Antimicrobial peptides (AMPs) are potent antibiotics of the innate immune system that have been extensively investigated as a potential solution to the global problem of infectious diseases caused by pathogenic microbes. A group of AMPs that are increasingly being reported are those that utilise pH dependent antimicrobial mechanisms, and here we review research into this area. This review shows that these antimicrobial molecules are produced by a diverse spectrum of creatures, including vertebrates and invertebrates, and are primarily cationic, although a number of anionic examples are known. Some of these molecules exhibit high pH optima for their antimicrobial activity but in most cases, these AMPs show activity against microbes that present low pH optima, which reflects the acidic pH generally found at their sites of action, particularly the skin. The modes of action used by these molecules are based on a number of major structure/function relationships, which include metal ion binding, changes to net charge and conformational plasticity, and primarily involve the protonation of histidine, aspartic acid and glutamic acid residues at low pH. The pH dependent activity of pore forming antimicrobial proteins involves mechanisms that generally differ fundamentally to those used by pH dependent AMPs, which can be described by the carpet, toroidal pore and barrel-stave pore models of membrane interaction. A number of pH dependent AMPs and antimicrobial proteins have been developed for medical purposes and have successfully completed clinical trials, including kappacins, LL-37, histatins and lactoferrin, along with a number of their derivatives. Major examples of the therapeutic application of these antimicrobial molecules include wound healing as well as the treatment of multiple cancers and infections due to viruses, bacteria and fungi. In general, these applications involve topical administration, such as the use of mouth washes, cream formulations and hydrogel delivery systems. Nonetheless, many pH dependent AMPs and antimicrobial proteins have yet to be fully characterized and these molecules, as a whole, represent an untapped source of novel biologically active agents that could aid fulfillment of the urgent need for alternatives to conventional antibiotics, helping to avert a return to the pre-antibiotic era.

Linearized esculentin 2 EM (E2EM-lin) from the frog, Glandirana emeljanovi was highly active against Gram-positive bacteria (minimum lethal concentration ≤ 5.0 μM) and strongly α-helical in the presence of lipid mimics of their membranes (> 55.0 %). The Nterminal α-helical structure adopted by E2EM-lin showed the potential to form a membrane interactive, tilted peptide with an hydrophobicity gradient over residues 9 to 23. E2EM-lin inserted strongly into lipid mimics of membranes from Gram-positive bacteria (maximal surface pressure changes ≥ 5.5 mN m -1 ), inducing increased rigidity (Cs -1 ↑), thermodynamic instability (ΔGmix < 0 → ΔGmix > 0) and high levels of lysis (> 50.0%). These effects appeared to be driven by the high anionic lipid content of membranes from Gram-positive bacteria; namely phosphatidylglycerol (PG) and cardiolipin (CL) species. The high levels of α-helicity (60.0%), interaction (maximal surface pressure change = 6.7 mN m -1 ) and lysis (66.0%) shown by E2EM-lin with PG species was a major driver in the ability of the peptide to lyse and kill Gram-positive bacteria. E2EM-lin also showed high levels of α-helicity (62.0%) with CL species but only low levels of interaction (maximal surface pressure change = 2.9 mN m -1 ) and lysis (21.0%) with the lipid. These combined data suggest that E2EM-lin has a specificity for killing Gram-positive bacteria that involves the formation of tilted structure and appears to be primarily driven by PG-mediated membranolysis. These structure / function relationships are used to help explain the pore forming process proposed to describe the membranolytic, antibacterial action of E2EM-lin. Highlights• E2EM-lin shows specificity and potent efficacy towards Gram-positive bacteria • PG-driven membranolysis promotes E2EM-lin action against Gram-positive bacteria • PE-driven membranolysis promotes E2EM-lin action against Gram-negative bacteria • CL-mediated mechanisms contribute to E2EM-lin action against both bacterial types

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.

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Relevance. Pasteurellosis is a group of zoonotic infectious diseases caused by Pasteurella multocida. According to the antigenic composition, P. multocida is divided into 5 serogroups (A, B, D, F and E). Pathogenic and virulent properties of various serogroups and serotypes of the pathogen in different animal species vary widely and are a marker for determining their role in the development of the disease. Typing of P. multocida strains by capsule groups is an important condition for a comprehensive assessment of the epizootic situation, including for solving the issue of specific disease prevention.Methods. 82 strains of P. multocida from the collection of FGBI «VGNKI», isolated in different years from various animals, were used in the work. Phenotypic typing of pasteurella strains by capsule groups according to Carter was carried out in a test for the detection of hyaluronic acid in a pasteurella capsule and by the type of reaction in a tripaflavin sample. Strains giving a positive reaction with staphylococcus hyaluronidase wereassigned to capsule group A. If the test culture did not belong to group A, but was positive when examined in a tripaflavin sample, it was assigned to capsule group D.Results. Discrepancies between the results of phenotypic typing of pasteurella strains by capsule groups and by PCR were established. The results of typing did not coincide between PCR and Carter typing for capsule group A with respect to 25 strains, for capsule group D with respect to 5 strains and for capsule group B with respect to 5 strains. The number of untyped or doubtful phenotypic properties of pasteurella strains was 29.73%, untyped by PCR 2.46%. Hyaluronidase and acriflavin tests, unlike the PCR-method, do not provide an opportunity for typing pasteurella groups E and F.