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. One of the largest concerns in public health is the continual emergence of multidrug-resistant bacterial pathogens, which severely limits treatment options. Staphylococcus aureus is a particularly problematic pathogen that is prevalent in human and animal populations. This organism commonly causes infections of the superficial skin, soft tissue, surgical wounds, and sometimes the bloodstream and lungs. Methicillin-resistant S. aureus (MRSA) and specific strains with reduced susceptibility to vancomycin could cause infections and diseases that are difficult to treat or resistant to the empirical antibiotics1. The global supply of antibiotics that are available for the treatment of infections that are associated with this microorganism is decreasing.Many studies have reported that medicinal herbs from different countries exhibited anti-MRSA activities, which was due to their phytochemical contents2. These plants could be employed as alternatives for drug development to stop, or control, or both MRSA infections.Cryptotanshinone (CT) is a fat-soluble extract from the traditional Chinese medicine Salvia miltiorrhiza (Danshen), which dilates blood vessels, and has antitumor and antiinflammatory activity3. In addition, CT inhibits the growth of S. aureus and MRSA in vitro4; however, the mechanism of action is unknown, which limits its further applications.Microarray platforms were recently employed to understand gene expression in bacteria that were treated by various Chinese medicines to identify some pathogen genes that are associated with antibacterial mechanisms5. In addition, the Gene Expression Omnibus (GEO) database offers methods for the downstream bioinformatics mining of gene expression profiles in a variety of bacteria.In this study, the differentially expressed genes (DEGs) were identified between strains of S. aureus that were treated with normal saline and CT by mining microarray datasets from the GEO database, which aimed to identify and confirm the mechanism associated with the antibacterial effects of CT.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 log2FC > 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.Figure 1Screening of DEGs in MRSA induced by CT treatment: (a) volcano plots; and (b) hierarchical cluster analysis (heatmaps) of the common DEGs between CT treated samples and normal controls in GSE 13203. Statistically significant DEGs were defined with p 1.0 as the cutoff threshold.GO and KEGG pathway of DEGs enrichment analysis (Fig. 2)Figure 2Functional and signaling pathway analysis of the overlapped DEGs according to BP, CC, MF, and KEGG pathways.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 of numerous genes6. 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 tuberculosis7,8,9,10. In addition, it has been reported that MRSA is inhibited in vitro by hampering the activity of PK11,12,13. 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 strainsThe 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 displays bacteriostatic action against MRSA, which agreed with previous reports14, which suggested that CT could be used to fight MRSA infection.Table 1 Specimen and tests information on MRSA strains.Figure 3MIC assay of MRSA strains: (a) MICs of CT to seven MRSA strains isolated from clinical specimens and standard MRSA strain ATCC 43300; and (b) MICs of vancomycin to seven MRSA strains isolated from clinical specimens and standard MRSA strain ATCC 43300.The effect of CT on the activity of MRSA PKIn 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.Figure 4Effect of CT on the activity of PK of eight MRSA strains. #Control group versus 1 × MIC group p < 0.05, *control group versus 2 × MIC group p < 0.05, **:1 × MIC group versus 2 × MIC groups p < 0.05.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 compounds15. 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 MRSA16. Plants that grow in the natural environment are affected by pathogenic microorganisms, such as bacteria and viruses. Some plants have developed two major strategies to defend against pathogenic microorganisms: (1) plants have a lot of epidermal hairs, a thick waxy stratum corneum, and small stomata to block the invasion of pathogenic microorganisms; and (2) plants produce antibiotic substances, such as tannins and alkaloids to inhibit or kill invading pathogenic microorganisms17. 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 tanshinones18. 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 agent19.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 reports14, which suggests that CT could be used to fight MRSA infection.Although thousands of herbal compounds have been listed as antimicrobial phytochemicals20, 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 CO2 are the products of aerobic growth conditions21. 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 MRSA13,22,23,24.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 mutations25, 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 factors26. Howeve
https://www.nature.com/articles/s41598-021-01121-9
Identification of the antibacterial mechanism of cryptotanshinone on methicillin-resistant Staphylococcus aureus using bioinformatics analysis
