Proteomics and Transcriptomics Analysis of  Emilia sonchifolia (L.) DC Antibacterial Activity against Methicillin-resistant Staphylococcus epidermidis

doi:10.21203/rs.3.rs-5071407/v1

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Proteomics and Transcriptomics Analysis of Emilia sonchifolia (L.) DC Antibacterial Activity against Methicillin-resistant Staphylococcus epidermidis

https://doi.org/10.21203/rs.3.rs-5071407/v1

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Background: Bloodstream infections (BSIs) are a public health concern and cause substantial morbidity and mortality. The pathogen Staphylococcus epidermidis causes a significant number of BSIs. Antibiotics targeting Staphylococcus epidermidis have been the mainstay in BSIs. However, conventional antibiotics have been eclipsed in combating with drug-resistant bacteria. Alternate ways of treating these antibiotic-resistant infections are thus urgently needed. Numerous studies have demonstrated that certain traditional Chinese medicine(TCM) exhibit notable antimicrobial activity and possess the ability to impede the development of bacterial resistance. Based on an extensive body of research in the field of TCM, it has been determined that the compositae plant exhibits a noteworthy anti-Methicillin-Resistant Staphylococcus Epidermidis (MRSE) effect. Thus, Emilia sonchifolia was used to explore the antibacterial activity againse MRSE.

Methods: Here, the objective of this study was to examine the antibacterial efficacy and underlying antibacterial mechanism of Emilia sonchifolia against methicillin-resistant Staphylococcus epidermidis( MRSE). The antibacterial activity of Emilia sonchifolia against MRSE was assessed through in vitro tests measuring minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC).On the other hand, a mouse bloodstream infections of MRSE was used to evaluate the antibacterial activity of Emilia sonchifolia against MRSE in vivo . Furthermore, based on proteomics and transcriptomics were investigated to explore the underlying antibacterial mechanisms of Emilia sonchifolia against MRSE.

Results: The results showed that MIC and MBC values of Emilia sonchifolia against MRSE were 5mg/mL and 20mg/mL, respectively. Meanwhile, Emilia sonchifolia can effectively treat MRSE induced bloodstream infections.In addition, proteomic and transcriptomic data revealed a significant down-regulation of purine metabolism,which were associated with oxidative stress and cell wall synthesis. Furthermore,We determined imp, AMP and GMP by Elisa. The results showed that the contents of these enzymes all decreased, indicating that purine metabolism was inhibited. At the same time, SEM results showed that bacterial cell wall was destroyed.

Conclusions: Emilia sonchifolia exerts antibacterial effects by affecting purine metabolism, promoting bacterial oxidative stress and inhibiting bacterial cell wall synthesis. Thus, the aforementioned observations have contributed novel insights into the mechanistic understanding of Emilia sonchifolia's efficacy against MRSE, thereby offering potential strategies for managing MRSE infections.

Health sciences/Diseases

Health sciences/Medical research

Methicillin-resistant Staphylococcus epidermidis

Bloodstream infections

Emilia sonchifolia

Purine metabolism

Bloodstream infections (BSIs) represent a significant global public health concern, characterized by a substantial mortality rate^[1]. 1200 000 episodes of BSIs each year in Europe were estimated and had long-term sequelae^[2]. BSIs were usually caused by catheter devices, including Hemodialysis catheter, central venous catheters and so on^{[3, 4]}. Catheter-related infections are frequently caused by skin commensals, with Gram-positive organisms accounting for 70% of cases^[4]. Among these, Staphylococcus epidermidis (S. Epidermidis) is the predominant pathogen associated with both implanted devices and bloodstream infections (BSIs)^[5]. More seriously, S. epidermidis infections frequently exhibit resistance to antibiotics and the occurrence of multi-resistance in nosocomial strains of S. epidermidis has been reported to reach as high as 70–85%^[8][9]. Meanwhile, a large proportion of S. epidermidis isolates is resistant to methicillin (MRSE) and showed the multi-resistant to β-lactam antibiotics^[9]..Vancomycin represents a restricted selection of available treatment options^[11]. However, it is important to note that vancomycin possesses limited biocidal properties, necessitating therapeutic drug monitoring, and may induce nephrotoxicity and/or ototoxicity in specific patient populations ^{[12, 13]}.Therefore, the current prevalence of MRSE BSIs has presented a significant challenge, prompting the necessity for ongoing research into more effective and secure therapeutic agents^[14].

“Traditional Chinesel medicines (TCM)”, derived from plants and historically employed in diverse cultures, are commonly perceived as safe when administered in appropriate dosages^[15]. Consequently, there is a renewed focus on exploring traditional or herbal medicines as potential alternatives or complementary treatments for emerging and re-emerging multi-drug resistant bacteria^[16]. Meanwhile, numerous traditional or herbal medicines have been studied for their antimicrobial activity^[17–20]. The aqueous extracts of Carum copticum and Albizia adianthifolia exhibited notable antibacterial effects against multidrug-resistant gram-negative human pathogenic bacteria, including E.coli, Pseudomonas, K. pneumonia, Salmonella, and Proteus species ^{[21, 22]}. Similarly, Patrinia scabiosaefolia had good antibacterial effect against MRSE invitro and in vivo in our previous study^[23]. Therefore we speculated that the other traditional or herbal medicines may have good antibacterial activity against MRSE BSIs.

Emilia sonchifolia has a well-documented history of use in China, as recorded in the "Lingnan Medicine Collection." It is primarily utilized as a traditional Chinese medicine and is known for its anti-inflammatory, analgesic, antibacterial, antiviral, antitumor, hypoglycemic, and immune-regulating properties^[24]. Further reports that Emilia sonchifolia has demonstrated significant antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Neisseria gonorrhoeae in vitro^[24]. However, its antibacterial activity and antibacterial mechanism against MRSE remains unknown.

In contemporary research, there has been a growing utilization of multi-omics in the study of microorganisms^[25–27]. Proteomics is employed within the realm of antibacterial research to elucidate antimicrobial mechanisms, conduct drug screening, and facilitate vaccine development^[26]. Similarly, transcriptomics can be utilized in the field of antibacterial investigation to explore antibacterial mechanisms, identify therapeutic targets, and foster the development of novel drugs through the analysis of gene expression profiles^[28]. The utilization of proteomics and transcriptomics technology significantly contributes to the comprehension of bacterial mechanisms^[29]. Hence, the concurrent utilization of proteomics and transcriptomics in the realm of antibacterial research enables a comprehensive comprehension of alterations in bacterial protein expression and gene regulation. This approach facilitates the elucidation of antibacterial mechanisms and the exploration of novel antibacterial targets.

Based on the above research, this study focuses on a systematic investigation of the in vitro and in vivo antibacterial activity of Emilia sonchifolia against MRSE. Additionally, we employed transcriptomic and proteomics methodologies to elucidate the potential MRSE mechanism of Emilia sonchifolia.

2.1 Culture conditions

The MRSE strains were stored at a temperature of -80℃ in Tryptic Soy Broth (TSB) supplemented with 20% (vol/vol) glycerol. Before each experiment, approximately 20 µL aliquots of the frozen cultures were transferred to tubes containing 5 mL of TSB and incubated overnight at 37℃. Subsequently, the cultures were inoculated onto Tryptic Soy Agar (TSA) and stored at 4℃ until they were needed for further experimentation

2.2 The Antibacterial Activity of Emilia Sonchifolia In Vitro

2.2.1 Determination of Minimum Inhibitory Concentration (MIC)

According to the Clinical and Laboratory Standards Institute of the United States of America, the MIC of Emilia sonchifolia was determined using the broth dilution method. In brief, a bacterial suspension cultured to the logarithmic phase was diluted to a concentration of 1×10⁶ colony-forming units per milliliter (CFU/mL). Then, 100 µL of the adjusted inoculum (1×10⁶ CFU/mL) was added to each well of a 96-well Microtiter plate. Subsequently, 100 µL of serial twofold dilutions of Emilia sonchifolia with tryptic soy broth (TSB) were dispensed in the wells, ranging from a concentration of 80 mg/mL to 0.0195 mg/mL. After incubation for 18 hours at 37℃, the MIC was defined as the lowest concentration of luteo.

2.2.2 Determination of Minimum Bactericidal Concentration (MBC)

The dilution in broth method was employed to determine the MBC for emilia sonchifolia^[30]. The culture suspensions (100 L) of MRSE treated with emilia sonchifolia at concentrations of 5 and 40 mg/mL, where no bacterial growth was observed, were evenly spread on tryptic soy agar (TSA) plates and incubated at 37℃ for 18 hours. The surviving colonies were subsequently observed. The MBC was defined as the lowest concentration of antimicrobial agent capable of inactivating more than 99.99% of the bacterial population (＜10 CFU/mL). The assay was performed in triplicate.

2.3 The Antibacterial Activity of Emilia Sonchifolia In Vivo

2.3.1 Animals conditioning

The mice were assigned to individual cages in a pathogen-free facility, with a maximum occupancy of six mice per cage. Prior to their utilization, the mice were subjected to a 12-hour light-dark cycle at a temperature of 22 ± 2°C for a duration of one week. Additionally, they were provided unrestricted access to both food and water.

2.3.2 Establishment of mouse immunosuppressive models

Based on the existing literature, a mouse immunosuppressive model was established through intraperitoneal injection of CP doses at 120 mg/kg of body weight for a duration of 4 days. Control mice were administered PBS. Subsequently, the thymus and spleen were extracted from both the immunosuppressive and control mice, and the thymus and spleen index was computed.

2.3.3 Treatment of Emilia Sonchifolia against MRSE-induced BSIs

The animals were randomly assigned to different groups prior to the experiment, including the immunosuppressive group, bloodstream infection group, Emilia sonchifolia group, and blank group. In the Emilia sonchifolia group, Emilia sonchifolia was administered orally at a dosage of 8g/kg, 7 days prior to infection. The other three groups received an equal volume of PBS.

The immunosuppression model was implemented three days prior to infection, following the aforementioned method, in the immunosuppressive group, bloodstream infection group, and Emilia Sonchifolia group, and the blank group were dealt with PBS. Furthermore, bloodstream infection group, Emilia sonchifolia group were infected at 7 d after Emilia sonchifoli oral administration by tail vein injection with 1× 10⁸cfu/mL MRSE, and the immunosuppressive group,blank group were dealt with PBS. The way of tail veins were consistent with the procedure outlined in Liu et al^[31].After that, at 12 h post infection (pi), blood samples were collected from tail veins in all groups. Meanwhile the above mouse were euthanized by 100mg/kg Pentobarbitol Sodium (IP). Subsequently, the aforementioned samples underwent serial dilution in phosphate-buffered saline (PBS) and were subsequently plated on tryptic soy broth (TSB) agar plates for a duration of 24 hours at a temperature of 37◦C. Ultimately, the enumeration of clones was conducted to assess the efficacy of the mouse bloodstream infection (BSI) models^[32].

The aforementioned mouse experiments were authorized by the University Committee on Use and Care of Animals at the Guizhou University of Traditional Chinese Medicine, in accordance with the Laboratory Animal – Guideline for Ethical Review of Animal Welfare (GB/T 35892—2018). We confirming that all experiments were performed in accordance with relevant guidelines and regulations.

2.4 Proteomics Study

TMT-based quantitative proteomics analyses were implemented as Liu described^[23]^. Three biological replicates were prepared for water extract , ethanol extract and control group, which were sent to LC-BIO Technologies Co., Ltd. (Hangzhou, Zhejiang, China) for proteomics analysis. Detailed protein extraction, purification, peptide tagging and reverse-phase chromatography and mass spectrometry were provided in Supporting Information 2. Protein identification, quantification, classification and interaction prediction were analyzed^[23]. The raw files generated by AQ Exactive Plus were converted using Proteome Discoverer 2.1 (Thermo Fisher Scientific), and the files were sent to OmicStudio tools (Lc-bio Technologies Co., Ltd.) for analysis^[33]. Differentially expressed proteins were defined as those with a fold change (FC) ≥1.2 or ≤1/1.2 -fold and a P-value＜0.05. The differentially expressed proteins were functionally classified by Gene Ontology (GO) terms (http://www.omicsbean.com). In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of altered proteins was categorized utilizing the same resource.

2.5 Transcriptomics Study

Transcriptome sequencing was performed as Cheng described^[34]. Three biological replicates were prepared for water extract , ethanol extract and control group, which were sent to LC-BIO Technologies Co., Ltd. (Hangzhou, Zhejiang, China) for transcriptome sequencing analysis. Detailed RNA isolation, purification, quantification and cDNA Library Construction were provided in Supporting Information 1.

After estimating the expression levels of all transcripts and analyzing expression levels for mRNAs, the differentially expressed mRNAs were selected with fold change >2 or fold change <0.5 with p value < 0.05 by DESeq2, Go and KEGG enrichment to the differentially activated pathways were analyzed.

2.6 Transcriptomic and proteomic integrative analysis methods

The transcriptomic and proteomic analyses were carried out to identify the expressions of genes, potential antibacterial mechanism of Emilia Sonchifolia against MRSE.

2.7 Effect of Emilia Sonchifolia on purine metabolism

The activities of IMP,GPM and AMP were evaluated in MRSE under emilia sonchifolia stress. MRSE in the logarithmic growth phase was introduced to Emilia sonchifolia at a concentration of 1 minimum inhibitory concentration (MIC). Subsequently, the mixtures were incubated at 37°C for 4 hours. A negative control was also prepared, consisting of phosphate-buffered saline (PBS) instead of emilia sonchifolia. The mixtures were then subjected to centrifugation at 12000 revolutions per minute for 5 minutes at 4°C, and the resulting supernatant was discarded. The cells were subsequently washed and suspended in PBS. Subsequently, the samples were subjected to ultrasonication at a concentration of 30% for a duration of 30 minutes (with an interval of 5 seconds) and then centrifuged at a speed of 12000 revolutions per minute for 5 minutes at a temperature of 4℃. Ultimately,the supernatant was divided into aliquots and used for the IMP,GPM and AMP assays by kits from Beijing APPLYGEN Gene Technology Co., LTD.

2.8 Effect of Emilia Sonchifolia on Antioxidant system

The activities of CAT and SOD were evaluated in MRSE under emilia sonchifolia stress. MRSE in the logarithmic growth phase was introduced to emilia sonchifolia at a concentration of 1 minimum inhibitory concentration (MIC). Subsequently, the mixtures were incubated at 37°C for 4 hours. A negative control was also prepared, consisting of phosphate-buffered saline (PBS) instead of emilia sonchifolia. The mixtures were then subjected to centrifugation at 12000 revolutions per minute for 5 minutes at 4°C, and the resulting supernatant was discarded. The cells were subsequently washed and suspended in PBS. Subsequently, the samples were subjected to ultrasonication at a concentration of 30% for a duration of 30 minutes (with an interval of 5 seconds) and then centrifuged at a speed of 12000 revolutions per minute for 5 minutes at a temperature of 4℃. Ultimately,the supernatant was divided into aliquots and used for the CAT and SOD assays by kits from Beijing Solarbio Science & Technology Co.,Ltd..

2.9 Effect of Emilia sonchifolia on morphological characterizations of MRSE

Morphological characterizations of MRSE treated with emilia sonchifolia were explored using Scanning electron microscopy (SEM) and Transmission Electron Microscopy (TEM).

The scanning electron microscopy (SEM) was conducted using a modified method that had been previously employed. In summary, Emilia sonchifolia (1 MIC) was introduced to a logarithmic phase bacterial suspension. The control group received an equal volume of phosphate buffer saline (PBS). All groups were incubated at 37℃ for a duration of 4 hours. Subsequently, the treated bacterial cells were harvested, washed, and subsequently fixed in a 3% glutaraldehyde solution overnight at 4℃. Next, the bacterial cells underwent dehydration through the application of ethanol with varying concentrations, followed by replacement with tertiary butyl alcohol. Subsequently, the samples were subjected to freeze-drying, sputter coated with gold, and examined through microscopic observations utilizing a scanning electron microscope. For the transmission electron microscopy (TEM) analysis, the MRSE were treated in a manner consistent with the SEM procedure. As per the report, the specimens were affixed to a fibrous carbon film and observed using TEM ^[35].

3.0 Statistical analysis

The values were reported as means ± standard deviations (SDs). To assess the statistical differences among the various groups, a one-way analysis of variance (ANOVA) was conducted. Significant differences between means were determined using Tukey's Honest Significant Difference test, with a significance level set at p < 0.05.

1. The Antibacterial Activity of Emilia Sonchifolia In Vitro

The antibacterial activities of Emilia sonchifolia against MRSE were mainly indicated by MIC and MBC. After 18 h of incubation at 37℃, turbidity was noticed in the well 2.5 to 0.039 mg/ml of Emilia sonchifolia. In concentrations ranging from 40 to 5 mg/ml, the absence of turbidity was observed, indicating the inhibitory effect on bacterial proliferation. According to CLSI, the the MIC value of Emilia sonchifolia against MRSE was 5 mg/mL (Table 1).

Meanwhile, MRSE showed resistance to oxacillin, confrming the efective resistance of MRSE. Furthermore, the suspension ranging from 40 to 5 mg/mL was introduced onto a TSA agar plate and subjected to an 18-hour incubation period. The resulting observation of less than 10 CFU/mL in the concentration range of 40 to 20 mg/mL serves as conclusive evidence of its bactericidal properties. So, the MBC value of Emilia sonchifolia was 20 mg/mL (Table 2).

2.The Antibacterial Activity of Emilia Sonchifolia In Vivo

Emilia Sonchifolia exhibited superior in vitro antibacterial activity against MRSE, as demonstrated by previous research. To further assess its antibacterial efficacy in vivo, Emilia Sonchifolia treatment was administered against MRSE-induced bloodstream infection(Figure 1A). Prior to the establishment of MRSE-induced bloodstream infection, an immunosuppression model was employed. Meanwhile, the spleen and thymus index were used to evaluate the immunosuppression model. The Figures 1B and C demonstrate a significant reduction in the spleen and thymus indices of the immunosuppressive group, bloodstream infection group, and Emilia Sonchifolia group, when compared to the blank group, which demonstrated that model was successfully established. Based on the immunosuppression model, a bloodstream infection was induced by MRSE. Additionally, the quantification of bacterial presence in the blood served as a standard measure to assess the antibacterial efficacy of Emilia Sonchifolia. In Figure 1D, a notable decrease in the number of clones in the blood was observed in the Emilia Sonchifolia. group compared to the bloodstream infection group, indicating a significantly superior in vivo antibacterial activity of Emilia Sonchifolia..

3.Proteomic analysis of MRSE upon Emilia sonchifolia treatment

The influence of Emilia sonchifolia treatment on MRSE at the protein level was evaluated by the TMT-based quantitative method. A total of 1684 proteins were

identified in MRSE in this study. Differentially expressed proteins (DEPs) were defined as follows: fold change (FC) > 1.20 or <0.80 and p < 0.05. The comparison of control and Emilia sonchifolia-treated MRSE revealed 113 significant DEPs (64 upregulated proteins and 49 downregulated proteins, volcano plots of FC values against p-values (two-tailed Student’s test), along with the number of differentially expressed proteins and genes(figure2A-D). GO functional enrichment analysis of the DEPs showed that the 15 most significantly enriched GO terms were mainly BPs and MFs,including oxidation-reduction process (GO:0055114),integral component of membrane(GO:0016021),cytoplasm(GO:0005737)) (p < 0.001 in figure2E. KEGG functional enrichment analysis of the DEPs showed that the most significantly enriched Metabolic pathways,Biosynthesis of secondary metabolites, Biosynthesis of antibiotics, Microbial metabolism in diverse environments, Carbon metabolism,

Biosynthesis of amino acids,Purine metabolism shows in figure 2F,

Moreover, KEGG pathway analysis revealed components of 21 KEGG pathways among the 33 down-regulated proteins, the majority of which were associated with Purine metabolism ,Cationic antimicrobial peptide (CAMP) resistance,Two-component system, Metabolic pathways（figure 2G) and the up-regulated of KEGG was associated with ribosome metabolism nitrogen metabolism(figure 2H)

4.Transcriptomic analysis of MRSE upon Emilia sonchifolia treatment

The effects of Emilia sonchifolia treatment on the MRSE transcriptome were determined by RNA-seq experiments.A total of 2353 mRNA were identified in this study Differentially expressed genes (DEGs) were defined as follows: corrected p-value <0.05 and |log2 FC| ≥ 1. The comparison of control and Emilia sonchifolia -treated MRSE revealed 511 DEGs (299 upregulated genes and 212 downregulated genes)（figure 3a, 3b & 3c)

GO annotation analysis of revealed 1421 functional terms, including 308 MFs, 28 CCs and 197 BPs. GO functional enrichment analysis of the DEGs showed that most of the top enriched terms were BPs and MFs, including phosphorylation(GO:0016310),integral component of membrane(GO:0016021),cytoplasm(GO:0005737),metal ion binding(GO:0046872)（figure3D). KEGG pathway annotation and enrichment analysis indicated that the DEGs were enriched in several pathways, Energy metabolism including Amino acid metabolism; Biosynthesis of other secondary metabolites,Carbohydrate metabolism,Metabolism of cofactors and vitamins（figure3E).

Moreover, KEGG pathway analysis revealed components of KEGG pathways among the 212 down-regulated proteins, the majority of which were associated with Metabolism including Arginine biosynthesis,Thiamine metabolism,Glyoxylate and dicarboxylate metab,Lysine biosynthesis(figure3 F); and environmental Information Processing including ABC transporters, Two-component system(figure3 G).

5.Transcriptomic and proteomic integrative analysis

Targeted transcriptomic data based on proteomic pathways were found to be related to pathways such as mapko00230(Purine metabolism), mapko02020(Two-component system), mapko01100(Metabolic pathways)figure 4, etc.

6.The Antibacterial Mechanism of Emilia sonchifolia In Vitro

6.1 Effect of Emilia sonchifolia on purine metabolism

Bacterial purine metabolism pathway plays an important role in maintaining bacterial survival and adapting to the environment. In recent years, researchers have discovered a number of new enzymes and metabolic pathways involved in bacterial purine metabolism such as GMP , AMP and IMP played important role in purine metabolism. In this study, we found that we found that the activities of GMP , AMP, IMP and ATP respectively decreased after MRESE exposed to MIC Emilia sonchifolia for 4h (Fig 5A-C).Such findings indicated that Emilia sonchifolia can inhibit the activities of GMP, AMP and IMP in MRSE.

6.2Effect of Emilia sonchifolia on antioxidant system

The levels of antioxidant can alleviate the harmful ROS^[36].In contrast, high antioxidant level is considered as the mechanism of antibacterial agents. The levels of antioxidant molecules, such as CAT and SOD played important role in antioxidant system. In this study, we found that we found that the activities of CAT and SOD respectively decreased by 73％and 90％after MRESE exposed to MIC Emilia sonchifolia for 4h (Fig 6a,6b).Such findings indicated that Emilia sonchifolia can inhibit the activities of SOD and CAT in MRSE.

6.3 SEM Analysis

In order to examine the alterations in cellular morphology of MRSE following treatment with Emilia sonchifolia, we conducted observations of MRSE utilizing scanning electron microscopy (SEM). For the control group (Fig 7A), the cells of MRSE presented regular shape, smooth surface, and intact surfaces. After subjecting the bacteria to an incubation period of 4 hours with a concentration of 5 mg/mL of Emilia sonchifolia, notable alterations in the morphology of the bacterial cells were observed, as depicted in Figure 7B. The surface of MRSE with Emilia sonchifolia displayed irregular and rugged characteristics, with certain areas exhibiting extensive fractures that resulted in the release of contents.

6.4 TEM Analysis

In order to examine the ultrastructural modifications in MRSE cells following treatment with Emilia sonchifolia, we utilized transmission electron microscopy (TEM). The control group exhibited an intact cell wall, wherein the cytoplasm remained undisturbed in conjunction with the bacterial cell membrane (Fig 8A). Nevertheless, notable alterations in MRSE morphology were observed subsequent to interaction with Emilia sonchifolia. Figure 8B exhibited the occurrence of cell wall lysis and the presence of a discontinuous cell membrane, resulting in the release of numerous intracellular components from the MRSE cells. Moreover, a small proportion of cells underwent dissolution.

S. epidermidis is one of the leading cause of BSIs^[37]. Currently, the emergence of antibiotic-resistant strains, particularly MRSE, has posed a significant challenge in the treatment of BSI. The theory of TCM believes that clearing heat and detoxifying TCM, which is cold in nature, can alleviate or eliminate the infection of pathogenic microorganisms such as bacteria and viruses^[38]. Pharmacology researches showed that clearing heat and detoxifying TCM such as Lonicera japonica Thunb., Forsythia suspensa, Scutellaria baicalensis Georgi, Coptis chinensis Franch. had good antibacterial activity^[39]. Meanwhile, Patrinia scabiosaefolia belonged to clearing heat and detoxifying TCM had strong antibacterial activity against MRSE in our previous study^[23]. In addition, the previous study showed that Emilia sonchifolia extracts had better antibacterial effect to Staphylococcus aureus, Pseudomonas aeruginosa and Neisseria gonorrhoeae^[24]. Therefore, this study examined the in vitro and in vivo antibacterial activity of Emilia Sonchifolia against MRSE

The results of MIC and MBC in our study demonstrated that Emilia Sonchifolia exhibited a strong antibacterial efficacy in vitro as presented in Table 1 and Table 2. To assess theantibacterial activity in vivo, the MRSE-induced BSIs of mouse was established. As the result of S. epidermidis being the opportunistic pathogen, the host immune response can eliminate them. So MRSE infection would occur in immunosuppression. On the basis of the above gist, the mouse immunosuppressive model was built. Furthermore, Emilia Sonchifolia was used to demonstrate the therapy to BSIs in mouse. The results showed that Emilia Sonchifolia had good antibacterial activity in vivo ( Fig. 1). The above results demostrated that Emilia Sonchifolia has good antimicrobial activity.

Recently, a deeper understanding of the complex mechanisms of antibacterial mechanism requires the integration of multi-omics data^[40]. The proteomic and transcriptomic data showed that the proteins of about purine metabolites are significantly down-regulated in the energy metabolic pathway. Purine metabolites play a vital role in growth of bacteria^[41], Research showed purine metabolism are involved in the regulation of the growth and metabolism of bacterial cells^[45]. Proteomics and transcriptomics were used to culture MRSE using Emilia sonchifolia and found that the proteins involved in purine metabolism were significantly down-regulated. Our results are consistent with a previous report showing that Tannic acid down-regulated purine metabolism proteins in Staphylococcus aureus^[29].

the down-regulation of purine metabolism suggests inhibition of bacterial growth, providing evidence that Emilia sonchifolia exhibit antibacterial properties through the inhibition of purine metabolism.

The process of purine metabolism encompasses metabolic pathways such as the synthesize, salvage pathway and the regulation of purine synthesis^[42]. The main components of purine metabolism include the aspartate pathway and the glutamate pathway. The aspartate pathway involves the synthesis of IMP (adenine monophosphate) from aspartic acid, as well as the synthesis of other nucleotides from IMP. The glutamate pathway, on the other hand, involves the synthesis of GMP (guanine monophosphate) from glutamate, followed by the synthesis of other nucleotides from GMP. The purine nucleotide salvage pathway is primarily facilitated by three enzymes: adenosine kinase (ADK),adenine phosphoribosyl transferase (APRT), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Generally, the complementary salvage pathway could satisfy the cellular requirements for purine^[43]. In the guanine nucleotide pathway, two enzymes are involved in converting IMP (inosinmonophosphat) to GMP^[44].Purines serve a vital function in bacterial organisms as organic compounds that constitute fundamental components of nucleic acids, specifically DNA and RNA. Within bacteria, purines are integral in the storage and dissemination of genetic material^[29]. Purines play a role in bacteria's energy metabolism by producing ATP, which is essential for sustaining their life activities^[46]. Researches show that Inosine monophosphate (IMP) is an important intermediate in de novo purine biosynthesis because it can be converted to either guanosine or adenosine monophosphate (GMP or AMP) through two parallel pathways. Enzymes in the GMP pathway include IMP dehydrogenase (IMPDH) and GMP synthase (GMPS)^[47]. Thus, in our study, he activities of GMP, AMP, and IMP were examined. Figure 6 demonstrates that Emilia sonchifolia effectively inhibits the activities of GMP, AMP,and IMP. These findings align with previous research^{[48, 49]}.

Research shows that when the three enzymes GMP, AMP, and IMP are inhibited, bacterial growth will also be inhibited(in Fig. 9). Through experimental verification, this subject found that the activities of these enzymes were indeed down-regulated, which proved that Emilia sonchifolia inhibited the three enzymes of the synthesis pathway and the salvage pathway, thereby inhibiting purine metabolism and thus exerting an antibacterial effect.

The regulatory mechanism governing bacterial purine metabolism relies on a Two-component system that precisely and efficiently modulates the expression of pertinent genes in response to purine levels, thereby facilitating the coordinated regulation of the entire purine metabolic pathway. Two-component system (TCS) is the main signal transduction system of bacteria, When the perception of external relief is decreased, the bacteria are subjected to external attacks, and the corresponding protein proteins are significantly down-regulated. Proteomics and transcriptomics were used to culture MRSE using Emilia sonchifolia and found that the proteins dltA involved in Two-component system were significantly down-regulated. dltA proteins are involved in the synthesis of bacterial cell walls. The cell wall and membrane serve as a physical barrier, effectively separating the internal cellular environment from the external surroundings and preventing the escape of intracellular components^[50]. The findings depicted in Figs. 1 and 2 demonstrate that Emilia sonchifolia has the ability to disrupt the cell wall and membrane of MRSE, thereby compromising the stability of the internal environment. Similar outcomes were observed when Aronia melanocarpa (Michx.), Chaenomeles superba Lindl., and Cornus mas L. leaf extracts were tested against bacterial cells^[51].

Some studies indicate that the utilization of AMP, IMP, and GMP could lead to an increased level of UA which is the final product of the purine metabolism pathway, associated with increased oxidative stress^{[52, 53]}. We further detected changes in defense mechanisms. The disruption of microorganism homeostasis caused by the oxidative burst of reactive oxygen species (ROS) was counteracted by the cellular defense mechanisms of the antioxidant enzyme system^[54]. This system comprises various antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and others^[55]. As seen in figure X, the activity of SOD and CAT were decreased by Emilia sonchifolia, illustrating Emilia sonchifolia can inhibit the defense mechanisms of MRSE. Similarly, curcumin-based photodynamic inactivation can amplify the oxidative damage to bacteria by inhibiting the activity of SOD and CAT^[54].

Data showed that Didianhong inhibits the purine metabolic synthesis pathway and salvage pathway by inhibiting AMP, IMP, and GMP, which in turn leads to enhanced oxidative stress capacity and ultimately exerts an antibacterial effect.

In comparison to conventional antibiotics, Emilia sonchifolia exhibits the advantageous characteristic of targeting multiple sites against MRSE. This attribute may account for the reduced likelihood of drug resistance development in TCM. The present investigation aims to provide a preliminary elucidation of the antibacterial activity and mechanisms of Emilia sonchifolia against MRSE by plays an antibacterial role by inhibiting purine metabolism, thereby affecting oxidative stress and damaging cell membranes and cell walls. Nevertheless, further research is warranted to comprehensively understand the specific mechanism of action employed by Emilia sonchifolia against MRSE.

In conclusion, the present study has demonstrated that Emilia sonchifolia exhibits a noteworthy antibacterial efficacy against MRSEboth in vitro and in vivo. Furthermore, proteomic and transcriptome data foreshadowed the antibacterial mechanism of Emilia sonchifolia against MRSE in several ways, which affected purine metabolism, promoting bacterial oxidative stress and inhibiting bacterial cell wall synthesis. Meanwhile, the results of enzyms in purine metabolism, antioxidant enzymes and cell morphology proved that the antibacterial mechanism of Emilia sonchifolia against MRSE inhibited purine metabolism which promoted bacterial oxidative stress and destroyed bacterial cell wall. The above results demonstrated that Emilia sonchifolia had great potential as antibacterial agent.

BSIs

Bloodstream infections

S.Epidermidis

Staphylococcus epidermidis

MRSE

Methicillin-resistant Staphylococcus epidermidis

CAT

catalase

SOD

Superoxide dismutase

TSB

Tryptic soy broth

TSA

Tryptic soy agar

MIC

Minimum inhibitory concentration

MBC

Minimum bactericidal concentration

SEM

Scanning electron microscopy

TEM

Transmission electron microscopy

PBS

Phosphate buffer saline

TCM

traditional Chinese medicine

GMP

Guanosine

AMP

adenosine monophosphate

IMP

inosine monophosphate

Ethics approval and consent to participate

This study was conducted after it is ethically reviewed and approved by the all participants.

Consent for publication

Not applicable

Availability of data and materials

The corresponding author can provide all data and materials of this work upon request.

Competing interests

The authors have explicitly stated that they do not possess any conflicting interests.

Acknowledgements：

This study were supported by the earmarked funding for National Nature Science Foundation of China under Grant number 82260824, Science and Technology Foundation of Guizhou Province under Grant number Qianke He Foundation-ZK [2021]General 08 ,Young scientific and technological talents project of Gui zhou Department of Education under Grant number Qianjiaohe KY [2022] 269. Guizhou Administration of Traditional Chinese Medicine Science and Technology Research Project on Traditional Chinese Medicine and Ethnic Medicine(project number:QZYY-2024-016)

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Table 1

MIC value of *E. sonchifolia* against MRSE isolates
Bacteria	Sample	MIC (mg/mL)	Concentration of antimicrobial agent (mg/mL)
MRSE	E. sonchifolia	5	160-0.078125

Table 2

MBC of *E. sonchifolia* value against MRSE
Bacteria	Samples	CFU/ml
MRSE	Control (TSB)	TNTC
MRSE	5mg/mL E. sonchifolia	TNTC
MRSE	10mg/mLE. sonchifolia.	7.3×10⁴
MRSE	20mg/mL E. sonchifolia	Nil
MRSE	40mg/mL E. sonchifolia	Nil
MRSE	80mg/mL E. sonchifolia	Nil
MRSE	160mg/mL E. sonchifolia	Nil

No competing interests reported.

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Proteomics and Transcriptomics Analysis of Emilia sonchifolia (L.) DC Antibacterial Activity against Methicillin-resistant Staphylococcus epidermidis

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