NH125 kills methicillin-resistant Staphylococcus aureus persisters by lipid bilayer disruption

Abstract

Background: NH125, a known WalK inhibitor kills MRSA persisters. However, its precise mode of action is still unknown.

Methods & results: The mode of action of NH125 was investigated by comparing its spectrum of antimicrobial activity and its effects on membrane permeability and giant unilamellar vesicles (GUVs) with walrycin B, a WalR inhibitor and benzyldimethylhexadecylammonium chloride (16-BAC), a cationic surfactant. NH125 killed persister cells of a variety of Staphylococcus aureus strains. Similar to 16-BAC, NH125 killed MRSA persisters by inducing rapid membrane permeabilization and caused the rupture of GUVs, whereas walrycin B did not kill MRSA persisters or induce membrane permeabilization and did not affect GUVs.

Conclusion: NH125 kills MRSA persisters by interacting with and disrupting membranes in a detergent-like manner.

Keywords: MRSA; NH125; antibiotics; giant unilamellar vesicle; two-component system.

Figure 1.. NH125 eradicates persisters formed by 11 clinical staphylococcal isolates.

Persister cells of 11 clinical staphylococcal isolates were treated with 0.1% DMSO (control) and 5 µg/ml NH125 for 4 h. Persister viability was measured by serial dilution and plating on TSA plates. The asterisks on the x-axis are below the level of detection (2 × 10² CFU/ml). Results are shown as means ± SD; n = 3.

Figure 2.. NH125 shows less hemolytic activity against human erythrocytes compared with 16-BAC.

1% human erythrocytes were treated with twofold serially diluted concentration of NH125 (solid circles) or 16-BAC (open circles) for 1 h at 37°C. A sample treated with 1% Triton-X 100 was used as the control for 100% hemolysis. Results are shown as means ± SD; n = 3.

Figure 3.. Chemical structure of compounds.

(A) NH125, (B) Walrycin B and (C) 16-BAC.

Figure 4.. Inhibition of the WalK/R system does not lead to death of MRSA persisters or rapid membrane permeabilization.

MRSA persisters were treated with 10X MIC NH125 (A), 10X MIC 16-BAC (B), 10X MIC walrycin B (C) and 0.1% DMSO (D). Persister viablity (solid circles) was measured by serial dilution and plating on TSA plates. Membrane permeability (open circles) was measured spectrophotometrically by monitoring the uptake of SYTOX Green (Ex = 485 nm, Em = 525 nm). The data points on the x-axis are below the level of detection (2 × 10² CFU/ml). Results are shown as means ± SD; n = 3.

Figure 5.. Time-lapse fluorescence micrograph showing effects of antimicrobial agents on giant unilamellar vesicles.

Giant unilamellar vesicles consisting of DOPC/DOPG (7:3) labeled with 18:1 Liss Rhod PE (0.05%) were treated with 10X MIC NH125, 10X MIC 16-BAC, 10X MIC Walrycin B and 0.1% DMSO. After adding compounds at t = 0 s, changes of giant unilamellar vesicles were observed for 100 s using a fluorescent microscope (40× objective, Ex = 460 nm, Em = 483 nm).