Abstract
Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.
Highlights
Antimicrobial peptides (AMPs) have remained an effective component of the innate immune system throughout evolutionary history and have been isolated from numerous and diverse organisms[1,2,3,4,5,6]
The spectrum of activity for aurein 2.5 is narrower with almost no detectable activity against either Pseudomonas aeruginosa isolate suggesting the two peptides have differing abilities to penetrate its outer membrane, the potency of each antimicrobial peptides (AMPs) is likely affected by interactions with the differing cell walls in each pathogen and/or may differ in their susceptibilities to bacterial proteases and/or efflux systems
We investigated whether two short, hydrophobic antimicrobial peptides, both derived from frog dorsal secretions and either known or predicted to adopt highly ordered α-helix conformations are identical or can be distinguished in their interactions with model membranes and whether such behaviour can be ascribed to specific amino acids in the primary sequences
Summary
Antimicrobial peptides (AMPs) have remained an effective component of the innate immune system throughout evolutionary history and have been isolated from numerous and diverse organisms[1,2,3,4,5,6]. Occurring AMPs have attracted considerable interest as a starting point for rational design processes aiming to enhance antimicrobial or immunomodulatory properties[3,4,5,6] Such AMPs are often found to be part of a larger family of very close-related peptides which share substantial primary sequence and physico-chemical similarities and seemingly have very similar functions. Temporin L and aurein 2.5 are each members of much larger families of AMP produced by each frog, they represent interesting examples of parallel evolution as, despite the lack of sequence similarity, they do share numerous physico-chemical properties (Table 1); they are both relatively short and hydrophobic, they carry a modest positive charge and are known to adopt ordered α-helix conformations in model membranes or membrane mimicking milieu due to their secondary amphipathicity[13,28]. The ability to describe the similarities and some key differences, between the two AMPs, at the molecular level, allows better understanding of the importance of specific molecular features – notably conformational flexibility and the role of individual hydrophobic amino acids – refining our appreciation of the requirements for potent antibacterial activity
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