Physalin H, physalin B, and isophysalin B suppress the quorum-sensing function of Staphylococcus aureus by binding to AgrA

Abstract

The virulence of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), depends on the expression of toxins and virulence factors controlled by the quorum-sensing (QS) system, encoded on the virulence accessory gene regulator (agr) locus. The aim of this study was to identify a phytochemical that inhibits Agr-QS function and to elucidate its mechanism. We screened 577 compounds and identified physalin H, physalin B, and isophysalin B--phytochemicals belonging to physalins found in plants of the Solanaceae family--as novel Agr-QS modulators. Biological analyses and in vitro protein-DNA binding assays suggested that these physalins suppress gene expression related to the Agr-QS system by inhibiting binding of the key response regulator AgrA to the agr promoters, reducing the function of hemolytic toxins downstream of these genes in MRSA. Furthermore, although physalin F suppressed gene expression in the Agr-QS system, its anti-hemolytic activity was lower than that of physalins H, B, and isophysalin B. Conversely, five physalins isolated from the same plant with the ability to suppress Agr-QS did not reduce bacterial Agr-QS activity but inhibited AgrA binding to DNA in vitro. A docking simulation revealed that physalin interacts with the DNA-binding site of AgrA in three docking states. The carbonyl oxygens at C-1 and C-18 of physalins, which can suppress Agr-QS, were directed to residues N201 and R198 of AgrA, respectively, whereas these carbonyl oxygens of physalins, without Agr-QS suppression activity, were oriented in different directions. Next, 100-ns molecular dynamics simulations revealed that the hydrogen bond formed between the carbonyl oxygen at C-15 of physalins and L186 of AgrA functions as an anchor, sustaining the interaction between the carbonyl oxygen at C-1 of physalins and N201 of AgrA. Thus, these results suggest that physalin H, physalin B, and isophysalin B inhibit the interaction of AgrA with the agr promoters by binding to the DNA-binding site of AgrA, suppressing the Agr-QS function of S. aureus. Physalins that suppress the Agr-QS function are proposed as potential lead compounds in the anti-virulence strategy for MRSA infections.

Keywords: Agr-QS modulator; AgrA-DNA inhibition; MRSA; anti-hemolytic activity; molecular docking; molecular dynamics simulation; physalins.

FIGURE 1

Physalin H suppresses Agr-QS function regardless of the subgroups. (A) The structure of physalin H. (B) Relative P3 promoter activity (solid line) and relative growth (dot line) when cultured for 24 h without (black) and with (red) autoinducer peptide (AIP) added to media containing serial concentrations of physalin H. P3 promoter activity is expressed relative to that in culture with 1% DMSO, considered as 100% (n = 3). Data are presented as the mean ± standard deviation (SD). (C) Normalized RNAIII, agrA, hla, and psm α expression during 8-h culture of LAC with 100 μM physalin H (red) and 1% DMSO (black) (n = 3). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, unpaired t-test. (D) Activity values for 50% hemolyzing red blood cells in culture supernatants of LAC cultured for 4, 8, and 24 h with 100 μM physalin H (red) and 1% DMSO (black) (n = 3). Data are presented as the mean. **p < 0.01, ns, not significantly different, unpaired t-test. (E) Growth curve for 24-h culture of LAC strain with 50 (green), 100 (blue), and 200 (red) μM physalin H and 1% DMSO (black). OD₆₂₀ of 24-h medium with 200 μM physalin H is indicated as a control medium containing physalin H (dot line). (F) Normalized RNAIII expression in the 8-h culture of Agr-I strain M1K051, Agr-I I strain M1K155, and Agr-III strain CN02 with 100 μM physalin H (red) and 1% DMSO (black) (n = 3). **p < 0.01, unpaired t-test.

FIGURE 2

Physalin F, physalin B, and isophysalin B can suppress Agr-QS expression. (A) Relative P3 promoter activity and growth during 24-h culture with 100 μM physalins (PS) two to nine% and 1% DMSO (n = 3). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ns, not significantly different, unpaired t-test. (B) Relative P3 promoter activity (solid line) and relative growth (dot line) during 24-h culture with serial concentrations of PS2 (physalin F, red), PS3 (physalin B, blue), and PS4 (isophysalin B, green). P3 promoter activity is expressed relative to activity in cultures without PSs, considered as 100% (n = 3). Data are presented as the mean ± SD. (C) Normalized RNAIII expression (bar) and growth (line) during 8-h culture of LAC with 50 μM PS2, 100 μM PSs3–9 and 1% DMSO (n = 3). Data are presented as the mean ± SD. **p < 0.01, unpaired t-test. (D) Activity values for 50% hemolyzing red blood cells in culture supernatants of LAC cultured for 24 h with 50 μM PS2, 100 μM PSs3–9 and 1% DMSO (n = 3). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, unpaired t-test. (E) OD₆₂₀ after 24-h culture with LAC strain at 2.5×10⁵ CFU/mL with serial concentrations of PSs2–9 and DMSO.

FIGURE 3

Physalins inhibit the binding of AgrAc to DNA. (A) (B) Effect of increasing concentrations of physalin H (31.25–500 μM) on His⁶-AgrAc–Cy5-labeled 100-bp (A) and 19-bp (B) oligonucleotide complex formation by electrophoretic mobility shift assay (EMSA). (C) Effect of 500 μM PSs1–9, on AgrAc–Cy5-labeled 100-bp oligonucleotide complex formation by EMSA.

FIGURE 4

Physalins that suppress Agr-QS interact in a different state of interaction with the site of AgrA from physalins without the ability of Agr-QS suppression. (A) In silico docking of physalin H to AgrAc from Staphylococcus aureus (PDB ID: 4G4K). Space-filled representation of the C-terminal AgrA binding domain (blue) bound to target DNA. Surface residues that directly interact with DNA are shown in dark blue. Physalin H is represented as a stick. An enlarged view of the boxed area shows the physalin H-docking site and surrounding residues. The dotted line indicates H-bound between ketone structure at C-1 and C-18 of physalin H, and R198 and N201 of AgrA. (B) Docking state A of four physalins with Agr-QS-suppression activity. Physalin H (green), PS2 (cyan), PS3 (magenta), and PS4 (yellow). (C) Two-dimensional (2D) interaction of AgrA and physalin H. (D) Docking state B of three physalins without Agr-QS-suppression activity. PS5 (green), PS6 (cyan), and PS7 (magenta). (E) 2D interaction of AgrA and PS5. (F) Docking state C of two physalins without Agr-QS-suppression activity. PS8 (green) and PS9 (cyan). (G) 2D interaction of AgrA and PS8.

FIGURE 5

Molecular dynamics analysis of three physalin conformations bound to AgrA. (A–C) The results of protein root mean square derivation (RMSD) (black line) and ligand RMSD (red line). (D–F) The root mean square fluctuation (RMSF) plot of residue index. The red position indicates the residue index of AgrA bound by the ligand. (G–I) The residues of AgrA interacted with physalins. The results of RMSD, RMSF, and interaction of physalin H (A, D, G), PS5 (B, E, H), and PS8 (C, F, I) bound to AgrA.

FIGURE 6

Simulated complex of the DNA binding region of AgrA in the presence of physalin H (A) and PS5 (B) at 0, 20, 40, 60, 80, and 100 ns.