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Virulence Inhibitors From Brazilian Peppertree Block Quorum Sensing and Abate Dermonecrosis in Skin Infection Models

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Virulence Inhibitors From Brazilian Peppertree Block Quorum Sensing and Abate Dermonecrosis in Skin Infection Models

Amelia Muhs et al. Sci Rep.

Abstract

Widespread antibiotic resistance is on the rise and current therapies are becoming increasingly limited in both scope and efficacy. Methicillin-resistant Staphylococcus aureus (MRSA) represents a major contributor to this trend. Quorum sensing controlled virulence factors include secreted toxins responsible for extensive damage to host tissues and evasion of the immune system response; they are major contributors to morbidity and mortality. Investigation of botanical folk medicines for wounds and infections led us to study Schinus terebinthifolia (Brazilian Peppertree) as a potential source of virulence inhibitors. Here, we report the inhibitory activity of a flavone rich extract "430D-F5" against all S. aureus accessory gene regulator (agr) alleles in the absence of growth inhibition. Evidence for this activity is supported by its agr-quenching activity (IC50 2-32 μg mL-1) in transcriptional reporters, direct protein outputs (α-hemolysin and δ-toxin), and an in vivo skin challenge model. Importantly, 430D-F5 was well tolerated by human keratinocytes in cell culture and mouse skin in vivo; it also demonstrated significant reduction in dermonecrosis following skin challenge with a virulent strain of MRSA. This study provides an explanation for the anti-infective activity of peppertree remedies and yields insight into the potential utility of non-biocide virulence inhibitors in treating skin infections.

Conflict of interest statement

C.L.Q. and J.T.L. are named inventors on a provisional patent application concerning the technology presented in this paper. The authors confirm that any competing interests do not alter their adherence to all the Nature Publishing Group policies on sharing data and materials.

Figures

Figure 1
Figure 1. An exotic pest plant to some, a valued source of medicine to others.
Schinus terebinthifolia Raddi is classified as a Category I pest plant by the Florida Exotic Pest Plant Council. Efforts to remove it from the United States have included the use of the herbicides triclopyr and glyphosate. On the other hand, its value as a medicinal plant has been broadly reported in South America. (a) S. terebinthifolia in fruit (Photo Credit: CL Quave). (b) Written historical records of the medicinal uses of Schinus sp. plant date back to 1648, appearing in the Historia Naturalis Brasiliae by Dutch naturalist, Willem Piso. A full translation of this section of text is provided (Suppl. Table 1). (c) Historical traditional medicinal uses and preparations of different tissues from S. terebinthifolia.
Figure 2
Figure 2. Isolation scheme of bioactive fraction 430D-F5.
(a) The bioassay-guided fractionation scheme is illustrated, demonstrating the path from crude fruit extract to refined bioactive fraction 430D-F5. Percent yields of extracts in relation to starting dry plant material are represented at each separation step. The most active fractions at each step are highlighted in bold. (b) The corresponding HPLC chromatograms for the most active fractions demonstrate an increase in relative abundance of peaks at a retention time range of 45–50 min.
Figure 3
Figure 3. 430D-F5 inhibits agr activity for all four alleles in a non-biocide manner.
(a) Data are represented as percent agr activity or growth of the vehicle (DMSO) control at 24 hours. The solid lines represent agr activity, measured by fluorescence, and the dashed lines represent growth, measured using OD600. See Suppl. Table 3 for additional data. (b) Minimum inhibitory concentrations (MICs) for 430 and lead fractions, reported as μg mL−1.
Figure 4
Figure 4. 430D-F5 inhibits δ-toxin and α-hemolysin production in a dose-dependent manner.
(a) Levels of δ-toxin were quantified by HPLC analysis of culture supernatant following treatment with sub-MIC50 concentrations of 430D-F5. S. epidermidis strain is NRS101, all others are S. aureus. Refer to Suppl. Table 5 for full strain details. Results are expressed as the peak area, normalized for optical density (600 nm) at the time of supernatant harvest. Statistical significance is denoted as *P-value < 0.05, P < 0.01, P < 0.001. (b) Western blot analysis displays dose dependent inhibition of α-hemolysin production in USA300 strain LAC (c) Red blood cells exposed to supernatants from 430, 430D and 430D-F5 treated cultures demonstrate dose-dependent inhibition of hemolysis by S. aureus (LAC) toxins. All treatments are significant in comparison to wild type vehicle control at P < 0.001. (d) Dose dependent reduction in hemolysis is also evident in 430D-F5 treated supernatants from a hla mutant strain (AH1589). All treatments are significant in comparison to wild type vehicle control at P < 0.001. No significant growth inhibition in comparison to the vehicle control was observed.
Figure 5
Figure 5. General toxicity of S. aureus supernatants to HaCaTs.
An immortalized line of human keratinocytes was treated with supernatants of S. aureus (NRS385) that were grown +/− 430D-F5 or vehicle (DMSO). Controls with either no supernatant added or staurosporine added were also examined. Both the (a) fluorescent microscopy (200X) and (b) LDH assay demonstrated that treated cultures lacked the suite of exotoxins in their supernatants, and thus did not impact HaCaT viability.
Figure 6
Figure 6. Impact of 430D-F5 on S. aureus biofilm formation and planktonic growth in a biofilm model.
USA 200 isolate UAMS-1 and its isogenic sarA mutant (UAMS-929) were used in the biofilm assay. (a) Images of crystal violet stained biofilm in 96-well plates. (b) The optical density (OD595nm) of the crystal violet eluent is plotted with the OD600nm for planktonic cells, measured by transfer of the well supernatants to a new 96-well plate. Statistical significance in comparison to the vehicle treated wild type control is denoted as *P-value < 0.05, P < 0.01, P < 0.001.
Figure 7
Figure 7. Extracts are non-toxic to human cells and mouse skin at the concentration required for quorum quenching activity.
(a) Extracts were tested for cytotoxicity using a LDH assay against HaCaT cells at a concentration range of 6–734 μg mL−1, with the vehicle (DMSO) not exceeding 1.5% of the well volume. The extracts were non-toxic at concentrations necessary for agr inhibition. The IC50 values for 430, 430D, and 430D-F5 were 734, 367, and 184 μg mL−1, respectively (data not shown). No IC90 values were established at this concentration range. (b) There was no significant difference in weight gains or losses between mice injected with 50 μg of 430D-F5 or saline. (c) No skin irritation or injury were grossly apparent following a 50 μg injection of 430D-F5 into healthy mouse skin (ruler in cm). Photo taken on Day 1.
Figure 8
Figure 8. 430D-F5 mediates quorum quenching in vivo and attenuates MRSA-induced dermatopathology in a murine model of skin and soft tissue infection.
(a) BALB/c mice were intradermally challenged with an inoculum mixture containing 1 × 108 CFUs of MRSA (LAC) along with either 50 μg of 430D-F5 or the vehicle control (DMSO). Representative images of the resulting cutaneous injuries sustained in 430D-F5 and vehicle control mice are shown for days 3 and 7 post-infection (scale in cm). (b) A single 50 μg dose of 430D-F5 profoundly attenuates dermatopathology following cutaneous MRSA challenge. (c) 430D-F5 reduces morbidity as measured by animal weight. (d) To determine if 430D-F5 inhibits quorum sensing in vivo, mice were challenged intradermally with an agr P3-lux reporter strain (AH2759) +/− 430D-F5 and agr-driven bioluminescence was measured at the indicated time points via IVIS imaging. Quorum sensing peaks at 3 hr post injection, and a single injection of 430D-F5 exhibits significant inhibition of the system for the first 4 h post-injection. (e) In vivo monitoring demonstrates that 430D-F5 quenches quorum sensing signaling. This image was taken at 3 hr post challenge, during the peak period for agr activity. Significant differences between treatment and vehicle are represented as: *P < 0.05; P < 0.01; P < 0.001.
Figure 9
Figure 9. Characterization of 430D-F5 major constituents.
(a) LC-FTMS ESI negative and positive base peak chromatograms for 430D-F5. All peaks correspond to data presented in Suppl. Table 4. (b) Putative structural matches are listed by peak number. Peak 2 was determined to be C30H17O10 and putative structural matches include: (2a) amentoflavone, (2b) agathisflavone, and (2c) robustaflavone. Peak 4 was determined to be C30H21O10 and putative structural matches include: (4a) chamaejasmin, (4b) tetrahydroamentoflavone, and (4c) tetrahydrorobustaflavone. Peak 14 was determined to be C30H45O4 and putative structural matches include: (14a) albsapogenin, (14b) (13α,14β,17α,20 R,24Z)-3α-hydroxy-21-oxolanosta-8,24-dien-26-oic acid, (14c) (13α,14β,17α,20 S,24Z)-3α-hydroxy-21-oxolanosta-8,24-dien-26-oic acid, (14d) (3α,13α,14β,17α,24Z)-3-hydroxy-7-oxo-lanosta-8,24-dien-26-oic acid, and (14e) mollinoic acid. Peak 19 was determined to be C30H45O4 and putative structural matches include (19) isomasticadienonalic acid.

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