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.
Antimicrobial peptides (AMPs) are small molecules consisting of 10–100 amino acid residues (Moretta et al., 2020) produced by all organisms. In the last few years, these compounds are being considered as new alternative therapeutic agents for their rapid bactericidal activity, their broad spectrum of action against both Gram-positive and Gram-negative bacteria, fungi and viruses, and their immunomodulatory activity (Amerikova et al., 2019).
AMPs are usually cationic, with a highly positive net charge (+2 to +9), and amphipathic molecules, as their structure includes both hydrophobic and hydrophilic moieties (Torres et al., 2019). They can display powerful antimicrobial activities against antibiotic-resistant bacteria acting at the level of bacterial membranes with several mechanisms, depending on the molecular properties of the peptides themselves and the lipid composition of the membranes (Zhang et al., 2021). Their specific mode of action differs from those of common drugs, thus not allowing the development of drug resistance (Moravej et al., 2018).
Biophysical studies led to hypothesize three models that can explain the membrane disruption by AMPs: barrel-stave, toroidal pore and carpet mechanisms (Di Somma et al., 2020). According to these mechanisms, antimicrobial peptides interact with bacterial outer membranes, perturbing their integrity and causing their disaggregation (Huang, 2006). Most of AMPs show pore formation after binding to the membrane surface. X-ray crystallization studies and spectroscopic analyses reported that Alamethicin, a channel-forming peptide, insert into the lipid bilayer to form a barrel-stave pore structure consisting of eight alamethicin helices (Qian et al., 2008). Melittin, a peptide isolated from bee venom, is a basic amphiphilic peptide, which mainly acts on the lipid matrix of membranes. Fluorescence studies with phospholipid vesicles reported the formation of pore coupled with the translocation of peptide across the lipid bilayer (Lohner and Blondelle, 2005).
Magainins, a group of AMPs derived from the African clawed frog Xenopus laevis, exhibit a net positive charge showing greater selectivity for negatively charged sites on the microbial membranes (McMillan and Coombs, 2020). Peptide binding is favoured by both hydrophobic interactions between non-polar amino acids and the hydrophobic nucleus of the membrane and electrostatic interactions between the positive charges of the peptides and the negative charges of lipids (Bahar and Ren, 2013). Although the mechanism of action is not yet fully understood at molecular level, it is known that Magainin-2 (Mag-2) exerts its antimicrobial activity according to a toroidal mechanism (Billah et al., 2022). The peptide induces a high curvature in the bacterial membrane leading to the formation of pores causing membrane dysfunction and the loss of essential contents from the inside of the cell, eventually leading to cell death (Dennison et al., 2014).
However, the molecular events underlying this mechanism are still far from being understood. Investigation of Mag-2 behaviour is mainly focused on the interaction of the peptide with lipid membrane components and the lipopolysaccharide (LPS) molecules (Ding et al., 2003). In this work, we elucidated the mechanism of action of Mag-2 on Escherichia coli used as a model with the aims to a) demonstrate that our approach was effective in defining the mechanism of action of this AMP at the molecular level and b) find a possible target to be exploited by antimicrobial compound(s) devoid of dangerous collateral effects. Functional proteomics experiments were exploited for the identification of Mag-2 protein targets identifying several membrane proteins as putative interactors. Among these, we demonstrated a specific interaction of the peptide with BamA, an outer membrane protein belonging to the BAM complex. In Gram-negative bacteria, the BAM complex is responsible for the folding and insertion of nascent β-barrel Outer Membrane Proteins (OMPs) in the outer membrane (Tomasek et al., 2020). The interaction of Mag-2 with BamA was predicted by molecular docking analysis, leading to the identification of peptide-protein interface involved in the binding. This portion of the BamA protein was produced in recombinant form and the peptide-protein interaction was confirmed in vitro by fluorescence assay Light Scattering Measurements. Finally, the functional role of Mag-2/BamA interaction in impairing the folding of the membrane protein OmpA was investigated by both western blot experiments and tandem mass spectrometry analyses carried out in Multiple Reaction Monitoring (MRM) scan mode. These approaches demonstrated a clear decrease in E. coli OmpA production following incubation with Mag-2. Since Mag-2 has significant toxicity against human cells, these observations can lead to the development of new peptides or peptidomimetic drugs based on the specific interaction with BamA, considered a potential target for novel antibiotics.
E. coli cells were inoculated in 10 ml of LB liquid medium (Luria-Bertani) and incubated at 37°C for 16 h under stirring. Then, bacterial cells were grown in 1 L at 37°C under stirring for 3 h. The pellet was recovered by centrifugation at 4°C for 15 min at 5,000 rpm, resuspended in 5 ml of Cell Lysis Buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 4 mM DTT, 1 mM PMSF) and subjected to mechanical lysis by sonication. The sample was centrifuged at 4°C for 30 min at 15,000 rpm to pellet unlysed cells and cellular debris and the recovered supernatant was ultracentrifuged for 2 h at 4°C at 54,000 rpm. The obtained pellet was resuspended in 1 ml per gram of pellet of membrane resuspension buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% Glycerol, 4 mM DTT, 1 mM PMSF, 6 mM 3 [(3-Cholamidopropyl) dimethylammonium]-1-propanesulfonate (CHAPS) under stirring at 4°C for 16 h. The sample was ultracentrifuged at 4°C for 2 h at 54,000 rpm. The supernatants containing the solubilized membrane proteins were collected (Newby et al., 2009).
The pull-down experiment was performed using 200 μL of dried avidin-conjugated agarose beads. A resin with free agarose beads was used for the pre-cleaning, and a resin incubated with a solution of 2 mg/ml of biotinylated Mag-2 for 30 min at 4°C under stirring was used for the pull-down assay. The supernatant was removed by centrifugation at 4°C for 10 min at 3,000 rpm and the resin equilibrated with five volumes of binding buffer at 4°C. On the pre-cleaning resin, 2.5 mg of membrane proteins were incubated at 4°C for 2 h under stirring to remove possible non-specific interactors. Indeed, only the supernatant containing the unbound membrane proteins was recovered by centrifugation at 4°C for 10 min at 3,000 rpm and then transferred on the pull-down resin for 3 h at 4°C under stirring. The beads were washed with five volumes of binding buffer and the peptide-interacting proteins were released by competitive elution with 500 µL of elution buffer containing an excess of biotin (2 mM) for 1 h at 4°C under stirring. Mag-2 putative protein interactors were fractionated by SDS-PAGE and the protein bands from both the sample and the control lanes were excised from the gel and subjected to in situ hydrolysis with trypsin. The resulting peptide mixtures were directly analyzed by Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) using an LTQ Orbitrap XLOrbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) and the obtained data were used to search for a non-redundant protein database using an inhouse version of the Mascot software, leading to the identification of the putative AMP protein interactors. Proteins identified both in the control and in the sample were discarded, whereas those occurring in the sample and absent in the control were considered as putative Mag-2 interactors. The putative peptide interactors were gathered within functional pathways by the bioinformatic tools DAVID (Huang da et al., 2009), KEGG (Kanehisa et al., 2017), and STRING (Szklarczyk et al., 2021).
The putative binding sites of Mag-2 peptide on BamA protein were determined through molecular docking calculations. Both peptide and protein have been modelled using I-TASSER Server (Roy et al., 2010; Yang et al., 2015; Yang and Zhang, 2015). The Magainin-2-BamA complex model has been obtained using PatchDock Server (Schneidman-Duhovny et al., 2005) and the structures have been refined with FireDock Server (Mashiach et al., 2008), which also gives the Global Energy, the Attractive and Repulsive Van der Waals (VdW) forces and the Atomic Contact Energy (ACE) values of the complex. All the interactions and the amino acids involved at the interface were determined using the PDBsum Server (Laskowski, 2001; Laskowski, 2009; De Beer et al., 2014). The Gibbs free energy, ΔG, and the dissociation constant, Kd, of the protein-peptide complex have been predicted using the PRODIGY webserver (Xue et al., 2016). All the figures have been generated through UCSF CHIMERA software (Pettersen et al., 2004).
6XHis-tagged E. coli BamAp5 consisting of the β-barrel domain and the fifth POTRA domain (residues 344–810) was expressed in the pet30b_BamA_PD5 plasmid in E. coli BL21 cells and then cells were used to inoculate 1L of LB culture media with 50 μg/ml kanamycin. The culture was incubated at 37°C until the ODλ = 600 reached 0.5 OD/mL value and then protein expression was induced by adding Isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The culture was incubated at 37°C for 3 h. Cells were then harvested by centrifugation for 15 min at 4°C at 5,000 rpm and the obtained pellets were resuspended in lysis buffer (150 mM NaCl,
