The antimicrobial peptide Magainin-2 interacts with BamA impairing folding of E. coli membrane proteins

Abstract

Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans.

ATP‐independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next‐generation therapeutics

“…The antimicrobial peptide magainin‐2 (Mag‐2) from Xenopus laevis binds BamA with high affinity, and impairs its function as the insertase for nOMPs (di Somma et al, 2022). Mag‐2 also disrupts the membrane by pore formation, triggering cell death (Billah et al, 2022; Maher & McClean, 2006).…”

“…Molecular docking analyses showed that Mag‐2 binds the lumen of BamA, thereby, either blocking OMPs from folding inside the BamA β‐barrel, or hindering the conformational changes of BamA required during OMP folding. OMP levels decrease after Mag‐2–BamA binding (di Somma et al, 2022). Mag‐2 however, displays cytotoxicity (Wade et al, 2022).…”

“…Hence, the essential nature and accessibility of the BAM complex makes it a novel and attractive target in today's scenario with widespread antibiotic resistance (Lewis, 2020; Lithgow et al, 2023; Waller et al, 2023). Both natural and synthetic inhibitors of the BAM complex (Overly Cottom et al, 2023) (Table 1 and Figure 6) have recently been tested as potential therapeutics (Di Somma et al, 2022; Ghequire et al, 2018; Hagan et al, 2015; Hart et al, 2019; Imai et al, 2019; Li et al, 2020; Luther et al, 2019; Miller et al, 2022; Overly Cottom et al, 2023; Steenhuis, van Ulsen, et al, 2021; Storek et al, 2018; Urfer et al, 2016; Xu et al, 2023). These target either (Nikaido, 2003) structural components of BamA accessible extracellularly, or (Bos et al, 2007) essential interactions between BAM complex components, such as BamA–BamD interactions (Figures 6 and 7) (Overly Cottom et al, 2023; Srinivas et al, 2010; Xu et al, 2023).…”

“…Given the resurgence of drug‐resistant Gram‐negative bacterial pathogens, the BAM complex is being actively examined as a novel therapeutic target for inhibitor design and screens of peptidotherapeutics (Di Somma et al, 2022; Diederichs et al, 2021; Doyle & Bernstein, 2022; Ghequire et al, 2018; Hagan et al, 2015; Hart et al, 2019; Imai et al, 2019; Li et al, 2020; Luther et al, 2019; Miller et al, 2022; Overly Cottom et al, 2023; Steenhuis, van Ulsen, et al, 2021; Storek et al, 2018; Tomasek & Kahne, 2021; Urfer et al, 2016; Xu et al, 2023). Most recent advances in the identification and design of inhibitors that specifically target this essential bacterial protein (Haysom et al, 2023; Miller et al, 2022; Overly Cottom et al, 2023; Seyfert et al, 2023; Steenhuis, van Ulsen, et al, 2021; Xu et al, 2023) now offer promise in successfully developing translational therapeutics for drug‐resistant bacterial pathogens.…”

Molecular Aspects of the Functioning of Pathogenic Bacteria Biofilm Based on Quorum Sensing (QS) Signal-Response System and Innovative Non-Antibiotic Strategies for Their Elimination

“…Antimicrobial properties of NAMPs, including gramicidin S from B. brevis [ 210 ], polymyxin B and A from B. polymyxa or vancomycin produced by S. orientalis [ 211 ], have been reported in several studies [ 207 , 212 ]. Similarly, magainin-2 extracted from amphibians, such as frog skin, showed antibacterial activity against MDR strains, protozoa, yeasts, and fungi [ 213 ]. Crotalicidin extracted from rattlesnakes killed 90% of E. coli and P. aeruginosa cells within 90–120 min and 5–30 min, respectively [ 214 ].…”