Identification of the antibacterial mechanism of cryptotanshinone on methicillin-resistant Staphylococcus aureus using bioinformatics analysis

Cryptotanshinone (CT) is an extract from the traditional Chinese medicine Salvia miltiorrhiza, which inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) in vitro. This study aims to determine the antibacterial mechanisms of CT by integrating bioinformatics analysis and microbiology assay. The microarray data of GSE13203 was retrieved from the Gene Expression Omnibus (GEO) database to screen the differentially expressed genes (DEGs) of S. aureus strains that were treated with CT treatment. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to identify the potential target of CT. Data mining on the microarray dataset indicated that pyruvate kinase (PK) might be involved in the antimicrobial activities of CT. The minimum inhibition concentrations (MICs) of CT or vancomycin against the MRSA strain ATCC43300 and seven other clinical strains were determined using the broth dilution method. The effects of CT on the activity of PK were further measured. In vitro tests verified that CT inhibited the growth of an MRSA reference strain and seven other clinical strains. CT hampered the activity of the PK of ATCC43300 and five clinical MRSA strains. CT might hinder bacterial energy metabolism by inhibiting the activity of PK.


Results
DEGs. The volcano and heatmaps plots were generated to show the down and upregulated genes in the GEO datasets (GSE13203). A total of 64 overlapping DEGs (absolute log 2 FC > 1 and FDR < 0.05) were identified, which included 33 downregulated and 31 upregulated genes, respectively (Fig. 1). The top ten up and downregulated DEGs are listed in Table 2.
GO and KEGG pathway of DEGs enrichment analysis (Fig. 2). Enriched biological process (BP), cellular component (CC), and molecular function (MF) analyses were used to better understand the biological functions of overlapping DEGs. The results indicated that the significantly enriched GO term for BP was pathogenesis, the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS) and translation. The significantly enriched GO terms for CC were the integral components of the membrane, plasma membrane, and cytoplasm. In addition, the significantly enriched GO terms for MF were metal ion binding, transmembrane transporter activity, and oxidoreductase activity.
In addition, the results of the KEGG pathway analysis revealed that these DEGs were primarily enriched in metabolic pathways, biosynthesis of secondary metabolite ABC transporters, and two-component systems.
Based on the results from the KEGG analysis, the effect of CT on S. aureus was concentrated on the metabolic pathways, moreover, the results of the GO-BP analysis indicated that the effect of CT on S. aureus concentrated on pathogenesis and the PEP-dependent sugar (PTS). PTS is a distinct system that is used by bacteria for sugar uptake when the energy source is phosphoenolpyruvate (PEP). In addition, PTS acts as a complex protein kinase system that regulates a wide variety of transport, metabolic, and mutagenic processes and the expression  www.nature.com/scientificreports/ of numerous genes 6 . Among the DEGs, the pyk gene, which encodes PK was significantly downregulated. PK catalyzes the irreversible conversion of adenosine diphosphate (ADP) and PEP into adenosine triphosphate (ATP) and pyruvic acid, which are crucial for cellular metabolism. Therefore, PK plays a key role in controlling metabolic flux and ATP production. Meanwhile, as a highly conserved enzyme from animals to humans, PK has recently been identified as an essential gene for the survival of bacteria such as Haemophilus influenzae, Streptococcus pneumoniae, and Mycobacterium tuberculosis [7][8][9][10] . In addition, it has been reported that MRSA is inhibited in vitro by hampering the activity of PK [11][12][13] . Following CT action on S. aureus, PTS could detect changes in the surrounding environment that might further affect S. aureus metabolism via PK.
MICs of vancomycin or CT against the MRSA strains. The MICs of vancomycin or CT against eight MRSA strains are given in Table 1 and Fig. 3. Among the seven clinical strains, five were derived from sputum and two were derived from blood, all of which were positive after a cefoxitin screening test. The MIC of vancomycin against ATCC43300 strain was 0.9 μg/mL, the MICs of vancomycin against seven other clinically isolated strains were between 0.45 and 1.9 μg/mL. This reflects the status of vancomycin as the first-line treatment for MRSA since the 1950s. The MIC of CT against a standard strain ATCC43300 was 1.9 μg/mL, the MICs of CT against seven other clinically isolated strains were between 0.9 and 3.9 μg/mL. This result showed that CT dis-   www.nature.com/scientificreports/ plays bacteriostatic action against MRSA, which agreed with previous reports 14 , which suggested that CT could be used to fight MRSA infection.
The effect of CT on the activity of MRSA PK. In this study, the impact of CT was examined on the activities of PK in a standard MRSA strain ATCC43300 and seven other clinical strains. As shown in Fig. 4, in ATCC43300, MRSA3, and MRSA6, CT at a concentration of double the MIC reduced the activity of PK (p < 0.05). However, CT at the concentration of the MIC did not show an effect on the activity of PK. In MRSA1 and MRSA4, CT at the concentration of MIC and double the MIC reduced the activity of PK significantly (p < 0.05), with no difference between both concentrations. In MRSA7, CT at the same concentration and double the MIC decreased the PK activity with a difference between both concentrations (p < 0.05). In MRSA2 and MRSA5, CT did not affect PK activity at either concentration.

Discussion
The evolution of MRSA demonstrates its genetic adaptation into a first-class multidrug-resistant pathogen. Following the introduction of penicillin and methicillin, S. aureus rapidly developed resistance to these β-lactam compounds 15 . In the 1950s, vancomycin was used to treat MRSA infections, but recently the emergence of MRSA strains that are less sensitive to vancomycin (VISA) or even resistant to vancomycin (VRSA) means that clinicians are less confident when dealing with MRSA. Therefore, there is an urgent requirement to explore effective drugs for MRSA 16 . Plants that grow in the natural environment are affected by pathogenic microorganisms, such  www.nature.com/scientificreports/ alkaloids to inhibit or kill invading pathogenic microorganisms 17 . Therefore, potential antimicrobial chemicals could be found in plants to treat pathogenic microorganisms, such as bacteria. Salvia miltiorrhiza (Danshen) has been widely used in traditional Chinese medicine to treat a variety of diseases, which include coronary artery disease, acute ischemic stroke, hyperlipidemia, chronic renal failure, chronic hepatitis, and Alzheimer's disease. In addition, Danshen has no serious adverse effects. The main bioactive constituents of S. miltiorrhiza include water-soluble phenolic acids and lipophilic diterpenoid tanshinones 18 . CT is one of the major tanshinones that were isolated from the roots of the Danshen plant. Recent studies have shown that CT has the potential to treat and prevent the previously mentioned diseases and it is a potent antibiotic agent 19 .
In this study, the antibacterial effect of CT on MRSA was tested in vitro, and the MICs of CT on MRSA reference strain ATCC43300 and seven clinical strains were detected using a two-fold serial dilution method. The results showed that CT displayed bacteriostatic action against MRSA, which was consistent with previous reports 14 , which suggests that CT could be used to fight MRSA infection.
Although thousands of herbal compounds have been listed as antimicrobial phytochemicals 20 , limited understanding of the mechanisms limits the application of these substances. To determine the mechanism of CT inhibition on the growth of MRSA, bioinformatics tools were used. In this study, the data from was retrieved from GSE13203 and systematically analyzed the differential gene expression of S. aureus were systematically analyzed between the CT treatment and control. Therefore, 64 DEGs were identified, which included 33 downregulated and 31 upregulated genes.
Then, GO and KEGG analysis of these DEGs revealed that CT had a greater impact on the metabolism of S. aureus. Because MRSA is a special species of S. aureus that carries a multidrug resistance gene, the metabolic pathways of MRSA and S. aureus are similar. As a facultative anaerobe, S. aureus uptakes a variety of nutrients that include glucose, mannose, mannitol, glucosamine, N-acetylglucosamine, sucrose, lactose, galactose, and beta-glucosides. The central pathways for glucose metabolism are the Embden-Meyerhof-Parnas pathway and the pentose phosphate cycle. Lactate is the end product of anaerobic glucose metabolism and acetate, and CO 2 are the products of aerobic growth conditions 21 . In the glucose metabolic pathway, PK is the rate-limiting enzyme of energy metabolism, which plays a central role in the carbohydrate metabolism of MRSA. It catalyzes the final rate-limiting step of glycolysis. During this irreversible process, the high-energy phosphate bond in the PEP molecule is transferred to ADP to generate ATP. The metabolites PEP and pyruvate are related to other biosynthetic pathways, and therefore, the potential inhibition of PK could obstruct MRSA energy metabolism. The potential inhibition of PK might result in the decreased metabolism of MRSA 13,[22][23][24] .
A PK test kit was used to detect the effects of CT on the activity of PK of MRSA in vitro. The results showed that except for in the MRSA2 and MRSA5 groups, CT hampered the PK activity of MRSA. PK was identified as a highly interconnected essential hub protein in MRSA, with structural features distinct from human homologs. Currently, the majority of antibiotics in use are directed at critical proteins that are unique to the bacteria and without human homologs to avoid mechanism based toxicity. In addition, based on the supposition that hub proteins are critical for bacterial survival and they should be very sensitive to mutations 25 , and therefore, targeting them should reduce the potential to develop resistant strains and species. Therefore, CT could be a valuable antibacterial candidate because of its effect on the PK of MRSA.
In this study, CT did not affect PK activity in MRSA2 and MRSA5, which were collected from a blood specimen and the five other strains were isolated from a sputum specimen. In different environments, S. aureus will utilize a variety of metabolic pathways, which depend on the oxygen content, source of carbohydrates, and other factors 26 . However, the MIC of CT was 0.9 μg/mL for MRSA2, and 1.9 μg/mL for MRSA5, which indicated that CT might have additional mechanisms that affect the growth of MRSA in addition to affecting the PK activity of both strains. As shown in Table 2, in addition to the pyk gene, a significant downregulation of isdB and isdG were observed, both of which belong to the iron-regulated surface determinant family. The GO-MF analyses revealed that CT affected the metal ion binding of S. aureus. Iron is vital for the growth and proliferation of nearly all organisms, including MRSA. In addition, iron is required for the colonization of host tissues by MRSA and subsequent pathogenesis 27 . Therefore, in addition to affecting PK activity, CT might inhibit MRSA growth by interfering with its iron metabolism, however, this requires further investigation.
The most significant limitation of this study was that only changes in the activities of PK MRSA were examined after treatment with CT. Future research should include measuring the expression of PK of MRSA after treatment with CT. In addition, only seven MRSA strains were isolated from a clinical environment; therefore, a larger number of samples is required in future research to verify the antimicrobial mechanism of CT on MRSA.
In summary, CT inhibited the growth of MRSA in vitro. To determine the mechanism of activity, multiple bioinformatics tools were used combined with a comprehensive analysis of gene expression profiles to identify the key signaling pathways. CT interfered with the activities of PK, which is the key rate-limiting enzyme during glycolysis in MRSA. The results of this study might provide new insights into the antibacterial mechanisms of CT. However, further research and studies with larger sample sizes are required to confirm these findings.

Methods
Acquisition of gene expression profiling data. The microarray data for GSE13203 that was deposited by Feng et al. 28 was retrieved from the GEO database(http:// www. ncbi. nlm. nih. gov/ geo/). The gene expression profile was generated using the Microarray Analysis Suite 5.0 (Affymetrix, Santa Clara, CA, USA). GSM333383, GSM333384, and GSM333385 were used for S. aureus specimens that were treated with CT for 45 m (intervention group). GSM333386, GSM333387, and GSM333388 were used are for the S. aureus specimens that were treated with saline for 45 m (control group). www.nature.com/scientificreports/ Raw data preprocessing and screening of DEGs. First, the chip data files were downloaded and the gene name information, sample number, and value were copied into a Microsoft Office Excel 2019 table (the  Excel table cell format selected the text format) to sort the data and delete incomplete data, followed by saving all data as "input.txt". Then, gene expression differences between the CT treated samples and the control group were identified using the limma R package with the Empirical Bayes method 29 . The Benjamini-Hochberg false discovery rate (FDR) was used to correct for p-value. In this study, adjusted p-value < 0.05 and absolute Log 2 fold change (FC) > 2.0 were set as the cutoff criteria to screen DEGs. Furthermore, a volcano map was plotted with the ggplot2 package according to the adjusted p-value and log 2 FC. In addition, hierarchical clustering analysis of DEGs was performed and visualized using the pheatmap package in R Language.

Functional and pathway enrichment analysis.
To explore the biological functions and the pathways involved in the significant up and downregulated DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the clusterProfiler package in R language 30 . GO is a comprehensive database that describes gene functions in three parts: BP, CC, and MF. GO function enrichment uses p-values < 0.05 as the threshold for significant enrichment. KEGG is a comprehensive database that integrates genomic, chemical, and system function information 31  www.nature.com/scientificreports/ diluted to a 0.5 turbidity standard bacterial suspension (1.5 × 10 8 cfu/mL). A 96-well plate that contained 50 μL of bacteria suspension diluted in MHB II 1:500 in each well were mixed gently and placed in a 37 °C constant temperature incubator for 12 h. After the 96-well plate was removed from the incubator, 1 × 10 5 cfu bacteria respectively were collected and centrifuged at 3000 rpm, at 4 °C for 5 m. After removing the supernatant, 100 μL of extract fluid (BC0540, Solarbio Science & Technology, Beijing, China) was added to the pellets and the bacterial cells were broken in an ultrasonic cell disruptor (Xinzhi JY92-IIN, Saide Electic, Hangzhou, Zhejiang Province, China). After centrifuging at 8000g, at 4 °C for 10 m, 30 μL of the supernatant was incubated with reagents (BC0540, Solarbio Science & Technology, Beijing, China) to initiate the reactions. PK activity proportional to the rate of change at 340 nm was expressed as specific activity (μmol/min/mg), which is defined as the amount of PK that catalyzes the formation of 1 μmol of either product per minute.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.