (ITC) studies of peptide-lipid bilayer interaction, we used POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline), either alone or in mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), to mimic the charge properties of eukaryotic and bacterial membranes, respectively, as well as in mixtures with lipid A, rough LPS, and smooth LPS as models of the outer membrane of E. coli. Peptide accumulation was determined by both electrostatic and hydrophobic interactions. The susceptibility of the lipid systems followed the order of POPC-smooth LPS >> POPC-rough LPS > POPC-lipid A ‫؍‬ POPC-POPG > POPC. Low peptide hydrophobicity and enhanced flexibility reduced binding. The influence of the other properties on the free energy of partitioning was low, but an enhanced hydrogen-bonding ability and dipole moment resulted in remarkable variations in the contribution of enthalpy and entropy. In the presence of rough and smooth LPS, the binding-modulating role of these parameters decreased. The highly differentiated activity pattern against E. coli was poorly reflected in peptide binding to LPS-containing membranes. However, stronger partitioning into POPC-smooth LPS than into POPC-rough LPS uncovered a significant role of O-antigen and outer core oligosaccharides in peptide transport and the permeabilization of the outer membrane and the anti-E. coli activity of the cyclic peptides.

What Goes around Comes around-A Comparative Study of the Influence of Chemical Modifications on the Antimicrobial Properties of Small Cyclic Peptides

“…Reduction in the size or positioning of cationic residues next to the hydrophobic molecular surface area would reduce amphipathicity. t R -measurements have successfully been applied to characterize amphipathic helices [24], β-structured peptides [25] and different sets of our small cyclic R-, W-rich peptides [23,26,27]. …”

“…Here , membrane permeabilisation, as monitored by PI influx, correlated with peptide hydrophobicity (see Figure 2). An increase in hydrophobicity of this class of cyclic peptides and a large number of other antimicrobial sequences was reported to be linked to a deeper peptide insertion into and subsequent enhanced disturbance of the lipid matrix of cellular membranes [7,24,26]. …”

The effect of glycine replacement with flexible ω-amino acids on the antimicrobial and haemolytic activity of an amphipathic cyclic heptapeptide

“…In order to do so, we introduced several flexible linkers in the structure of 1 and we monitored the effects of such substitutions on its antimicrobial and haemolytic activity. Many of the structural investigations aimed at characterizing membrane-active AMPs focus on the amphipathicity-driven, early stages of membrane association [26,32,[38][39][40]. This is presumably due to established spectroscopic and calorimetric methods being best suited for the investigation of these thermodynamic events [41].…”

Novel antimicrobial peptide discovery using machine learning and biophysical selection of minimal bacteriocin domains

“…This observation is in contrast to the activities of the high-scoring Peptides 3 and 4, whose antimicrobial activities were higher against E. coli (Tables 1 and 2). Therefore, it may be possible to tune antimicrobial specificity by modifying the biophysical scores (Bagheri, Keller, & Dathe, 2011;Datta et al, 2016).…”