The efficacy of the food-grade antimicrobial xanthorrhizol against Staphylococcus aureus is associated with McsL channel expression

Abstract

Background: The emergence and spread of multidrug-resistant Staphylococcus aureus strains demonstrates the urgent need for new antimicrobials. Xanthorrhizol, a plant-derived sesquiterpenoid compound, has a rapid killing effect on methicillin-susceptible strains and methicillin-resistant strains of S. aureus achieving the complete killing of staphylococcal cells within 2 min using 64 μg/mL xanthorrhizol. However, the mechanism of its action is not yet fully understood.

Methods: The S. aureus cells treated with xanthorrhizol were studied using optical diffraction tomography. Activity of xanthorrhizol against the wild-type and mscL null mutant of S. aureus ATCC 29213 strain was evaluated in the time-kill assay. Molecular docking was conducted to predict the binding of xanthorrhizol to the SaMscL protein.

Results: Xanthorrhizol treatment of S. aureus cells revealed a decrease in cell volume, dry weight, and refractive index (RI), indicating efflux of the cell cytoplasm, which is consistent with the spontaneous activation of the mechanosensitive MscL channel. S. aureus ATCC 29213ΔmscL was significantly more resistant to xanthorrhizol than was the wild-type strain. Xanthorrhizol had an enhanced inhibitory effect on the growth and viability of exponentially growing S. aureus ATCC 29213ΔmscL cells overexpressing the SaMscL protein and led to a noticeable decrease in their viability in the stationary growth phase. The amino acid residues F5, V14, M23, A79, and V84 were predicted to be the residues of the binding pocket for xanthorrhizol. We also showed that xanthorrhizol increased the efflux of solutes such as K⁺ and glutamate from S. aureus ATCC 29213ΔmscL cells overexpressing SaMscL. Xanthorrhizol enhanced the antibacterial activity of the antibiotic dihydrostreptomycin, which targets the MscL protein.

Conclusion: Our findings indicate that xanthorrhizol targets the SaMscL protein in S. aureus cells and may have important implications for the development of a safe antimicrobial agent.

Keywords: MscL; Staphylococcus aureus; food-grade antimicrobial; mutants; xanthorrhizol.

Figure 1

Time-kill kinetic curves of xanthorrhizol against MRSA ATCC 33592 or against MSSA ATCC 29213 and ATCC 29213ΔmscL. Exponentially growing cultures of ATCC 33592 (A) or ATCC 29213 and its mscL null mutant (B) were exposed to 64 μg/mL (A) and 32 μg/mL (B) xanthorrhizol and incubated at 37°C for 1 h. Error bars represent standard deviations from three independent experiments.

Figure 2

Maximum RI projection images (A) and quantitative analysis results (B–E) for S. aureus ATCC 29213 cells treated with xanthorrhizol or DMSO over time. The mean RI (B), cytoplasm concentration (C), cell volume (D) and cellular dry mass (E) for the control (n = 11) and xanthorrhizol (n = 12) groups were determined over time. The error bars indicate the standard errors.

Figure 3

Effect of SaMscL protein deficiency or overproduction on the viability of S. aureus ATCC 29213 and S. aureus ATCC 29213ΔmscL in the presence of xanthorrhizol. Growth inhibition by increasing concentrations of xanthorrhizol was shown for exponentially growing S. aureus ATCC 29213, S. aureus ATCC 29213ΔmscL, S. aureus ATCC 29213ΔmscL (pBEP) and S. aureus ATCC 29213ΔmscL (pBEP-SaMscL) cultures (A). The decrease in growth is reflected as the percentage of the OD₆₀₀ of the treated cultures to that of the untreated control. The error bars indicate the standard errors. Time-kill kinetic curves of xanthorrhizol against exponentially growing S. aureus ATCC 29213ΔmscL cells carrying the plasmids pBEP or pBEP-SaMscL were obtained 1 h after IPTG induction (B), as described in the Materials and Methods section. The viability of stationary-phase cultures of S. aureus ATCC 29213, S. aureus ATCC 29213ΔmscL, S. aureus ATCC 29213ΔmscL (pBEP) and S. aureus ATCC 29213ΔmscL (pBEP-SaMscL) was studied after treatment with xanthorrhizol for 6 h (C), where *p < 0.01 (n = 3); **p < 0.002 (n = 6) by a 2-tailed, paired Student’s t test.

Figure 4

Xanthorrhizol increases the potency of DHS in a MscL-dependent manner. The concentration-dependent effects of DHS (A) and xanthorrhizol in the presence of DHS (8 μg/mL) are shown (B) for exponentially growing S. aureus ATCC 29213 and S. aureus ATCC 29213ΔmscL cells. The decrease in growth is reflected as the percentage of the OD₆₀₀ of the treated cultures to that of the untreated control. The error bars indicate the standard errors. The viability of stationary-phase cultures of S. aureus ATCC 29213 and S. aureus ATCC 29213ΔmscL (C) was studied after treatment with 50 μg/mL DHS or a combination of DHS (50 μg/mL) and xanthorrhizol (64 μg/mL) for 6 h, where *p < 0.01 (n = 3); *p < 0.05 (n = 3) by a 2-tailed, paired Student’s t test.

Figure 5

The decrease in the steady-state levels of glutamate and K⁺ in S. aureus ATCC 29213ΔmscL cells treated with xanthorrhizol is associated with the SaMscL protein The glutamate and K⁺ levels in S. aureus ATCC 29213ΔmscL cells harboring the empty expression vector pBEP or pBEP-SaMscL after overnight incubation with xanthorrhizol are shown as percentages of those in the untreated controls. Error bars reflect standard deviations from three independent measurements for glutamate, **p < 0.002; for K⁺, *p < 0.01 by a 2-tailed, paired Student’s t test. The viability of the bacterial cells was measured at the same time and is displayed as a percentage of the viability of the untreated controls. Error bars represent standard deviations from three independent measurements, **p < 0.002 by a 2-tailed, paired Student’s t test.

Figure 6

Molecular modeling of EcMscL with the docking site for xanthorrhizol. Xanthorrhizol binding pocket (A) in the EcMscL complex and an enlarged view of xanthorrhizol relative to the positions of residues F7 and K97 (B). Sequence alignment of the MscLs from E. coli and S. aureus with the corresponding residues (C).

Figure 7

Detection of key amino acid residues involved in the interaction of SaMscL with xanthorrhizol. Time-kill kinetic curves of xanthorrhizol against exponentially growing S. aureus ATCC 29213ΔmscL cells carrying a plasmid (pBEP) with or without mutated mscL genes (A,B). The viability of stationary-phase cultures of S. aureus ATCC 29213ΔmscL carrying the empty vector or expressing wild-type and mutants of SaMscL was studied after treatment with 64 μg/mL xanthorrhizol for 6 h (C).