Gymnema saponin-induced lipid flip-flop identifies rigid membrane phenotype of methicillin resistant S. aureus and enhances it's antibiotic susceptibility

Our previous study revealed that lipid flip-flop inducing phytochemicals from Gymnema sylvestre increase membrane permeability of antimicrobials in S. aureus. However, their lipid flipping and membrane permeabilizing effect on methicillin resistant S. aureus (MRSA) membrane that has intrinsically higher aminoacylated lipid content compared to methicillin sensitive S. aureus (MSSA) is poorly characterized. Gymnema saponins, gymnemic acid I and IV significantly increased the antibiotic susceptibility in both MSSA and MRSA. MRSA exhibited a rigid membrane with lipid diffusion coefficient 0.0002 μm²/s compared to the MSSA membrane lipids with diffusion coefficient 1.48 μm²/s. Further, unlike MSSA, MRSA cells inhibited fusion of fluid liposomes with their plasma membrane. In vitro assay on reconstituted membrane vesicles revealed that Gymnema saponins induced 60 % lipid flipping in MSSA membrane compared to only 20 % lipid flipping in MRSA, indicating significantly lower Gymnema saponin-induced trans-bilayer lipid mobility in MRSA. Gymnema saponins induced significantly lower crystal violet uptake, release of cellular protein, cell shrinkage and lysis in MRSA compared to MSSA. Gymnema saponins led to dose-dependent inhibition of lipid-aminoacylation in both MSSA and MRSA making their membranes more negative compared to untreated control cells. In silico analysis reveals binding of both gymnemic acid I and IV to multiple peptide resistance factor (binding energy ∼ 7.5 kCal), the protein responsible for lipid aminoacylation in S. aureus. For the first time, our study reveals that MRSA membrane with higher aminoacyl-PG compared to MSSA shows significantly lower rate of diffusion and trans-bilayer flip-flop of lipids. Further, gymnemic acids are useful probes for identification, characterization and drug sensitization of rigid membrane MRSA phenotypes.