Insights into the potential dual-antibacterial mechanism of Kelisha capsule on Escherichia coli

Traditional Chinese medicine (TCM), AYURVEDA and Indian medicine are essential in disease prevention and treatment. Kelisha capsule (KLSC), a TCM formula listed in the Chinese Pharmacopoeia, has been clinically proven to possess potent antibacterial properties. However, the precise antimicrobial mechanism of KLSC remained unknown. This study aimed to elucidate the dual antibacterial mechanism of KLSC using network pharmacology, molecular docking, and experimental validation. By analyzing the growth curve of Escherichia coli (E. coli), it was observed that KLSC significantly inhibited its growth, showcasing a remarkable antibacterial effect. Furthermore, SEM and TEM analysis revealed that KLSC damaged the cell wall and membrane of E. coli, resulting in cytoplasmic leakage, bacterial death, and the exertion of antibacterial effects. The network pharmacology analysis revealed that KLSC exhibited an effect on E. coli ATP synthase, thereby influencing the energy metabolism process. The molecular docking outcomes provided evidence that the active compounds of KLSC could effectively bind to the ATP synthase subunit. Subsequently, experimental findings substantiated that KLSC effectively suppressed the activity of ATP synthase in E. coli and consequently decreased the ATP content. This study highlighted the dual antibacterial mechanism of KLSC, emphasizing its effects on cell structure and energy metabolism, suggesting its potential as a natural antibacterial agent for E. coli-related infections. These findings offered new insights into exploring the antibacterial mechanisms of TCM by focusing on the energy metabolism process.


Introduction
Bacterial infections have become a major factor affecting public health issues [1].At present, the use of antibiotics is a simple and common treatment for infections.However, the increasing and widespread use of antibiotics exacerbated the development of drug-resistant bacteria.Drug-resistant bacterial infections cause approximately 700,000 deaths worldwide each year [2].Drug-resistant pathogens have caused a serious threat to global health and antimicrobial treatment [3,4].
To address an unfavorable situation, one way is to develop new and more potential antibacterial drug BMC Complementary Medicine and Therapies candidates.Recent studies have shown that newly synthesized cadmium (II) complexes exhibit strong antibacterial and antifungal effects [5].Furthermore, iron nanoparticles synthesized using novel and green synthetic techniques have demonstrated potential antibacterial activity against E. coli in 2022 [6].In 2023, a review elucidates in detail that some newly synthesized pyridine compounds show good antimicrobials and can inhibit multidrug-resistant bacteria [4].Another approach is to find a promising alternative strategy for treating bacterial infectious diseases [7].Combination drug therapy is one of the treatment methods.Encouragingly, the essential oil of Copaifera spp.presents a promising alternative treatment for infections caused by multidrug-resistant Staphylococcus aureus when combined with conventional antibiotics or used in conjunction with LED lights [8].
At the same time, traditional Chinese medicine (TCM) with multi-target antibacterial modes is emerging as one of the effective antibacterial agents against bacteria [9].Numerous studies have highlighted the significant antimicrobial properties of natural compounds and medicinal plants [10][11][12][13][14].In vitro experiments have conclusively shown that extracts of Elatostema papillosum Wed.exhibit bacteriostatic effects when using different extracting solvents [15].Medicinal plants have been proven to damage the cell wall structure as part of their antibacterial mechanisms, as evidenced by numerous studies.Rose essential oil disrupted the cell walls and membranes of Pseudomonas putida, as observed through scanning electron microscope (SEM) and transmission electron microscope (TEM) observation [16].This disruption caused the leakage of intracellular substances, thereby exerting antibacterial effects [17][18][19].
Affecting the energy metabolism process is another way for TCM to inhibit bacteria.Bacterial ATP synthase is a potential therapeutic target for developing a new solution to treat bacterial infections [20].ATP synthase, also called F 0 F 1 ATP synthase, plays a vital role in cellular bioenergetics by facilitating the production of ATP from ADP and inorganic phosphate (Pi) [21].ATP energy is essential for the functioning of all organisms, and disruption of cellular bioenergetics could result in cell death [22].For example, Ocimum gratissimum L. essential oil could exert an antibacterial effect by inhibiting ATPase activity and affecting its metabolic capacity [23].And various natural active molecules have been proven to inhibit the ATP synthase activity of bacteria, such as resveratrol, thymoquinone and Quercetin [24].However, there is limited research on how TCM inhibits bacterial ATP synthase activity or impacts the energy metabolism pathway in terms of its antibacterial mechanism.
Kelisha capsule (KLSC) is a Traditional Chinese Medicine (TCM) commonly used in China.Its potent antibacterial properties have been demonstrated, yet its specific antibacterial mechanism has not been thoroughly explored, particularly at the molecular level.Network pharmacology, a novel discipline for studying drug mechanisms [25], offers a valuable bioinformatics tool for elucidating the synergistic therapeutic effects of TCM [26,27].While existing literature predominantly focuses on drug mechanisms in the human body, this study employs network pharmacology in a novel manner to investigate the action mode of KLSC on bacteria, specifically Escherichia coli (E.coli).
To gain a comprehensive understanding of the antibacterial mechanism of KLSC against E. coli, the SEM and TEM were employed to observe the morphological alterations in E. coli cells.Subsequently, this study investigated the impact of KLSC on the E. coli energy metabolism process based on network pharmacology combined with molecular docking.The study revealed the dual antibacterial mechanism of KLSC, involving disruption of bacterial cell structure and energy metabolism pathways.The research findings contribute to exploring the antimicrobial mechanism of TCMs through the energy metabolism pathway, providing valuable insights into potential therapeutic strategies.

Materials and reagents
The KLSC utilized in this study was provided by Zhejiang Sukean Pharmaceutical Co., Ltd.It consisted of a combination of 12 Chinese medicinal ingredients, which were finely powdered and encapsulated into capsules.The key ingredients of KLSC were the medicinal herb clove and Piper longum L, which were recognized as the official botanical remedies in AYURVEDA and the Indian medicine system.And the TCM information contained in KLSC was shown in

Antibacterial effect of KLSC on E. Coli Preparation of KLSC solution
The powdered KLSC was accurately weighed and transferred to a centrifuge tube.Subsequently, the LB medium was added, and the mixture was subjected to ultrasound treatment for 60 min.After the ultrasound, the solution was centrifuged at 10,000 rpm for 10 min.And the resulting KLSC solution was filtered through a 0.45 μm microporous membrane to obtain the final solution.

Determination of MIC and MBC
The minimum inhibitory concentration (MIC) was determined using the microdilution method [28,29].Twofold serial dilutions of KLSC solution were prepared in LB medium in 96-well microtiter plates.Bacterial suspensions were added to each well.Next, the bacterial organisms were cultured for 18 h at 37 ℃ and 60% RH.After cultivation, 20 µL 5% 2,3,5-Triphenyl-2 H-Tetrazolium Chloride (TTC) solution was added to each well and cultured at 37 ℃ for 30 min in the dark.The viable E.coli cells could reduce the colorless TTC to pink or red 1,3,5-triphenyl formazan (TPF) [30].Therefore, the MIC was identified as the lowest concentration at which no color change occurred.
To determine the minimum bactericidal concentration (MBC), the 100 µL E. coli organisms were spread onto LB medium plates and incubated for 24 h at 37 ℃ and 60% RH.The MBC was the lowest concentration at which no visible bacterial colonies were observed [31,32].

Measurement of the E. Coli growth curve
The bacterial growth curve was made to assess the impact of KLSC on E. coli growth [33,34].An overnight E. coli culture was added to the 48-well microtiter plate with varying concentrations of KLSC solutions.The KLSC solutions were at final concentrations of 1/2 MIC, MIC and 2 MIC.Next, the bacterial suspensions were inoculated at 37 °C and 60% RH.Finally, the growth of E. coli was monitored at different time points (0, 2, 4, 6, 8, 10, 12, and 24 h) by measuring the optical density at 600 nm (OD600) using a microplate reader (Tecan Spark).Control experiments were conducted using E. coli cells in the LB medium.All experiments were repeated three times.

Effect of KLSC on the structure of E. Coli Scanning electron microscopy observation
E. coli was grown in LB broth until reaching the logarithmic growth phase.Subsequently, the 20 mL E. coli suspensions were mixed with 20 mL KLSC solutions (128 mg/mL), and inoculated at 37 °C and 60% RH for 18 h.A control group was established by adding an equivalent volume of LB broth to E. coli suspensions.After cultivation, the E. coli cells from different experimental groups were collected by centrifugation at 4200 rpm for 4 min at 4 ℃ (Eppendorf centrifuge AG5424, Hamburg, Germany).The E. coli samples were prepared according to a published paper [35].Then, the SEM (Hitachi SU8100, Japan) was applied to observe the morphology of E. coli samples.

Transmission electron microscopy (TEM) observation
For TEM observation, the E. coli cells were collected using the same method described in Sect.2.4.1.The samples were processed following the procedure outlined in the literature [35], involving fixation, dehydration, embedding, slicing, and staining.Subsequently, the morphologies of various E. coli samples were analyzed by TEM (Hitachi HT7800, Japan).

Measuring permeability of cell wall and cell membrane
To estimate the permeability of the cell wall, alkaline phosphatase (ALP) leakage was utilized as an indicator to evaluate the bacterial cell wall damage [36].The E. coli suspensions in the logarithmic growth phase were treated with KLSC solutions in MIC and 2 MIC concentrations, respectively.These bacterial suspensions were then incubated at 37 °C and 60% RH.At 0, 2, 4, 6, and 8 h, the E. coli suspensions were centrifuged, and the supernatants were collected.ALP assay kit (lot: 101822230103, Beyotime Biotech.Inc. Shanghai, China) was used to measure the ALP leakage in the supernatants at different time points.The LB medium was served as the normal control.

Detection of electrical conductivity
Furthermore, the detection of electrical conductivity was conducted to evaluate cytoplasmic leakage from the bacterial cells into the LB medium [37].The experimental procedure involved obtaining E. coli suspensions in the exponential growth phase, treating them with KLSC solutions at 1 MIC and 2 MIC concentrations, and then incubating the suspensions at 37 °C and 60% RH.At specific time intervals, the bacterial suspensions were collected.Following centrifugation at 10,000 rpm for 10 min, the conductivity of the supernatant was measured by a conductivity meter (DDBJ-350, Hangzhou Qi Wei Instrument Co., Ltd.Hangzhou, China).The LB medium was considered as the normal control.All experiments were repeated three times.

Effect of KLSC on the ATP metabolism process of E. Coli Compounds screening and putative protein targets of KLSC
In the Traditional Chinese Medicine System Pharmacology Database (TCMSP, https://old.tcmsp-e.com/tcmsp.php#), the main chemical compounds of medicinal herbs were searched.The bioactive phytoconstituents of each herbal medicine were collected based on oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18) [38,39].Active components of mineral medicine in KLSC were screened from the HERB database (http://herb.ac.cn/).And some active ingredients were supplemented by relevant literature.The protein targets that acted on E. coli were predicted according to the STITCH database (http://stitch.embl.de/).The information on protein targets such as gene names or gene IDs was transformed through the UniprotKB database (https://www.uniprot.org/).A network encompassing "drug-compound-target-E. coli" was constructed using Cytoscape 3.9.1 software, with key active compounds being identified based on their degree values [40].

Construction and analysis of protein-protein interaction network
The intersection protein targets between KLSC and E. coli were considered potential antibacterial targets.Next, the intersection targets were imported into the STRING database (https://cn.string-db.org/) to construct a protein-protein interaction (PPI) network.The resulting PPI network was saved in a TSV format file and further analyzed using Cytoscape 3.9.1.Finally, the hub gene within the network was identified by the MCODE plug-in in the Cytoscape software.

Gene ontology and KEGG pathway enrichment analysis
To delve deeper into the pathway and biological implications of the network, GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis were conducted using the STRING database.GO enrichment analysis included Molecular function (MF), Biological Process (BP), and Cellular Component (CC) [41].In the pathway enrichment analysis, the statistical significance threshold was set to p < 0.05.

Simulating validation of molecular docking
To confirm the interaction between active compounds of KLSC and E. coli protein targets, key proteins were used for molecular docking simulation.The active compound structures and core protein targets were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and Protein Data Bank (PDB) (https://www.rcsb.org/),respectively.Following processing based on published papers [42,43], Autodock Vina1.5.7 was employed for docking core targets with key active medicinal molecules.And the outcomes after molecular docking were visualized by PyMOL software.

Measurement of ATP content
To validate the network pharmacology results, the intracellular ATP content of E. coli was measured.E. coli suspensions in the logarithmic growth phase were treated with KLSC solutions at MIC and 2 MIC concentrations, respectively, then incubated at 37 °C and 60% RH.Bacterial cells were collected by centrifugation at 4200 rpm for 10 min at 0, 0.5, 1.0, 1.5, and 2.0 h.Based on the instructions of the ATP Assay Kit (Lot: 072022220930, Beyotime Biotech.Inc. Shanghai, China), the intracellular ATP contents of E. coli were measured.The bacterial cells treated with LB medium served as the normal control group.

Detection of ATP synthase activity
In order to explore the effects of KLSC on ATP synthesis, ATP synthase activity was detected.According to the culture method in Sect.2.6, the E. coli cells treated with KLSC solutions were collected at various times.Then, the ATP synthase activities were detected according to the instructions of the ATP synthase Assay Kit (Lot: 20230625, Shanghai Jianglai Industrial Limited by Share Ltd, Shanghai, China).In this experimental test, the LB medium was considered as the normal control group.

Data analysis
In this study work, all experiments were repeated three times.The experimental data were expressed as average values ± standard deviations.The significant differences were tested by One-way analysis of variance (ANOVA).And the threshold value for the significant level was set at p < 0.05.

Antibacterial activity of KLSC against E. Coli
In this work, the MIC and MBC of KLSC against E. coli were initially determined using the microdilution method and TTC solution staining.The MIC was 64 mg/ mL.After the bacterial suspensions were spread into LB medium plates for incubation, it was found that no bacterial colonies were grown at the concentration of 64 mg/ mL.It indicated that the MBC was 64 mg/mL.
In order to further study the antibacterial activity against E. coli, the growth curves of E. coli were measured at 1/2 MIC, MIC and 2 MIC within 24 h.It could be found from Fig. 1 that the E. coli exhibited normal proliferation in the LB medium.However, the lag phase of the E. coli was postponed at 1/2 MIC, and the growth curve of E. coli decreased significantly.It was confirmed the inhibitory effect of KLSC against E. coli.At 1 MIC and 2 MIC, the E. coli growth was completely inhibited.It demonstrated that the KLSC had significant antibacterial effects.

Effect of KLSC on the structure of E. Coli The integrity of E. Coli cell wall
ALP, positioned between the cell wall and cell membrane, was not leaked into the extracellular environment [16].Monitoring ALP activity in the extracellular environment served as an indicator of cell wall damage.The study measured ALP activity in the extracellular E. coli, with results depicted in Fig. 2. The ALP activity remained relatively stable in the LB medium for 8 h.It suggested that the E. coli cell wall maintained its normal structure.However, the ALP activity was significantly increased within 2 h at concentrations of 1 MIC and 2 MIC, respectively.It indicated that the KLSC could damage the cell wall within this timeframe.This damage led to the leakage of ALP into the LB medium, resulting in an elevation in extracellular ALP activity.To visually assess cell wall changes, the SEM was used to examine the E. coli morphology.The results were displayed in Fig. 3. Figure 3 (a) showed that the untreated E. coli cells were intact and normal morphology, while those treated with KLSC showed evident differences.It could be seen from Fig. 3 (b) that the cell wall was cracked and damaged.The structure morphology was deformation and shrinkage.The morphological changes of E. coli were in accordance with the results of ALP activity.Based on the results of SEM and extracellular ALP activity, it was collectively confirmed that the KLSC could destroy the cell wall of bacteria, potentially leading to changes in the internal microenvironment and hastening bacterial cell death.

The integrity of E. Coli cell membrane
Maintaining the integrity of the cell membrane is one of the crucial conditions for the survival of bacterial cells [36].The TEM images clearly illustrated that E. coli cells in the LB medium had intact cell structures (Fig. 3 (c)).The cytoplasm presented a dense and homogeneous state.Conversely, in the KLSC group (Fig. 3 (d)), intracellular cavities were observed, indicating cytoplasm leakage.Therefore, cytoplasmic leakage was bound to alter the conductivity of the LB medium, as Fig. 4 evidenced by a significant increase in conductivity in the KLSC group compared to the normal group.The elevated conductivity indirectly suggested the leakage of various cellular components into the medium, implying irreversible damage to the E. coli cell structure caused by KLSC.
The cell membrane of bacteria serves as a crucial protective barrier that is essential for maintaining the viability and integrity of the cell.Disrupting the integrity of the cell wall and membrane can cause changes in membrane permeability, leading to the leakage of nucleic acids and proteins from the cell [44].These changes disrupt the balance of the internal environment and physiological processes to some degree, ultimately resulting in a loss of cell viability and hastening the bacteria's death.In essence, the findings suggest that KLSC has the ability to break down the cell wall of E. coli, representing one of its antibacterial mechanisms.

Effect of KLSC on the energy metabolism of E. Coli
ATP is crucial for various biological cellular processes and is essential for the survival of E. coli [24].This study employs network pharmacology to reveal the effect of KLSC on ATP metabolism in E. coli.

Identification of active components and hub targets
Initially, 82 active ingredients meeting the criteria of OB ≥ 30% and DL ≥ 0.18 were collected from the database.Since eugenol had an intense inhibitory activity against E. coli, it was added as an active pharmaceutical ingredient for network pharmacological analysis [45,46].Subsequently, a total of 83 active ingredients were examined, revealing that 22 of these interacted with 85 proteins in E. coli, as shown in Fig. 5 (a).Notably, the top ten compounds with larger degrees were identified as active components based on the "drug-compound-target-E. coli" network.The active molecules included kaempferol, bis(2-ethylhexyl) phthalate, quercetin, arsane, sesamin, eugenol, supraene, piperine, realgar, arsenic trisulfide, respectively.Detailed information on these active ingredients was listed in Table 2.
The hub genes associated with ATP metabolism, such as atpA, atpB, atpC, atpD, atpE, atpF, atpG, atpH, and nuoC, were identified through PPI network analysis using the STRING database and Cytoscape 3.9.1, as shown in Fig. 5 (b).These findings suggest that the active compounds in KLSC primarily interact with the ATP synthase of E. coli.

GO and KEGG analysis
In addition, the GO and KEGG enrichment analyses were conducted to investigate the underlying antibacterial mechanisms of KLSC.The GO enrichment was usually applied to analyze the functions of genes from the perspective of biological process (BP analysis), cellular component (CC analysis), and molecular function (MF analysis) [27].Go analysis revealed that there were 23 items correlated with BP (p < 0.05) and three items CC (p < 0.05).The 24 items involved in MF (p < 0.05).And the results were displayed in Fig. 6.In the GO analysis, the CC was involved in the proton-transporting ATP synthase complex (GO: 0045259), proton-transporting ATP synthase complex, catalytic core F(1) (GO: 0045261) and proton-transporting ATP synthase complex, coupling factor F(0) (GO: 0045261).The BP was also involved in monovalent inorganic cation transport (GO: 0015672) and inorganic cation transmembrane transport (GO: 0098662).And the MF was involved in proton-transporting ATP synthase activity-rotational mechanism (GO: 0046933), proton transmembrane transporter activity (GO: 0015078) and proton-transporting ATPase activity-rotational mechanism (GO: 0046961).Based on the GO analysis, the KLSC could affect the activity of the ATP synthase complex through hindering the transmission of protons.Through KEGG enrichment analysis, the signaling pathways included seven items.As shown in Table 3, the pathways were related to metabolic pathways (eco01100), oxidative phosphorylation (eco00190), and tryptophan metabolism (eco00380).These results demonstrated that KLSC may exert antibacterial effects through various pathways and multiple targets.

Molecular docking investigation
Based on the findings from network pharmacology screening, the validation of interactions between active compounds and key targets was conducted using molecular docking.Generally, the binding energy between ligand and receptor was less than − 5 kcal/mol, indicating a good bind activity [25,47].Lower binding energies signified higher affinity and greater stability of the conformation.The results illustrated in Fig. 7 demonstrated that the binding energies between ligands and receptors were predominantly below − 5 kcal/mol.It indicated that these active compounds in KLSC had high affinities with the various ATP synthase subunits.
The molecular docking outcomes revealed that piperine exhibited the lowest binding energy (-9.8 kcal/mol) with atpB among all interactions, suggesting a strong interaction with atpB.Additionally, quercetin and atpD (-9.0 kcal/mol), kaempferol and atpA (-8.1 kcal/mol), kaempferol and atpD (-8.8 kcal/mol), quercetin and atpA (-8.1 kcal/mol), and eugenol and atpB (-6.3 kcal/mol) displayed notable binding affinities, as depicted in Fig. 8.These results indicated that the compounds in KLSC could effectively interact with ATP synthase subunits.Given that ATP synthase is crucial for ATP synthesis, the inhibition of ATP synthase activity by KLSC ultimately led to the death of E. coli cells.Furthermore, the integration of network pharmacology analysis suggested that KLSC might impact the energy metabolism process of bacteria by inhibiting the ATP synthase activity.

Effects of ATP contents and ATP synthase activity
Based on the outcomes derived from network pharmacology and molecular docking analyses, it was observed that KLSC had an impact on the ATP metabolism process.Subsequently, KLSC was utilized as the subject of experimentation to validate the predictions made through network pharmacology and molecular docking.As illustrated in Fig. 8, the bioactive components present in KLSC were found to interact with ATP synthase   In conclusion, the collective evidence from network pharmacology, molecular docking, and experimental investigations indicated that KLSC could influence ATP synthase activity and ATP content.Given that ATP serves as the fundamental energy source in E. coli, ATP deficiency could result in cell death.Therefore, KLSC might exhibit antibacterial properties by modulating ATP synthesis.

Discussion
TCM often contains multiple antibacterial components with multiple targets, and can exert its antibacterial effect through a combination of multiple processes.Therefore, bacteria are less likely to develop resistance to TCM.TCM plays a very important role in the treatment of drug-resistant bacterial infections [48].As studied in this article, KLSC can damage the cell membrane system of E. coli and affect its energy metabolism process.Therefore, KLSC can exert antibacterial modes through the synergistic effect of multiple processes.The maintenance of cell viability relies on the integrity of the cell wall.Disrupting the cell wall is a common antibacterial mechanism in traditional Chinese medicines (TCMs).For instance, compounds like eugenol and kaempferol have been discovered to possess antibacterial effects by destroying the cell wall [45,49].KLSC contains these active compounds as key components.Therefore, it can be inferred that some active molecules in KLSC have the potential to disrupt the bacterial cell wall.SEM and TEM studies support this hypothesis.The disruption of the bacterial cell wall by pharmacological molecules may involve the inhibition of penicillin-binding proteins (PBPs), which are crucial for peptidoglycan synthesis.Peptidoglycan is an essential component of bacterial cell walls [50].The inactivation of PBPs can impede peptidoglycan formation, thereby hindering the development of cell walls [51] and ultimately leading to bacterial death.Therefore, PBPs are a major target for antibiotics [52].Through molecular docking, a large number of papers have reported that natural active molecules can be bound to PBPs [53][54][55][56].Natural active molecules like quercetin, eugenol and kaempferol have been reported to bind effectively to PBPs [57,58].It is worth noting that kaempferol exhibits higher specificity toward PBPs by interacting with crucial amino acids [59].This suggests that the small molecules in KLSC have the potential to disrupt E. coli cell wall formation and exhibit antimicrobial properties by targeting PBPs.However, these hypotheses lack experimental validation, representing a limitation of this study.
ATP synthase, an extremely conserved enzyme embedded in the bacterial plasma membrane, is considered an effective pharmaceutical target for treating bacterial infections [60].Energy metabolism plays a crucial role in an organism's survival, molecular function, and biological process [61].Oxidative phosphorylation is a key pathway for energy production, with ATP synthase being a critical component in this process, as shown in Fig. 10 (a) [62].ATP synthase generates ATP during the terminal step of oxidative phosphorylation, which is crucial for various cellular functions [63].Its inactivation can cause a reduction in ATP supply within bacterial cells, ultimately leading to bacterial death.Therefore, ATP synthase plays a vital role in bacterial survival.KLSC contains active compounds such as piperine, eugenol, and quercetin, which have been identified through network pharmacology analysis.Literature reports suggest that these compounds can inhibit ATP synthase activity to varying degrees [64][65][66].Experimental evidence has shown that KLSC can inhibit ATP synthase activity and reduce intracellular ATP levels.Through network pharmacology, molecular docking, and literature findings, it is suggested that these active compounds may impact energy metabolism by targeting ATP synthase.For ATP synthase, proton gradient-driven clockwise rotation of the subunit is essential for ATP synthesis [67], as shown in Fig. 10 (b).Based on the GO analysis, KLSC may interfere with proton transmission, potentially disrupting proton gradients and impeding ATP synthase rotation, thereby reducing ATP synthesis.Antibacterial mechanism of KLSC involves the modulation of energy metabolism, as supported by the available evidence from network pharmacology and experimental studies.
Revealing the action mode of antimicrobial agents is an important prerequisite for understanding the mechanism of drug-resistant bacteria [5].A promising solution for drug-resistant bacteria is the development of new antibacterial entities that act on novel targets of bacteria.Due to the different structures of ATP synthase between bacteria and mammalian [68], selectively targeting bacterial ATP synthase offers a safe and effective antibacterial approach that does not harm mammalian cells.Betaquiline is a successful ATP synthase inhibitor against multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains without causing toxic effects on ATP production in mammalian cells [20,69].Developing ATP synthase inhibitors is therefore viewed as a promising potential solution to overcome drug resistance.KLSC has multiple components and targets, and inhibits the activity of E. coli ATP synthase.Therefore, KLSC is a promising candidate for development as antibacterial agent against multidrug-resistant bacteria.

Conclusions
In this work, it was shown to be remarkable antibacterial efficacy of KLSC against E. coli.KLSC treatment disrupted the cell wall and membrane structure of E. coli.This accelerated the leakage of intracellular substances, thereby inducing bacterial death.Additionally, network pharmacology and molecular docking proved that the KLSC exerted antibacterial effects through affecting the energy metabolism process.Experimental findings further confirmed that KLSC could inhibit ATP synthase activity and decrease intracellular ATP levels.Thus, the dual antibacterial mechanism of KLSC was elucidated, involving disruption of the membrane system and affecting the energy metabolism process.

Fig. 3
Fig. 3 The structure of E. coli.(a) SEM micrographs of E. coli untreated with KLSC; (b) SEM micrographs of E. coli treated with KLSC; (c) TEM micrographs of E. coli untreated with KLSC; (d) TEM micrographs of E. coli treated with KLSC.

Fig. 8
Fig. 8 Molecular docking of active compounds and key targets

Table 1
. Escherichia Coli All ingredient information of KLSC

Table 2
The top ten active compounds of KLSC

Table 3
The KEGG enrichment analysis results Fig. 7 The heat map of Molecular docking binding energy.(Behp represents bis(2-ethylhexyl) phthalate)