Antibacterial Mechanisms of Constituents from Galla chinensis Revealed by Experimental and Virtual Screening-Based Studies

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Introduction
Antibiotics are one of the most signifcant medical advances, and their discovery had a great efect on the treatment of human diseases.However, widespread excessive dispensing and inappropriate use of antibiotics have led to the creation of resistant strains [1].Antimicrobial resistance in bacterial pathogens has been a major cause of high morbidity and mortality [2].Terefore, new antibacterial agents are crucially needed to overcome resistant bacteria.Many secondary metabolites of plants with the potential of inhibiting drug-resistant bacteria have been found and their pharmacological efects have been gradually explored to seek ingredients with strong antibacterial activity [3][4][5].
Te overall therapeutic efect of TCMs in clinical antiinfective treatments has been witnessed in its long-term clinical application [6].Many Chinese medicines have been confrmed to have antibacterial activities [6][7][8].However, compared with Gram-positive bacteria, Gramnegative ones are more resistant to antibiotics due to their impermeable barriers and cell membranes.In order to explore active natural ingredients with a signifcant activity against Gram-negative bacteria, 14 TCMs used to treat diseases associated with bacterial infections, such as diarrhoea, dysentery, and skin infections, were selected for minimal inhibitory concentration (MIC) and total antibacterial activity (TAA) screening (Table 1).
Galla chinensis, a traditional Chinese medicine, is a gall formed mainly by the aphid Melaphis chinensis (Bell) Baker
Based on the antibacterial activity, the TCM with the strongest activity against Gram-negative bacteria represented by E. coli was selected to systematically explore antibacterial active ingredients and target proteins, molecular docking, and inhibition of target protein activity.

Bacterial Strains and Culture Conditions.
Staphylococcus aureus ATCC6538 and E. coli ATCC 8739 were obtained commercially from Guangdong Institute of Microbiology (Guangzhou, China).Te bacterial solution stored at −4 °C was thawed at room temperature and 100 μL of the bacterial solution was taken in 5 mL of sterilised hydrolysed casein peptone medium and incubated overnight in a 37 °C incubator.

2.3.
Extraction. 100 g of pulverized sample was immersed in 95% ethanol for 48 h.Te immersion was repeated 3 times in the ratio of herbs to ethanol 1 : 3 (v/v).Te fltrations were mixed and concentrated under reduced pressure to obtain the ethanol extract and weighed.
2.5.Antibacterial Activity Assay.Te MICs of the ethanol extract against the two bacteria were determined utilizing 96-well plate serial dilution assays [43].Te bacterial culture medium after overnight incubation was formulated to an appropriate bacterial suspension concentration by ultraviolet spectrophotometry.Generally, the optical density (OD) value at the wavelength of 600 nm was reduced to 0.065-0.075,which was equivalent to a bacterial suspension concentration of S. aureus and E. coli (10 5 -10 6 CFU/mL).Te 14 test samples diluted with dimethyl sulfoxide (DMSO) to a certain concentration, PGG, and control substances (vancomycin hydrochloride and kanamycin sulfate) were diluted with DMSO to 100 mg/mL, 0.1 mg/mL, 1 mg/mL respectively.Ten, 20 μL of each was seeded into the frst row of the 96-well plate, with 2 parallel wells for each.Next, 180 μL of the resazurin-bacterial liquid mixture was loaded into the frst row, while 100 μL of the resazurin-bacterial liquid mixture was loaded into the other rows.After blending the frst row, 100 μL of its solution was transferred to the second row to mix evenly, and the operations of the remaining rows were the same as above.By this method, certain gradient concentrations were made by 2-fold dilution; E. coli was observed after culturing in a constant temperature incubator at 37 °C for 6 hours, and S.aureus was observed after culturing for 8 hours.Te MIC value was determined based on the colour change and turbidity of the solution in the well of the plate.Te experiment was independently repeated 3 times.

Target Prediction of 49 Chemical Components of Galla chinensis.
Te structural fles of 49 ligands prepared above were imported into the Pharm Mapper database (https:// lilab-ecust.cn/pharmmapper/index.html) to obtain the potential protein target fles.Te scores from the 49 predicted target fles were ranked from high to low, and then, the top (https://www.uniprot.org/uploadlists/).Te protein names were converted into standard gene names, and the relevant fles of the predicted proteins belonging to the E. coli species (ECOLI) were downloaded.

Construction of Galla chinensis Component-Target
Networks.Based on the 49 chemical components of Galla chinensis and the genes corresponding to the proteins belonging to the E. coli species as predicted above, the componenttarget network was constructed via Cytoscape3.7.2 [45].

1V4A Protein Construction. E. coli expression vector
PET-28a-1V4A-His was constructed by Nco I/Xho I Enzymatic escharotomy.All the clones were constructed by PCR-based strategy, and protein expression and purifcation were performed according to previous methods [46].Briefy, the exogenous DNA fragment was constructed and subcloned into the PET-28a expression vector.Te protein was expressed and purifed from E. coli BL21 (DE3) under natural circumstances.
2.6.6.PGG Inhibits 1V4A Protein Activity Assay.To assess inhibition of 1V4A enzymatic activity by PGG, the deadenylylation of glutamine synthetase (GS)-AMP was monitored as the formation of c-glutamylhydroxamate utilizing the c-glutamyl transferase activity of GS as previously described with slight modifcations [46].Te c-glutamyl transferase activity was measured using a GS activity assay kit (Solarbio) following the manufacturer's protocol.
After incubating at 30 °C for 30 min in a water bath, an aliquot of 10 μL was withdrawn from the 1V4A reaction mixture and added to the test tubes for the GS activity assay (100 μl reaction system).Another 10 μL was taken for the blank tubes (100 μL reaction system) [46].Following the instructions of the GS activity assay kit (Solarbio), reagents II and III were added sequentially to the test tubes, while reagents I and III were added sequentially to the control tubes.Both sets were mixed thoroughly and incubated at 30 °C for an additional 30 min.Te reaction was stopped by adding the stop reagents.Te quantity of c-glutamylhydroxamate produced was determined by measuring the absorbance at 540 nm: ΔA 540 � A test 540 − A blank 540 .
2.6.7.Statistical Analysis.Experiments were replicated 3 times and the mean was calculated.TAA was calculated with the formulate (mL/g) � the extraction yield ×1000/MIC value (mg/mL) [43].Te TAA value indicates how much water or solvent can be added to 1 g of the extract to still inhibit pathogen growth.Te half-maximal inhibitory concentration (IC 50 ) of PGG was calculated using the log (inhibitor) vs. response -Variable slope (four parameters) equation in Prism V8 (GraphPad).

Galla chinensis Exhibited the Greatest Antimicrobial
Activity against E. coli.First, we assessed the extraction yields of 14 TCMs.As shown in Table 2, the highest extraction yield was observed in Galla chinensis (32.7%), followed by Cassia fstula L (27.27%) and Rheum ofcinale Baill (22.66%).
Furthermore, we assessed the antibacterial activity of 14 TCMs.In Table 2, the ethanol extracts of Galla chinensis, Paulownia fortunei (Seem.)Hemsl., and Coptis chinensis Franch.displayed strong activities against E. coli with MIC values in a range of 0.625-1 mg/mL, and their TAA values were 327 mL/g, 185.6 mL/g, and 167 mL/g, respectively.Meanwhile, the ethanol extracts of Coptis chinensis Franch., Houpoea ofcinalis Rehder & E.H.Wilson, and Paulownia fortunei (Seem.)Hemsl.exhibited signifcant activities against S. aureus with MIC values in a range of 0.06-0.08mg/mL, and their TAA values were 2672 mL/g, 1820 mL/g, and 1484.8 mL/ g, respectively.
By comparing the antibacterial activity, the activities of 14 TCMs against Gram-positive bacteria (S. aureus) were determined to be markedly stronger than their activities against Gram-negative bacteria (E.coli), which was consistent with the previous introduction.Strikingly, Galla chinensis exhibited the greatest antimicrobial activity against E. coli.
Target prediction of 49 chemical components from Galla chinensis against E. coli was carried out using a pharmacophore matching platform, Pharm Mapper server.A total of 63 putative E. coli targets were predicted for 49 chemical components (Supplementary materials Table 2).

Docking and Visualizing of Galla chinensis Chemical
Components toward Target Protein of E. coli.To better understand the plausible antimicrobial activity mechanism of Galla chinensis, the top ten score target proteins of E. coli (PDB IDs: 2NYA, 2OPX, 1NLM, 2GFP, 2ZM5, 2IMO, 2NRO, 2WDV, 1V4A, and 3CA8) were chosen to further dock with 49 chemical components via Schrödinger suit [50].Te new top ten docking results were listed in Table 3. Te results revealed that PGG (WBZ4) and 1v4a-TGG (WBZ3) both exhibited promising binding interactions for 1V4A with docking score below −7.7.1V4A represents the crystal structure of the N-terminal domain of E. coli adenylyl transferase (ATase) that catalyses the deadenylylation and subsequent activation of GS, a key enzyme in response to signals of nitrogen and carbon status [51].
To further reveal the outstanding afnities of PGG and TGG binding to target protein 1V4A, detailed docking and visualizing were performed.Te interactions between TGG and 1V4A are shown in Figures 3(a) and 3(b), the hydroxyl of PGG engaged in eight direct hydrogen-bonding interactions with GLU306, GLU327, ARG324, GLN331, GLU254, and ASP223 of target protein 1V4A.In Figures 3(c) and 3(d), the hydroxyl of PGG engaged in ten direct hydrogen-bonding interactions with GLU231, GLU327, GLU306, TRP253, ASP173, and ARG225 of target protein 1V4A.Terefore, we hypothesized that TGG and PGG might exert antibacterial efects by inhibiting the activity of the 1V4A.

PGG Inhibits the Activity of the 1V4A.
1V4A represents the N-terminal domain of the E. coli ATase, which is accountable for the deadenylylation process catalyzing GS-AMP to active GS.To evaluate the inhibition of 1V4A enzymatic activity by PGG, initially, the 1V4A protein from E. coli was synthesized and purifed using established procedures [46].Subsequently, the deadenylylation of GS-AMP was closely monitored as the formation of c-glutamylhydroxamate utilizing the c-glutamyl transferase activity of GS as previously described with slight modifcations [46].Te c-glutamyl transferase activity was determined via utilizing a commercial GS activity assay kit to monitor the formation of c-glutamyl isohydroxamic acid.In this work, we determined that PGG exhibited inhibition of ATase-1V4A activity with IC 50 value of 37.01 μg/mL (Figure 3(e)).Although PGG is present in both the ATase-1V4A activity assay and the GS activity assay reaction systems, the concentration of PGG in the GS activity assay reaction system is 1/10 of that in the ATase-1V4A activity assay reaction system [46].To rule out the inhibitory efect of PGG on GS activity, we tested the impact of the maximum concentration of PGG (10 μg/mL) on GS activity.Te results indicate that 10 μg/mL PGG does not signifcantly inhibit GS activity, suggesting that PGG directly inhibits ATase-1V4A activity.4, PGG exhibited signifcant antibacterial activity against the Gram-negative bacteria (E.coli) with an MIC value of 250 μg/mL.Vancomycin hydrochloride and kanamycin sulfate were employed as positive controls for the antibacterial activity.

Journal of Chemistry
Te content of PGG in ethanol extract was found to be 1.95% (w/w) with PGG standard sample as control, which demonstrated that Galla chinensis contains a high content of PGG.

Discussion
Te overuse and misuse of antibiotics have led to antibiotic resistance, resulting in signifcant morbidity and mortality.Gram-negative bacteria present a more substantial challenge in the medical feld compared to Gram-positive bacteria due to their increased resistance to traditional antibiotics.Unlike Gram-positive bacteria, Gram-negative bacteria possess an outer membrane composed of phospholipids and glycolipids, serving as a permeability barrier that makes them less susceptible to treatment.Consequently, very few substances have been identifed as efective against Gram-negative bacteria.To address the escalating incidence of Gram-negative infections, it is imperative to explore new targets and drugs specifcally designed to combat these types of infections.
In this study, we found that Galla chinensis exhibited the most potent anti-E.coli activity among the 14 Chinese herbs tested.Galla chinensis, a widely used traditional Chinese medicine, is known for its antidiarrheal, antiparasitic, and antibacterial properties.Despite having a normal MIC value, Galla chinensis demonstrated the highest extraction yields (32.7%) and the highest total antibacterial activity (TAA) value (327 mL/g) against E. coli.Tis outcome suggests that the exceptional antibacterial efectiveness of Galla chinensis is attributed to its high content of specifc antibacterial components.
Galla chinensis tannin is a type of hydrolyzable tannin that consists of a central glucose core, surrounded by several gallic acid units, and further gallic acid units can be attached.Chemical fngerprinting showed gallic acid (WBZ12) is the highest peak in Galla chinensis's ethanol extract, which means the highest content.Meanwhile, PGG (WBZ4) has been identifed in the Galla chinensis ethanol extract with a high content of 1.95% (w/w).
To better understand the potential antimicrobial ingredients and action mechanism of Galla chinensis, the component-target network was constructed between 49 chemical components and 63 predicted E. coli targets.Docking and visualizing of Galla chinensis chemical components toward top ten target proteins showed that PGG exhibited notable docking scores for 1V4A.PGG is a naturally occurring glucose pentagallic acid ester that belongs to the hydrolysable tannins and is chemically classifed as a gallotannin.Unfortunately, the highest constituent, gallic acid, might not have been screened through molecular docking due to its potentially small molecular weight.Gallic acid is commonly considered to be an efective natural antibacterial agent [52].
In our study, PGG inhibited bacterial growth in accordance with PGG's previously reported broad-spectrum activity, with MICs in the 50-500 μg/mL range [53].PGG's antibacterial activity has been attributed to its chelation of iron and its binding with lipopolysaccharide [54], but this  study is the frst to our knowledge to attribute its antibacterial activity to its afnity with ATase.ATase, encoded by glnE, is a signal transduction enzyme widely distributed in bacteria, and in some bacteria is essential for cellular viability [55].Te enzyme regulates the activity of glutamine synthetase by adenylylation and deadenylylation in response to intracellular signals of nitrogen status.Docking analysis demonstrated PGG exhibited notable docking scores to ATase-1V4A of E. coli mainly due to forming ten hydrogen bonds with key residues located at the active site pocket of the enzyme (Figures 3(c), 3(d)).It is speculated that PGG may act on active sites and hinder the chelation of Mg 2+ with the conserved nucleotidyltransferase domains, resulting in inhibition of the bimetallic mechanism of ATase and followed glutamine synthetase activity, and ultimately repressing nitrogen assimilation, protein synthesis, and life activities [51].Terefore, ATase may be an important potential target for PGG's antibacterial activity.Previous studies have indicated that PGG also inhibits other proteins in the enzyme inhibition assay.PGG exhibited a strong inhibitory efect on H + , K + -ATPase (IC 50 : 166 nM) and relatively weak inhibitory efects on Mg 2+ -ATPase (IC 50 : >10 μM) [56].Additionally, PGG was identifed as a dual inhibitor of urokinase-type plasminogen activator and plasmin, with IC 50 values of 6.861 μM and 149.0 μM, respectively.Tese studies suggest that PGG is a compound with broad inhibitory activity against enzymes, but it exhibits some level of selectivity in its inhibition of diferent enzymes [57].
In present study, PGG could be considered a promising anti-E.coli natural product that can suppress ATase catalytic activity with an IC 50 value of 37.01 μg/mL, and we believe that the structure and mechanistic insights presented here would support the development of potent Gram-negative inhibitors.

Conclusions
Based on the present results, Galla chinensis and its key active ingredient PGG exhibit a strong antibacterial activity against E. coli, and PGG exhibits a signifcant inhibitory activity against ATase of E. coli, which may become one of the important potential targets for anti-Gram-negative bacteria.

Figure 2 :
Figure2: Te component-target network (pink regular octagon refers to Galla chinensis, yellow regular hexagon refers to 49 components from Galla chinensis, and blue diamond refers to potential target genes).

Figure 4 :
Figure 4: Chemical fngerprint of the Galla chinensis ethanol extract.Blue numbers represent identifed compounds.Te positions of peaks corresponding to compounds are indicated by orange arrows and numbers (retention time).

Table 1 :
Ethnic traditional uses of 14 medicinal plants.

Table 2 :
MIC and TAA of the ethanol extracts of 14 TCMs.

Table 3 :
Te top 10 putative target proteins identifed by molecular docking via Schrödinger suit.