Lateral membrane organization as target of an antimicrobial peptidomimetic compound

Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.</p

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1–3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1–3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1–3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates

AFM images and AFM force curves characterizing S. aureus lipid membrane upon interaction with antimicrobial peptidomimetic AMC-109, Mass Spectroscopy data of S. aureus lipid extracts: Experimental data for Lateral membrane organization as target of an antimicrobial peptidomimetic compound 2023

Atomic force microscopy (AFM) data characterizing the interaction of antimicrobial peptidomimetic AMC-109 with the lipid membrane extracted from bacteria Staphylococcus aureus. All experiments were done on membranes deposited on mica surface in PBS buffer at room temperature. AFM data were measured using JPK Nano Wizard Ultra Speed AFM. AFM raw images are presented in their JPK software format. Each AFM force-distance curve was calibrated according to a hard-response calibrating curve collected prior to each indentation of the membrane. These calibrated curves were exported in TXT format. Each TXT file contains 2 data sets corresponding to the approach and retract force-indentation curve one after another, organized in 2 columns showing "Vertical Tip Position" and "Vertical Deflection". Mass Spectroscopy data of S. aureus lipid extracts in their raw format gathered using an Ultra-high performance liquid chromatography-mass spectroscopy instrument consisting of Accela1250 UHPLC system (Thermo Fisher Scientific) coupled to a Thermo Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with an ESO ion-source in negative ionization mode. Detailed description of the individual folders: Folder "Figure 1": AFM raw images of S. aureus lipid membrane before and after step by step addition of antimicrobial peptidomimetic AMC-109. Folder "Figure S2": AFM raw images of S. aureus lipid membrane that is expanding in time after treatment with antimicrobial peptidomimetic AMC-109. Folder "Figure S3": AFM raw images of S. aureus lipid membrane before and after addition of antimicrobial peptidomimetic AMC-109 in a concentration above its minimal inhibitory concentration . Folder "Figure S5": AFM force-indentation curves gathered on S. aureus lipid membranes without any treatment and after treatment with subcritical concentrations of the antimicrobial AMC-109. The force-indentation curves were used to characterize the mechanical properties of the membrane upon the antibiotic treatment. The folder "Figure S5" contains three subfolders that contain individual force-indentation curves measured on S. aureus lipid membranes exposed to no AMC-109 (0_ug-ml_AMC-109), 0.5 ug/ml AMC-109 (0p5_ug-ml_AMC-109), and 1 ug/ml AMC-109 (1_ug-ml_AMC-109). Folder "Figure S7": AFM raw images of AMC-109 layer forming on the mica support. Mica was exposed to the PBS buffer with 2 ug/ml AMC-109, this layer was scratched by contact imaging with the AFM tip. The AFM images show the recovery of the AMC-109 layer on mica. Folder "Figure S14": AFM raw images of the S. aureus lipid membrane before and after the exposure to 0.023 vol% of DMSO. Folder "Table 1 and Figure S1": Mass Spectroscopy data of S. aureus lipid extracts in their raw format

Lateral membrane organization as target of an antimicrobial peptidomimetic compound

Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.</p