Structural insights into the combinatorial effects of antimicrobial peptides reveal a role of aromatic–aromatic interactions in antibacterial synergism

Abstract

Edited by Wolfgang PetiThe recent development of plants that overexpress antimicrobial peptides (AMPs) provides opportunities for controlling plant diseases. Because plants employ a broad-spectrum antimicrobial defense, including those based on AMPs, transgenic modification for AMP overexpression represents a potential way to utilize a defense system already present in plants. Herein, using an array of techniques and approaches, we report on VG16KRKP and KYE28, two antimicrobial peptides, which in combination exhibit synergistic antimicrobial effects against plant pathogens and are resistant against plant proteases. Investigating the structural origin of these synergistic antimicrobial effects with NMR spectroscopy of the complex formed between these two peptides and their mutated analogs, we demonstrate the formation of an unusual peptide complex, characterized by the formation of a bulky hydrophobic hub, stabilized by aromatic zippers. Using three-dimensional structure analyses of the complex in bacterial outer and inner membrane components and when bound to lipopolysaccharide (LPS) or bacterial membrane mimics, we found that this structure is key for elevating antimicrobial potency of the peptide combination. We conclude that the synergistic antimicrobial effects of VG16KRKP and KYE28 arise from the formation of a well-defined amphiphilic dimer in the presence of LPS and also in the cytoplasmic bacterial membrane environment. Together, these findings highlight a new application of solution NMR spectroscopy to solve complex structures to study peptidepeptide interactions, and they underscore the importance of structural insights for elucidating the antimicrobial effects of AMP mixtures.Despite progress in the field of antimicrobial therapeutics (1, 2), plant production remains profoundly affected by microbial disease outbreaks caused by pathogens of Xanthomonas and Pseudomonas species, leading to considerable losses in crop production and challenges for global food security (3). Finding new approaches for the management of microbe-borne diseases in plants remains a challenge, further complicated by the increasing occurrence of multidrug-resistant pathogens ("superbugs") that are immune to available treatments (4, 5). Additionally, commonly used bactericides and fungicides pose considerable environmental constraints (6), necessitating the development of sustainable management techniques that can effectively combat such pathogens.Antimicrobial peptides (AMPs) 3 represent the oldest domain of the evolutionary tree, being structurally and functionally conserved across all forms of life (7-10). While having attracted research interest for several decades, peptide-based antimicrobial therapy has seen accelerated attention during the last few years, motivated by its potential applicability against drug-resistant strains (11)(12)(13)(14)(15). The direct interaction of AMP with bacterial membranes and membrane components provides broad-spectrum antimicrobial effects (16), and for many AMPs also anti-inflammatory and oth...

Antimicrobial peptides: mechanism of action, activity and clinical potential

“…The combination of magainin 2 and tachyplesin 1 also enhances the bacterial membrane recognition by constituting the oligomeric structures before contacting the anionic bacterial membrane surface [ 378 ]. In addition, a recent study from Bhunia’s group reported that two AMPs, VG16KRKP and KYE28, exhibited synergistic antimicrobial effects against plant pathogens and proteases through formatting an unusual peptide complex [ 379 ].…”

Thanatin: An Emerging Host Defense Antimicrobial Peptide with Multiple Modes of Action

“…Three-D structures of AMPs can be determined in solutions of LPS micelles using tr-NOESY NMR [ 61 , 62 , 63 , 64 ]. Also, the LPS-interacting residues of AMPs are determined at atomic resolution by STD-NMR [ 65 , 66 , 67 , 68 ].…”

Effect of Secondary Structure and Side Chain Length of Hydrophobic Amino Acid Residues on the Antimicrobial Activity and Toxicity of 14‐Residue‐Long de novo AMPs

“…[19][20] AMPs are present ubiquitously in nature as the first line of defence in several life forms from invertebrates to the plants or mammals. [21][22][23][24] Natural AMPs show broad-spectrum antimicrobial activity against microbes like bacteria, fungi, protozoa and even several classes of pathologically relevant viruses. [25] AMPs have an additional strength over the conventional antibiotics in having diverse modes of action in contrast to the fixed intracellular targets for antibiotics.…”

“…AMPs are present ubiquitously in nature as the first line of defence in several life forms from invertebrates to the plants or mammals [21–24] . Natural AMPs show broad‐spectrum antimicrobial activity against microbes like bacteria, fungi, protozoa and even several classes of pathologically relevant viruses [25] .…”