Abstract
BackgroundPlant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays.ResultsAtr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors.ConclusionsThis study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.
Highlights
Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity
Atr-DEF2(G39-C54) was screened for the ability to disrupt membranes using an NPN-uptake assay (Fig. 4a). These results indicated that the sub-lethal 37 μM concentration of Atr-DEF2(G39-C54) used for the proteomic analysis would have been sufficient to perturb the outer membrane (OM) of E. coli and contribute to the adaptive responses observed in the proteome
Plant defensins are a well-studied family of Antimicrobial peptide (AMP) whose antibacterial activity has been largely unexplored
Summary
Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. The significant length and complexity of defensins can hinder synthetic approaches to obtain sufficient quantities of defensins for extensive biological characterization To circumvent this limitation and expedite the screening processes, previous studies have designed truncated defensins which are smaller (~ 1.2 kDa), less structurally complex (no disulfide bonds) and more synthetically tractable [8, 15, 17,18,19,20]. These analogs include the γ-core motif (GXCX3-9C, where Xn is the number of residues between cysteines) of target full length defensins [15, 17,18,19,20] (Fig. 1b). Previous activity comparisons between full length defensins and γ-core motif analogs suggest that the γ-core motif can exhibit approximately 10–40% of the activity of full length defensins and can serve as an effective proxy for full length peptides during initial activity screening and characterization [3]
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