Linearized esculentin-2EM shows pH dependent antibacterial activity with an alkaline optimum

Erum Malik,Sarah R Dennison,Timothy J Snape,Jaipaul Singh,David A Phoenix,Leslie H G Morton,Frederick Harris

doi:10.1007/s11010-021-04181-7

Abstract

Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m−1) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with − < µH > increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

Highlights

It is well established that pH plays an important physiological role in humans that is tightly regulated by acid–base homeostasis; unregulated changes in pH can impact on human health via multiple routes [1]
Statistical analysis showed that there was a significant difference between the minimum lethal concentration (MLC) of E2EM-lin for these Gram-negative and Gram-positive bacteria (F3, 11 = 74,217; p = 0.000), and in combination, these observations indicated that the peptide has a strong preference for Gram-positive bacteria, which is consistent with data reported by previous authors [52]
The peptide was circa 30-fold times less effective against E. coli and P. aeruginosa (≥ 75.0 μM, Table 2) and in combination, these results clearly show that E2EM-lin has a strong preference for Gram-positive bacteria, which is consistent with previous studies [37, 53, 58]

Summary

Introduction

It is well established that pH plays an important physiological role in humans that is tightly regulated by acid–base homeostasis; unregulated changes in pH can impact on human health via multiple routes [1]. In addition to low pH, a number of disorders and diseases are associated with high pH [2, 3, 12]; for example, pathological skin conditions such as psoriasis [13], acne [14] and atopic dermatitis [15] In these cases, alkaline conditions are produced by the collaborative action of a variety of exogenous and endogenous factors, such as skin type and dysregulated skin buffering, respectively [16,17,18]. Alkaline conditions are produced by the collaborative action of a variety of exogenous and endogenous factors, such as skin type and dysregulated skin buffering, respectively [16,17,18] These pH conditions are believed to decrease the production and potency of endogenous antimicrobials, thereby inducing inflammation, dysbiosis of the skin microbiome and microbial colonization of the skin [19,20,21,22]

Methods

Results

Conclusion