Analogs of the Frog-skin Antimicrobial Peptide Temporin 1Tb Exhibit a Wider Spectrum of Activity and a Stronger Antibiofilm Potential as Compared to the Parental Peptide

Abstract

The frog skin-derived peptide Temporin 1Tb (TB) has gained increasing attention as novel antimicrobial agent for the treatment of antibiotic-resistant and/or biofilm-mediated infections. Nevertheless, such a peptide possesses a preferential spectrum of action against Gram-positive bacteria. In order to improve the therapeutic potential of TB, the present study evaluated the antibacterial and antibiofilm activities of two TB analogs against medically relevant bacterial species. Of the two analogs, TB_KKG6A has been previously described in the literature, while TB_L1FK is a new analog designed by us through statistical-based computational strategies. Both TB analogs displayed a faster and stronger bactericidal activity than the parental peptide, especially against Gram-negative bacteria in planktonic form. Differently from the parental peptide, TB_KKG6A and TB_L1FK were able to inhibit the formation of Staphylococcus aureus biofilms by more than 50% at 12 μM, while only TB_KKG6A prevented the formation of Pseudomonas aeruginosa biofilms at 24 μM. A marked antibiofilm activity against preformed biofilms of both bacterial species was observed for the two TB analogs when used in combination with EDTA. Analysis of synergism at the cellular level suggested that the antibiofilm activity exerted by the peptide-EDTA combinations against mature biofilms might be due mainly to a disaggregating effect on the extracellular matrix in the case of S. aureus, and to a direct activity on biofilm-embedded cells in the case of P. aeruginosa. Both analogs displayed a low hemolytic effect at the active concentrations and, overall, TB_L1FK resulted less cytotoxic toward mammalian cells. Collectively, the results obtained demonstrated that subtle changes in the primary sequence of TB may provide TB analogs that, used alone or in combination with adjuvant molecules such as EDTA, exhibit promising features against both planktonic and biofilm cells of medically relevant bacteria.

Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and selectivity by fundamentally altering membrane activity

“…None of the three temporin B peptides were effective against Pseudomonas aeruginosa PAO1 in the conditions used here. The results reported here for temporin B KKG6A are in disagreement with those reported previously, which showed up to 8-fold improvement in the activity against Gram-positive bacteria 16,18 and 16fold against P. aeruginosa 16 when compared to temporin B. However different bacterial strains and protocols were applied in the previous work.…”

“…For temporin B L1FK, the optimization involved the substitution of Leu1 with Phe1 to render the N-terminus more hydrophobic, the addition of a lysine residue at the C-terminus and the deletion of Asn7. 16 These modifications improved the activity of the peptide inducing a 4.5-fold increase in potency against uropathogenic Escherichia coli NCTC9001 and a two-fold increase against Staphylococcus aureus Newman. To create temporin B KKG6A, two lysines are added to the N-terminus, increasing the nominal positive charge and Gly6 is substituted with alanine.…”

“…Temporin B L1FK maintains the physicochemical properties of the native peptide, such as hydrophobicity and amphipathic profile, but it has a broader spectrum of activity at low concentration, including against Gram-negative bacteria, as well as a higher potency in eradication of biofilms. 16,17 Temporin B KKG6A was instead obtained by further optimization of an analogue obtained through alanine scanning of the temporin B sequence. The properties of temporin B KKG6A differ from temporin B, with a higher positive charge, a different amphipathic profile and a higher helical content, but is reported to have improved antimicrobial activity against Gram-negative and Gram-positive bacteria, a low haemolytic activity and a strong tendency to bind to lipopolysaccharide.…”

“…The properties of temporin B KKG6A differ from temporin B, with a higher positive charge, a different amphipathic profile and a higher helical content, but is reported to have improved antimicrobial activity against Gram-negative and Gram-positive bacteria, a low haemolytic activity and a strong tendency to bind to lipopolysaccharide. 16,18 To distinguish between the mode of interaction of the three temporin B analogues we combined both time-resolved and steadystate biophysical methods including all atom molecular dynamics simulations (using NMR structures of the three temporin B peptides solved in anionic detergent as a starting point), with circular dichroism and solid-state NMR to determine conformational and topological differences during the initial stages of membrane interaction and in the steady state. In addition, electrophysiology measurements of the current transitions on model membranes obtained with the patch-clamp technique reveal how these impact on the ability of each peptide to permeate analogous model membranes and enable understanding of the antibacterial outcome.…”

“…Temporin B L1FK was obtained from a computational statistical model designed to predict the toxicity of the peptide as well as the antimicrobial activity. Temporin B L1FK maintains the physicochemical properties of the native peptide, such as hydrophobicity and amphipathic profile, but it has a broader spectrum of activity at low concentration, including against Gramnegative bacteria, as well as a higher potency in eradication of biofilms (19,20). Temporin B KKG6A was instead obtained by further optimization of an analogue obtained through alanine scanning of the temporin B sequence.…”