Investigation of the Role of Aromatic Residues in the Antimicrobial Peptide BuCATHL4B

Abstract

Background: Antimicrobial Peptides (AMPs) are an attractive alternative to traditional small molecule antibiotics as AMPs typically target the bacterial cell membrane. A Trp-rich peptide sequence derived from water buffalo (Bubalus bubalis), buCATHL4B was previously identified as a broad-spectrum antimicrobial peptide. Objective: In this work, native Trp residues were replaced with other naturally occurring aromatic amino acids to begin to elucidate the importance of these residues on peptide activity. Methods: Minimal Inhibitory Concentration (MIC) results demonstrated activity against seven strains of bacteria. Membrane and bilayer permeabilization assays were performed to address the role of bilayer disruption in the activity of the peptides. Lipid vesicle binding and quenching experiments were also performed to gain an understanding of how the peptides interacted with lipid bilayers. Results: MIC results indicate the original, tryptophan-rich sequence, and the phenylalanine substituted sequences exhibit strong inhibition of bacterial growth. In permeabilization assays, peptides with phenylalanine substitutions have higher levels of membrane permeabilization than those substituted with tyrosine. In addition, one of the two-tyrosine substituted sequence, YWY, behaves most differently in the lowest antimicrobial activity, showing no permeabilization of bacterial membranes. Notably the antimicrobial activity is inherently species dependent, with varying levels of activity against different bacteria. Conclusion: There appears to be little correlation between membrane permeabilization and activity, indicating these peptides may have additional mechanisms of action beyond membrane disruption. The results also identify two sequences, denoted FFF and YYW, which retain antibacterial activity but have markedly reduced hemolytic activity.

The Uniqueness of Tryptophan in Biology: Properties, Metabolism, Interactions and Localization in Proteins

“…A large number of diverse temporins and aureins, secreted from the skin of several frog species, protect these amphibians from infections in the wild. In another example, short Trp-rich AMPs provide antimicrobial defense to the water buffalo ( Bubalus bubalis ) [ 96 ] and have been subjected to mutational analysis. An optimally designed variant, WRK-12 (WRLRWKTRWRLK), was shown to efficiently target LPS and bacteria-specific phospholipids on the membrane [ 97 ], and as with many other AMPs, the placement of the Trp residues on one face of the helix ( Figure 7 A) was crucial for amphiphilicity and membrane interaction.…”

Biomimetic antimicrobial polymers—Design, characterization, antimicrobial, and novel applications

“…For example, Locock and coworkers have demonstrated that cationic methacrylate polymers penetrate the cell membrane of S. mutans and bind to DNA in the cytosol (Michl et al, 2020). Some AMPs were reported to target the intercellular components in bacteria including DNA (D'Souza et al, 2021;Le et al, 2017;Necelis et al, 2021). While these mechanisms are reported for specific AMPs, it is likely that any AMPs can simultaneously bind to different sites in bacteria including the cell wall, membrane, and intracellular proteins and DNA/RNA, and this promiscuous targeting may individually or collectively contribute to killing of bacteria (Mahlapuu et al, 2020).…”

Influence of Different Aromatic Hydrophobic Residues on the Antimicrobial Activity and Membrane Selectivity of BRBR-NH2 Tetrapeptide

“…According to the literature, tryptophan (W) has a higher membrane insertion tendency than the other 19 natural amino acids. Peptides containing tryptophan (W) and arginine (R), such as WRWRWR, VRRFPWWWPFLRR, , AIPWIWIWRLLRKG (BuCATHL4B), WWWLRRRW, and RRRWWWWV, are considered to have the most attractive properties suitable for antimicrobial agents. It has been reported that phenylalanine (F) acts as a membrane anchor in some AMPs .…”