Mechanisms of Rapid Bactericidal and Anti-Biofilm Alpha-Mangostin  Activity against

Staphylococcus aureus is considered one of the leading pathogens of hospital and community-acquired infections worldwide and causes acute diseases, such as pneumonia, endocarditis, and bacteremia (Tong et al. 2015). Moreover, S. aureus commonly forms biofilms on artificial implants, such as indwelling catheters, orthopaedic implants, and heart valves (Donlan 2001; Tande and Patel 2014; Cyphert and von Recum 2017; Hall and Mah 2017; Otto 2018). A systematic review of wound studies found a 78.2% prevalence of biofilms in chronic wounds and established biofilm infection as a significant cause of delayed wound healing (Roy et al. 2020). The most common bacterial strains in these infected wounds were S. aureus and Pseudomonas aeruginosa (Ghoreishi et al. 2022). Established biofilms are greatly recalcitrant to antibiotic therapy and tremendously increase medical cost and morbidity (Mah and O’Toole 2001; Stewart and Franklin 2008; Kalil et al. 2016). Due to the high incidence rate of biofilm-related S. aureus infections, it is urgent to develop novel antibacterial agents, especially anti-biofilm antimicrobials.

Recently, many natural products have been utilized as antimicrobials against S. aureus infections (Iinuma et al. 1996; Gibbons 2008; Kali 2015). For example, α-mangostin obtained from pericarps of mangosteen, has bright potential and prospects owing to its remarkable biological activities, such as anticancer, anti-allergy, antiviral, antibacterial, antioxidant, and anti-inflammatory (Chen et al. 2018; Chavan and Muth 2021). α-Mangostin has been demonstrated to exhibit rapid bactericidal and anti-biofilm activities against S. aureus (Phuong et al. 2017; Felix et al. 2022).

At present, many researchers have explored that α-mangostin has bactericidal activities against S. aureus by increasing the permeability of cell membrane, but it is still unclear which genes or proteins are included in this process (Koh et al. 2013; Lin et al. 2020; Meah et al. 2020; Felix et al. 2022). Many genes are associated with the formation of persister cells and biofilms of S. aureus, such as norA, norB, dnaK, groE, and mepR, which are down-regulated by α-mangostin (Felix et al. 2022). While another study found that the expression of ebpS and fnbB was regulated by α-mangostin, but noticeable differences were also observed between different S. aureus strains (Nguyen et al. 2021). Therefore, the present research intends to investigate mechanisms of α-mangostin against S. aureus through genome sequencing and proteomics analysis.

Experimental

Materials and Methods

Bacterial isolates and culture conditions. There were 328 non-duplicate S. aureus clinical isolates comprised of 138 methicillin-resistant S. aureus (MRSA) and 190 methicillin-sensitive S. aureus (MSSA) isolates used in this study. These isolates were obtained from Shenzhen Nanshan People’s Hospital, China, from January 2010 to December 2017. All clinical isolates were simultaneously identified by the BD Phoenix^™ 100 automated microbiology system (BD, USA) and mass spectrometer (IVD MALDI Biotyper^®, Germany). S. aureus isolates were grown in cation-adjusted Mueller-Hinton broth (CAMHB) at 37°C with shaking for antimicrobial susceptibility and time-killing assays. For biofilm assay, S. aureus isolates were grown in TSBG (tryptic soy broth with 0.5% glucose) at 37°C. The Ca²⁺ (50 mg/l) was added to CAMHB media in the experiments with daptomycin.

Chemicals and natural products. Vancomycin hydrochloride (catalogue no. HY-17362), linezolid (catalogue no. HY-10394), daptomycin (catalogue no. HY-B0108), and α-mangostin (catalogue no. HY-N0328) were purchased from MedChemExpress (China). Propidium iodide (PI) (catalogue no. P4170) and bis(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3)) (catalogue no. D8189) were purchased from Sigma-Aldrich (China).

Antimicrobial susceptibility test. The minimum inhibitory concentration (MICs) of α-mangostin against S. aureus isolates was determined by the broth macro dilution method in CAMHB according to the Clinical and Laboratory Standards Institute guidelines (CLSI-M100-S30) (CLSI 2020) and the previous study (Song et al. 2020). Briefly, α-mangostin or other antimicrobials were two-fold diluted with CAMHB and mixed with S. aureus suspensions (1.5 × 10⁶ CFU/ml). After 18 h incubation at 37°C, the MICs were defined as the lowest concentrations of antibiotics in tubes with no visible growth of bacteria. All experiments were performed in triplicate.

Bacterial growth curve and time-killing assay. The effect of α-mangostin, daptomycin, vancomycin, linezolid, and their synergistic combinations on the growth of S. aureus planktonic cells was explored based on a previous study (Wang et al. 2021). S. aureus suspension (OD₆₀₀ = 0.2) with α-mangostin or antimicrobials (at ½ × MIC) was inoculated into 96-well polystyrene microtiter plates (300 μl/well) and cultured for 24 h at 37°C. The optical density at 600 (OD₆₀₀) was measured with a Bioscreen C system (Lab Systems Helsinki, Finland). All experiments were performed in triplicate. The time-killing assay aimed to measure the rapid bactericidal activity of α-mangostin against S. aureus (Zheng et al. 2019). Briefly, this assay was conducted in a final volume of 4 ml CAMHB with α-mangostin or antimicrobials (at 4 × MIC). The samples were incubated at 37°C for 24 h. After 1, 3, and 24 h of time-killing assay, 1 ml aliquots were sampled, and the number of CFU/ml was determined. All experiments were performed in triplicate.

The effect of α-mangostin on S. aureus established biofilms. The eradicating effect of α-mangostin on established biofilms of S. aureus was measured by crystal violet staining (Zheng et al. 2020). The overnight S. aureus cultures were diluted with fresh TSBG (200 μl/well) and inoculated into 96 polystyrene microtiter plates. After static incubation at 37°C for 24 h (mature biofilms formed), the supernatant was discarded and washed, and then fresh TSBG containing α-mangostin was added. After static incubation at 37°C for 24 h, the supernatant was discarded, and plates were washed three times with 0.9% saline. The biomass of the remaining biofilms of S. aureus still attached to the wells was determined by crystal violet staining. All experiments were performed in triplicate.

Induction of α-mangostin non-sensitive clones in vitro. The α-mangostin non-sensitive S. aureus clones were selected in vitro (Zheng et al. 2021). S. aureus SA113 (MSSA) and YUSA145 (MRSA) strains were subcultured in CAMHB with α-mangostin. The initial concentration of α-mangostin was equal to ½ × MIC, then successively increased to a high concentration of 64 × MIC. Individual bacteria clones from the last passage of each concentration were selected and cultured without α-mangostin for two passages. Then the bacterial species were identified by mass spectrometry, and the MIC of α-mangostin was determined again. Finally, the α-mangostin non-sensitive clones (MIC of α-mangostin: ≥ 12.5 μM) were stored at –20°C for further analysis.

Whole-genome sequencing. The genomic DNA of the parental S. aureus isolate YUSA145 and α-mangostin non-sensitive clone YUSA145-L12 was obtained by the DNeasy Blood and Tissue Kit (QIAGEN, Germany). The genomic DNA per sample (1 μg) was used, and sequencing libraries were generated with the NEBNext^® Ultra^™ DNA Library Prep Kit for Illumina^® (New England Biolabs^®, Inc., USA). The whole genome sequencing was performed in the Illumina^® HiScan^™SQ system (Illumina^®, USA) based on our previous research (Zheng et al. 2021). Briefly, the coding genes, repetitive sequences, and other sequences were predicted by the following software or tools: GeneMarkS and RepeatMasker (http://www.repeatmasker.org) (Smit et al. 2013). Gene functions were analyzed by the following databases: GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes). Genomic alignments and SNPs analysis between the α-mangostin non-sensitive clone YUSA145-L12 genome and the parental YUSA145 genome (subcultured in MHB containing no α-mangostin) and reference genome (USA300 FPR3757, GenBank: CP000255.1) were completed with MUMmer and LASTZ.

Proteomics analysis. To illuminate the antibacterial mechanism of α-mangostin against S. aureus, the proteomics analysis was conducted in S. aureus YUSA145 with the treatment of α-mangostin (at ½ × MIC) for 4 h. Bacterial cultures were harvested at OD₆₀₀ ∼ 0.8 and transferred to a precooled 2 ml screw-cap tube. 1.5 volumes of zirconia/silica beads (Biospec, 0.1 mm) and RIPA lysis buffer (Beyotime Biotechnology, China) were added to the tube. The cells were lysed with a cell disruption device, and the protein concentrations were measured using a commercial BCA assay. The pretreatment of harvested protein samples (100 μg) before LC-MS detection was performed according to our previous study (Wen et al. 2022). Briefly, the harvested protein samples were reduced with 10 mM DTT (Sigma-Aldrich, USA), then alkylated with 50 mM iodoacetamide (IAA; Sigma-Aldrich, USA), avoiding light. The samples were desalted and washed with 0.5 M ammonium bicarbonate with Amicon^® Ultra Centrifugal Filters (10 kDa cutoff; Merk Millipore, USA), and digested with trypsin (Promega, USA).

LC-MS/MS detection was conducted in the UltiMate^™ 3000 RSLCnano system coupled to a Q Exactive^™ Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, USA). Peptides (250 mm) were loaded on 75 μm Acclaim™ PepMap^™ C18 reverse-phase analytical column (Thermo Scientific, USA), and eluted with 0.1% acetic acid (containing ~ 53% acetonitrile). The full scan was performed in the Orbitrap (m/z 300–1500) with a resolution of 70,000. The corresponding MS2 spectra for MS detection were at the resolution of 17,500 (maximally 50 ms). The different abundance proteins were determined with the Proteome Discoverer 2.4 base with the Sequest HT, according to the Uniprot reference proteome of S. aureus (strain NCTC 8325/PS47). The minimum unused score of 1.3 (equivalent to 95% confidence) and the false discovery rate (FDR) of less than 1% were used for all reported proteins (decreasing false-positive identification results). The different abundance proteins were analyzed with the OMICSBEAN database (http://www.omicsbean.com) for GO annotation, KEGG pathway analysis, and PPI networks.

Membrane integrity assay. Both propidium iodide staining and bis(1,3-dibutylbarbituric acid) trimethine oxonol uptake assay were performed following the protocol of Fan et al. (2019). The PI staining method mainly aimed to evaluate the integrity of the S. aureus cell membrane. S. aureus suspension (OD₆₀₀ = 0.2) was diluted 100-fold with PBS and pipetted into a 24-well plate. α-Mangostin was added into each well at final concentrations 1 × MIC and 4 × MIC, respectively. Sodium chloride solution and DMSO were used as the negative control group, and 1% Triton was used as the positive control group. PI solution (7.5 μg/ml) was added to each well and kept the plate at room temperature for 30 min. A microplate reader was used to detect fluorescence intensity at excitation and emission wavelengths of 535 and 615 nm, respectively. DiBAC₄(3) was a fluorescent dye and sensitive to membrane potential. Briefly, S. aureus suspension (OD₆₀₀ = 0.2) was added into a black, opaque, flat-bottomed 96-well plate, followed by the incubation with DiBAC₄(3) (1 μM) at 37°C in the dark for 30 min. Likewise, α-mangostin has added into each well with the final concentrations of 1 × MIC and 4 × MIC, respectively. Subsequently, the fluorescence intensity was detected at 492 nm and 515 nm. The controls were chosen as described above. Results were expressed in relative fluorescence units. All experiments were performed in triplicate.

Statistical Analysis. The results were shown as mean±SD and were compared by Student’s t-test or one-way analysis of variance (ANOVA). All the data were analyzed by the IBM^® SPSS^® software package (version 19.0, USA), and the p < 0.05 was determined as statistically significant.

Results

The rapid bactericidal activity of α-mangostin on planktonic cells of S. aureus. The MICs of α-mangostin against 328 clinical S. aureus isolates (190 MSSA and 138 MRSA) ranged from 1.56 μM to 6.25 μM, both with the MIC₅₀/MIC₉₀ of 3.13/3.13 μM, respectively (Table I). This study found that the low dose of α-mangostin combined with vancomycin (½ × MIC) had a significant inhibitory effect on the growth of S. aureus SA113 planktonic cells at a logarithmic phase (Fig. 1). Moreover, the growth of S. aureus SA113 planktonic cells could be markedly inhibited by the combination of α-mangostin with linezolid (Fig. 1).

Table I

Staphylococcus aureus susceptibility to α-mangostin.

S. aureus	The MICs (μM) of α-mangostin
S. aureus	1.56	3.13	6.25	MIC₅₀/MIC₉₀
MSSA (n = 190)	16	163	11	3.13/3.13
MRSA (n = 138)	13	117	8	3.13/3.13

MIC – minimum inhibitory concentration,

MSSA – methicillin-sensitive S. aureus,

MRSA – methicillin-resistant S. aureus,

MIC₅₀/MIC₉₀ – the MICs for 50% or 90% of bacterial growth inhibition

The effect of sub-MIC of α-mangostin on the growth of Staphylococcus aureus planktonic cells. S. aureus SA113 was treated with α-mangostin, daptomycin, vancomycin, linezolid, and their synergistic combinations (½ × MIC), and the growth of planktonic cells was detected by optical density at 600 nm (OD₆₀₀). The data presented was the average of three independent experiments (mean ± SD). MIC, minimum inhibitory concentration.

Interestingly, this research demonstrated that highdose of α-mangostin (4 × MIC) had a rapid bactericidal effect on S. aureus SA113 planktonic cells and was more effective (at least 2-log₁₀ CFU/ml) than daptomycin, vancomycin, and linezolid after 1 and 3 h of the timekilling test. However, the combination of α-mangostin with vancomycin, daptomycin or linezolid did not increase the bactericidal activity of α-mangostin against S. aureus (Fig. 2).

The antibacterial effect of α-mangostin on the planktonic cells of Staphylococcus aureus. S. aureus SA113 during logarithmic growth phase was treated with α-mangostin, daptomycin, vancomycin, linezolid, and their synergistic combinations (4 × MIC), the remaining planktonic cells were enumerated. The data presented was the average of three independent experiments (mean ± SD). MIC, minimum inhibitory concentration.

α-Mangostin significantly reduces established biofilms of S. aureus. To evaluate the eradicating effect of α-mangostin on established biofilms of S. aureus, the strong biofilm producer S. aureus SA113 and YUSA145 isolates were chosen, and the biofilm biomass was determined by crystal violet staining. The present study indicated that established biofilms of S. aureus could be notably eradicated by high-dose of α-mangostin in a concentration-dependent manner (≥4×MIC) (Fig. 3).

Effect of different concentrations of α-mangostin on Staphylococcus aureus biofilms.

A) S. aureus SA113 and B) YUSA145 formed mature biofilms, then were treated with different concentrations of α-mangostin for 24 h. The remaining biofilm biomass was determined by crystal violet staining. The data presented was the average of three independent experiments (mean ± SD). Compared with control: *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t-test. MIC – minimum inhibitory concentration

The genetic mutations in α-mangostin non-sensitive S. aureus clones. To further explore the resistance mechanism or potential target gene of α-mangostin in S. aureus, the α-mangostin non-sensitive S. aureus isolates were finally selected by in vitro induction of wild-type strains. Then whole-genome sequencing was performed to detect mutations. In the beginning, S. aureus SA113 and YUSA145 isolates were induced, but only YUSA145 isolate after about 160 days of arduous induction, resulting in the increased MICs of α-mangostin (≥ 12.5 × MIC). The genetic mutations in the α-mangostin non-sensitive clone YUSA145-L12 were detected by whole-genome sequencing. This study indicated that there were 58 SNPs in the S. aureus YUSA145-L12 clone (Table SI), of which 35 SNPs were located on both sides of the sarT gene, and 10 were located on the sarT gene, including one non-synonymous mutation and nine synonymous mutations (Fig. 4).

Schematic illustration of the SNPs in the sarT gene of the α-mangostin non-sensitive Staphylococcus aureus isolate. There were 35 SNPs located on both sides of the sarT gene, 10 SNPs in the sarT gene included one non-synonymous mutation (in red) and nine synonymous mutations.

The different abundance of proteins in α-mangostin treated S. aureus isolate. A total of 147 proteins with different abundances were determined (identified peptides > 2, p-value < 0.05, with fold-change > 2 or < 0.5) in α-mangostin treated S. aureus isolate, and of which the increase was observed for 91 proteins, whereas decrease for 56 proteins (Table SII). According to the GO annotation, the predominant cellular components of different abundance proteins were involved in oxidoreductase activity, nickel cation binding, and protein binding (Fig. 5A). Interestingly, the abundance of a variety of proteins in pathways related to the synthesis and transportation of cell membrane was reduced, which might result in the injury to the integrity of bacterial cell membranes (Fig. 5B). The protein-protein interaction network of different abundance proteins in this study was analyzed through STRING database (Fig. 6).

The different abundance proteins in-α-mangostin-treated Staphylococcus aureus isolate.

A) The molecular functions of different abundance proteins were classified by the GO analysis; B) different abundance proteins related to cell membrane synthesis and transport of biological process.

Protein-protein interaction network of different abundance proteins in α-mangostin-treated Staphylococcus aureus isolate. The protein-protein interaction network of different abundance proteins was analyzed through STRING database.

Interestingly, the abundance of regulatory proteins SarX and SarZ was elevated, while the abundance of SarT and IcaB was significantly reduced. The abundance of cell membrane proteins VraF and DltC in the cationic antimicrobial peptide (CAMP) pathway of S. aureus was augmented. However, the abundance of cell membrane proteins UgtP was detected a remarkably decreased (100-fold).

The disruption of membrane integrity in S. aureus treated with α-mangostin. To further evaluate the impact of α-mangostin on the membrane integrity of S. aureus, PI (Fig. 7A) and DiBaC₄(3) uptake assays (Fig. 7B) were performed. The results indicated that fluorescence intensity recorded in these assays was significantly increased in the α-mangostin treated group.

The fluorescence intensity was significantly increased in α-mangostin-treated Staphylococcus aureus isolate. S. aureus SA113 was treated with α-mangostin, and staining with A) propidium iodide or B) bis(1,3-dibutylbarbituric acid) trimethine oxonol to evaluate the integrity of S. aureus cell membrane. The results were expressed in a relative fluorescence units. The data presented was the average of three independent experiments (mean ± SD).

Discussion

S. aureus is considered the most frequent cause of infections on indwelling medical devices and tends to develop biofilm, facilitating resistance to antibiotics and host immune defenses (Otto 2018). This study revealed a remarkable reduction of established S. aureus biofilms was observed in the groups given high-dose of α-mangostin. To explore the mechanism of α-mangostin against S. aureus biofilms, the genetic mutations in the α-mangostin non-sensitive isolates were detected by whole-genome sequencing. This study showed that multiple SNPs occurred located on or near the genes sarT and sarU of S. aureus exposed to highdose (≥ 12.5 × MIC) of α-mangostin. When exposed to a low dose (½×MIC) of α-mangostin, the abundance of SarT protein decreased significantly. SarT is a regulator and is involved in the hla gene expression. It also downregulates RNAIII via SarU (indirect) in S. aureus participating in regulating S. aureus virulence and biofilm formation (Manna and Cheung 2003; Ciulla et al. 2018; Schilcher and Horswill 2020). Therefore, it is speculated that α-mangostin reduced established biofilms of S. aureus by inhibiting the function of SarT.

In this study, a total of 147 proteins with different abundances were identified by proteomics analysis. Significantly, many proteins in pathways related to cell membrane synthesis and transport were downregulated by α-mangostin through GO annotation. It has been noted that the abundance of UgtP protein is extremely low. The protein UgtP is responsible for anchoring lipoteichoic acid (LTA) on the cell membrane. Deleting the gene for UgtP protein impacts cell growth and division (Gründling and Schneewind 2007; Hesser et al. 2020).

The protein-protein interaction analysis showed that the abundance of IcaB was significantly reduced. The icaADBC system regulates the synthesis of polysaccharide intercellular adhesion (PIA), a dominant component of S. aureus biofilm (Arciola et al. 2015; Schilcher and Horswill 2020). As reported by Phuong et al. (2017), the MSSA strains were more sensitive to α-mangostin than MRSA. The possible reason was that in their study, the dominant component of MSSA biofilm was tested as PIA, while proteins comprised the central part of the MRSA biofilms. However, more evidence is still needed to confirm the relationship between the biofilm eradication activity of α-mangostin and the regulation of icaADBC.

The protein-protein interaction analysis also indicated that the abundance of cell membrane protein DltC and VraF, the two important members in the cationic antimicrobial peptide (CAMP) pathway, was remarkably increased. CAMP is a class of peptides with antimicrobial activity, which interacts with negatively charged cell membranes and destroys the permeability and integrity of cell membranes (Falord et al. 2012; Yang et al. 2012). In S. aureus, the GraSR/VraFG two-component system (TCS) controls CAMP resistance through d-alanylation of teichoic acids, mediated by the DltABCD enzymes, as well as MprF-dependent lysination of phosphatidylglycerolto increases the positive surface charge and prevents CAMP binding by electrostatic repulsion (Falord et al. 2012; Cho et al. 2021). Therefore, α-mangostin might be a membrane-target agent that exhibits a bactericidal action similar to CAMP, causing the up-regulation of DltC and VraF after treatment with α-mangostin.

Propidium iodide (PI) staining and DiBaC₄(3) uptake assay were performed to evaluate the membrane integrity in the present research. It is well known that PI is a membrane-impermeable dye and DNA intercalating agent. When the integrity of the inner membrane is disrupted, PI may bind to DNA in the cell and emit more intense fluorescence. DiBaC₄(3) is a cell membrane potential dye. When the membrane potential is disturbed and depolarized, the fluorescence intensity increases. PI staining assay and DiBaC₄(3) uptake assay results confirmed that α-mangostin is an excellent membrane target bactericide. Koh’s research also verified the direct membrane targeting mechanism through molecular dynamic simulations (Koh et al. 2013). However, the proteomics results revealed that the upstream GraS and GraR were not differentially expressed in the α-mangostin treatment group. Whether the mechanism of α-mangostin is related to the CAMP and GraSR TCS requires more experiments to clarify further.

Conclusion

In conclusion, this study reveals that α-mangostin is an effective antibacterial agent against S. aureus by targeting the cell membrane. The anti-biofilm effect of α-mangostin may be related to the inhibition of SarT and IcaB.

eISSN:: 2544-4646
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Mechanisms of Rapid Bactericidal and Anti-Biofilm Alpha-Mangostin In Vitro Activity against Staphylococcus aureus

Article Category: ORIGINAL PAPER

Published Online: Jun 14, 2023

Page range: 199 - 208

Received: Jan 27, 2023

Accepted: Apr 16, 2023

DOI: https://doi.org/10.33073/pjm-2023-021

Keywords
alpha-mangostin, biofilm, cell membrane, SarT

© 2023 Xiangbin Deng et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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