Targeting catabolite control protein A in Staphylococcus aureus with auranofin

Abstract

The emergence of antibiotic-resistant Staphylococcus aureus poses a huge threat to public health. Therefore, novel strategies to overcome antibiotic resistance are urgently needed. Auranofin, a marketed metallodrug for rheumatoid arthritis, has been recognized as a promising agent against multiple clinical isolates of S. aureus. However, its antibacterial mechanism is not yet well understood. Herein, we verified that the catabolite control protein A (CcpA) in S. aureus is an important target of auranofin. Auranofin was found to directly bind to CcpA via two cysteine residues. Importantly, both in vitro and animal infection model assays demonstrated that auranofin could disrupt the biological activity of CcpA, which attenuated bacterial growth, inhibited toxin secretion and enhanced the efficacy of aminoglycoside antibiotic. Together, these findings further revealed the bactericidal mechanism of auranofin against S. aureus.

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1 Introduction

As a notorious Gram-positive pathogen, Staphylococcus aureus (S. aureus) is responsible for significant morbidity and mortality worldwide.^1,2 It persists in the nasal passages of approximately 30% of humans and also colonizes the skin, throat, armpits, groin, and intestines. As a multi-host opportunistic pathogen, S. aureus can cause a series of human diseases, ranging from minor skin infections to life-threating sepsis.³ Notably, the overuse or abuse of antibiotics has led to the emergence of multi-drug resistant S. aureus, posing a great threat to global health.⁴ Worse yet, methicillin-resistant S. aureus (MRSA), also known as a superbug, has developed resistance to “last resort” marketed antibiotics, such as vancomycin and linezolid.^5,6

In S. aureus, catabolite control protein A (CcpA) acts as one of the principal regulatory factors for carbon catabolite repression, mainly working by binding to catabolite-responsive element (cre) DNA sequences.⁷ When abundant carbon sources are present in the environment, CcpA inhibits the activation of other metabolic pathways, thereby optimizing energy utilization and enhancing the survival capability of the bacteria.^8,9 Importantly, this regulatory mechanism enables S. aureus to effectively adapt to diverse survival environments, augmenting its pathogenic potential.^10,11 In addition, the critical role of CcpA as an important global regulator of bacterial virulence has been fully validated. The function of S. aureus CcpA is closely related to biofilm formation, toxin expression and resistance to antibiotics^12,13 Notably, we have also developed antibacterial agents targeting CcpA in our previous studies and confirmed that these inhibitors markedly reduced S. aureus pathogenesis.^13,14

Auranofin is a typical gold-based organogold drug (Fig. 1A), which was approved by the Food and Drug Administration (FDA) in 1985 for clinical application in the treatment of rheumatoid arthritis.¹⁵ Interestingly, it has been widely reported that auranofin also possesses a variety of clinically relevant biological activities, such as antifungal, antiparasitic, antibacterial and anticancer properties.^16–20 Especially in antimicrobial applications, auranofin has shown significant antibacterial activity against various drug-resistant pathogens, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE).²¹ In addition, auranofin is not only less prone to resistance but also shows better efficacy than linezolid, a drug specifically used to treat drug-resistant enterococcal infections.^22,23 However, to date, the inhibitory mechanism of auranofin is only partially known.^24,25 It is reported that its antibacterial mechanism includes the inhibition of the thioredoxin (Trx) system and cell wall synthesis.^26,27 Herein, we demonstrated that CcpA is one of the important targets for auranofin against S. aureus. Auranofin can directly bind to CcpA via two cysteine residues, and then disrupts the biological activity of CcpA in vitro and in vivo. Auranofin attenuates bacterial growth, inhibits the secretion of toxins and enhances the efficacy of the aminoglycoside antibiotic (Fig. 1B).

Fig. 1 (A) Chemical structure of auranofin. (B) Schematic of auranofin targeting CcpA via two cysteine residues, thereby disrupting its biological activity.

2 Results and discussion

2.1 Characterization of auranofin binding to CcpA in vitro and living bacterial cells

As a typical organogold drug, auranofin is composed of a linear double-coordinating gold(I) centre, a tetraacetyl glucothiolate ligand and a triethylphosphonic gold (TP-gold) ligand.²⁸ Based on this structure, auranofin is highly thiophilic. It is also demonstrated that auranofin can directly bind to the sulfhydryl group on thioredoxin reductase, and then inhibit its activity.²⁹ Notably, CcpA from S. aureus contains two cysteine residues (Cys216 and Cys242). These two residues are important for S. aureus to resist the innate immunity of its host.³⁰ In addition, we have confirmed that Ag⁺ can target CcpA via its two cysteine residues, and then abolishes its DNA binding capability.³¹ We therefore investigated whether auranofin could do the same thing. At first, a fluorescence-based protein thermal shift assay (FTSA)³² was employed to explore the interaction between auranofin and the purified CcpA protein. As depicted in Fig. 2A, the transition midpoint (T_m) of purified CcpA decreased by 7.31 °C in the presence of auranofin, indicating the binding of auranofin to the CcpA protein. Next, in order to examine whether auranofin binds to CcpA via the two cysteine residues, the CcpA^2CS protein (in which Cys216 and Cys242 were mutated to serine) was constructed and purified. Interesting, auranofin failed in affecting the midpoint (T_m) of purified CcpA^2CS (Fig. 2B), suggesting that it cannot bind to the CcpA^2CS protein.

Fig. 2 Characterization of auranofin binding to CcpA in vitro. Fluorescent thermal shift analysis of CcpA (A) or CcpA^2CS (B) stability after auranofin (AF) treatment. Fluorescence signals are plotted against temperature, and the melting temperatures were calculated. CcpA^2CS refers to the CcpA protein in which two cysteines were mutated to serine.

Lastly, a cellular thermal shift assay (CETSA) (Fig. 3A), which relies on the change of protein thermal stability to verify the target of drugs inside the cell,^33,34 was performed to further investigate whether auranofin binds CcpA in living bacterial cells. As indicated in Fig. 3B, the thermal stability of CcpA in S. aureus was remarkably decreased after exposure to auranofin, suggesting the binding of auranofin to CcpA in bacterial cells. Taken together, these results validated that auranofin can directly bind to CcpA in vitro and in living bacterial cells.

Fig. 3 Characterization of auranofin binding to CcpA in living bacterial cells. Cellular thermal shift assay (CETSA) (A) of S. aureus with or without 0.1 μg mL⁻¹ auranofin treatment. The soluble fractions of intracellular CcpA protein were quantified by western blotting (B). The band intensities at different temperatures are normalized to that at 36.6 °C. All experiments were performed in triplicate.

2.2 Auranofin disrupts the biological activity of CcpA in living bacterial cells

Taken together, the in vitro and in vivo results demonstrate that auranofin can bind to CcpA. Thus, we wondered whether auranofin could also disrupt the biological activity of CcpA in living bacterial cells. As a global transcription factor, CcpA works mainly by binding to a variety of cre promoter sequences, including pckA and hla promoters (Fig. S1†), which encode the phosphoenolpyruvate carboxykinase or α-toxin, respectively.^35,36 Subsequently, a β-galactosidase (β-Gal) assay was employed to examine the effects of auranofin on CcpA function in living bacterial cells. In brief, a reporter plasmid (which fuses the pckA or hla promoter with the lacZ gene) was constructed, and then transformed into S. aureus. Therefore, the β-Gal activity in cell lysates can directly reflect the function of CcpA. The results indicated that 0.2 μM auranofin treatment led to an obvious decrease of β-Gal activity in two S. aureus strains, which bear the pckA::lacZ (Fig. 4A) or hla::lacZ (Fig. 4B) reporter gene, suggesting that auranofin disrupted the DNA-binding capacity of CcpA. Given that the β-galactosidase (β-Gal) assay confirmed that auranofin inhibits the hla-binding capability of CcpA and the hla gene encodes the α-hemolysin, it is reasonably speculated that the secretion of α-hemolysin from S. aureus could be inhibited by auranofin. The amount of α-hemolysin secreted from S. aureus was found to remarkably decrease after being incubated with auranofin (Fig. 5). Collectively, these results validated that auranofin can disrupt the biological activity of CcpA in living bacterial cells.

Fig. 4 Auranofin disrupts the DNA-binding capability of CcpA in vivo. β-Galactosidase activities of the (A) pckA::lacZ and (B) hla::lacZ reporters in the S. aureus RN4220 strain incubated with 0.2 μM auranofin. All experiments were performed in triplicate. The results are shown as mean ± SD.

Fig. 5 Auranofin inhibits the secretion of toxins in S. aureus. The amount of α-hemolysin secreted by S. aureus with auranofin treatment was monitored by the lysis rate of rabbit erythrocytes.

2.3 CcpA is one of the important targets of auranofin against S. aureus infection

Since CcpA is the major gene regulator of the central metabolism in S. aureus, the inhibition of CcpA was found to retard bacterial growth, attenuating toxin secretion. We therefore further validated whether CcpA is involved in the treatment of S. aureus infections via auranofin. Firstly, the ccpA gene in S. aureus was replaced by a double cysteine mutant gene ccpA^2CS, and then a mutant strain was obtained (denoted as S. aureus ccpA::ccpA^2CS), in which two cysteines were not present in the expressed CcpA protein. Next, the growth of both S. aureus and the ccpA::ccpA^2CS mutant were detected after the addition of 0.9 μg mL⁻¹ auranofin at the exponential-growth phase. As exhibited in Fig. 6A and B, auranofin exhibited a weaker inhibition effect on S. aureus ccpA::ccpA^2CS than on S. aureus. These results clearly indicated the multi-target effect of auranofin, where CcpA is one of the important targets of auranofin. In addition, this phenomenon was further validated in an animal infection model. In brief, Galleria mellonella (G. mellonella) was infected with S. aureus or S. aureus ccpA::ccpA^2CS mutant, respectively. After 1 hour, the infected G. mellonella was then treated with auranofin. As shown in Fig. 6C and S2,† above 80%, the infected G. mellonella died after 5 days. However, the survival rate of G. mellonella infected with S. aureus was obviously elevated by auranofin, and went from 10% to 60%, suggesting the robust anti-infective efficacy of auranofin against S. aureus. However, auranofin failed to do the same thing in the case of the S. aureus ccpA::ccpA² mutant infection. Taken together, these results confirmed that CcpA is one of the important targets of auranofin against S. aureus infection, and auranofin targeted CcpA mainly via the two cysteine residues.

Fig. 6 Identification of CcpA as a target of auranofin against S. aureus infection. The inhibitory effect of auranofin on the bacterial growth of wild-type S. aureus (A) and ccpA::ccpA^2CS mutant strains (B). (C) The anti-infective efficacy of auranofin was examined though the G. mellonella wax worm infection model. The G. mellonella was infected with S. aureus or ccpA::ccpA^2CS strains. and then treated with auranofin. The survival rate of the infected G. mellonella was recorded over a duration of 120 h.

2.4 Auranofin enhanced the efficacy of antibiotics by targeting CcpA in vitro

In the previous studies, we have demonstrated that the small-molecule inhibitors of CcpA all exhibited synergism with aminoglycoside antibiotics against S. aureus.¹³ In consideration of auranofin targeting CcpA via the two cysteine residues in vitro and in vivo, we speculate that auranofin should be effective in enhancing the efficacy of aminoglycoside antibiotics against S. aureus, but not against the S. aureus ccpA::ccpA^2CS mutant. Subsequently, a checkerboard assay (Fig. 7A) was employed to explore the synergism between auranofin and three aminoglycosides antibiotics, including gentamicin, kanamycin and amikacin. Synergism was defined as a fractional inhibitory concentration index (FICIs) of ≤0.5. Satisfyingly, the heat plots of the checkerboard assays clearly indicated that auranofin can efficiently potentiate the efficacy of three aminoglycosides antibiotics against S. aureus. In contrast, when applied to S. aureus ccpA::ccpA^2CS mutant strains, auranofin failed to do the same (Fig. 7B–G). In addition, the FICIs values and the isobologram analysis were calculated and plotted. As shown in Fig. 8, when applied to S. aureus, the FICIs values of the three antibiotics in combination with auranofin were less than 0.5, indicating the synergism effect. In contrast, auranofin failed in enhancing the potency of the three antibiotics against the S. aureus ccpA::ccpA^2CS mutant, whose FICIs were higher than 0.5.

Fig. 7 Identification of the synergistic effects of auranofin with a checkerboard assay. Schematic of the synergy checkerboard assay (A). Heat plots of the checkerboard assays for auranofin in combination with three antibiotics against S. aureus Newman or S. aureus ccpA::ccpA^2CS strains (B–G).

Fig. 8 Identification of the synergistic effects of auranofin with FICI values. The fractional inhibitory concentration indices (FICI) of antibiotics in combination with auranofin against S. aureus (A) or S. aureus ccpA::ccpA^2CS strains (B). The isobologram analysis of the synergistic effects of auranofin with 3 antibiotics against S. aureus (C) or S. aureus ccpA::ccpA^2CS strains (D).

Next, a combined time-killing assay was conducted to further examine the synergism effect. In brief, S. aureus was co-incubated with a sub-lethal dosage of antibiotics, auranofin or the combination of antibiotics and auranofin. Then, the viable bacteria were detected at one-hour intervals. As shown in Fig. 9, both antibiotics and auranofin showed very weak bactericidal potency when they were used alone. However, the combined use of antibiotics and auranofin resulted in a significant decrease in the number of viable bacteria, which was consistent with the checkerboard assay. Taken together, these data indicated that auranofin can enhance the efficacy of antibiotics by targeting CcpA in vitro.

Fig. 9 Identification of the synergistic effects of auranofin with the killing curves. The killing curves of kanamycin (A), gentamicin (B) or amikacin (C) against S. aureus when used in combination with auranofin for a duration of 3 hours.

2.5 Auranofin enhanced the efficacy of antibiotics by targeting CcpA in vivo

Encouraged by the potentiation effect of auranofin on aminoglycoside antibiotics in vitro, two animal infection models were established to further validate this synergism activity in vivo. At first, the G. mellonella infection model was established through infecting S. aureus or the S. aureus ccpA::ccpA^2CS mutant, respectively (Fig. 10A), then antibiotics or auranofin was administrated at dose of 10 mg kg⁻¹. As shown in Fig. 10B and S3,† both kanamycin and auranofin showed a poor therapeutic effect, only resulting in a 20% and 10% survive rate of the infected G. mellonella, respectively. However, all infected G. mellonella survived after treatment with a combination of kanamycin and auranofin. Since auranofin cannot target CcpA in the S. aureus ccpA::ccpA^2CS mutant, it should fail in enhancing the efficacy of kanamycin when applied to S. aureus ccpA::ccpA^2CS mutant-infected G. mellonella. In fact, the results are exactly as we expected. Compared to that in the drug-alone group, the survival rate of the infected G. mellonella was not elevated after treatment with the combination of kanamycin and auranofin (Fig. 10C and S4†). In addition, a similar effect of auranofin on gentamicin was observed. That is, auranofin can enhance the efficacy of gentamicin against S. aureus in the G. mellonella infection model, but failed when applied to the S. aureus ccpA::ccpA^2CS mutant (Fig. 10D, E, S5 and S6†).

Fig. 10 Validation of CcpA as a target for auranofin to enhance the efficacy of antibiotics in a G. mellonella infection model. (A) Schematic of the G. mellonella infection model. The survival rate of the S. aureus-infected G. mellonella model after treatment with kanamycin (B) or gentamicin (D) in the presence of auranofin for a duration of 5 days. The survival rate of a S. aureus ccpA::ccpA^2CS-infected G. mellonella model after treatment with kanamycin (C) or gentamicin (E) in the presence of auranofin for a duration of 5 days.

Next, a mouse skin infection model was employed to further validate the potentiation effect of auranofin on antibiotics in vivo. To begin, a wound was cut on the back of the mice and the bacterial suspension was introduced to develop abscesses at the wound site. Then, the drugs were applied to the wound twice a day, and the number of viable bacteria in the infected tissue was detected on the fifth day (Fig. 11A). As shown in Fig. 11B, either kanamycin or auranofin alone at 50 μg mL⁻¹ did not significantly remove bacteria from the infected tissues, showing a similar viable bacteria load (CFU mL⁻¹) to the control group. In contrast, the bacterial load (CFU mL⁻¹) in the infected tissue was remarkably decreased after treatment with the combination of kanamycin and auranofin, indicating that auranofin enhanced the efficacy of kanamycin. However, this synergism effect disappeared when they were applied to the wound infected with the S. aureus ccpA::ccpA^2CS mutant (Fig. 11C), suggesting that auranofin enhanced the efficacy of antibiotics mainly by targeting CcpA. In addition, the same was true when auranofin was used in combination with gentamicin; that is, auranofin is only effective in S. aureus, not in S. aureus ccpA::ccpA^2CS mutant strains (Fig. 11D and E). Collectively, these results clearly validated that auranofin can efficiently enhance the efficacy of antibiotics by targeting CcpA via the two cysteine residues in vivo.

Fig. 11 Validation of CcpA as a target for auranofin to enhance the efficacy of antibiotics in a mouse infection model. (A) Schematic of the mouse infection model. The viable bacterial load (CFU mL⁻¹) in the wound on the S. aureus-infected mouse after treatment with kanamycin (B) or gentamicin (D) in the presence of auranofin. The viable bacterial load (CFU mL⁻¹) in the wound on a S. aureus ccpA::ccpA^2CS-infected mouse after treatment with kanamycin (C) or gentamicin (E) in the presence of auranofin.

2.6 Transcriptomics analysis validated the targeted effect of auranofin on CcpA

Lastly, we performed RNA-Seq to analyze the overall genetic changes in S. aureus treated with auranofin at the transcriptome level. The heatmap and hierarchical clustering depicted that the gene expression pattern induced by auranofin was significantly different from that of the control group (Fig. 12A). Generally, 467 genes were downregulated and 467 genes were upregulated (Fig. 12B). KEGG pathway analysis showed that multiple categories were highly enriched, such as carbon metabolism, which included carbon metabolism (TCA cycle, glycolysis/gluconeogenesis, pyruvate metabolism, glyoxylate and dicarboxylate metabolism), virulence-related pathways (bacterial secretion system, protein export and quorum sensing), and amino acids-associated metabolic pathways (biosynthesis of amino acids, glycine, serine, threonine metabolism and lysine degradation) (Fig. 12C), suggesting that metabolism may play an important role in the bactericidal of auranofin.

Fig. 12 The transcriptome profiles of S. aureus exposed to auranofin. (A) Heatmap of the clustering analysis of gene expression patterns in S. aureus under auranofin treatment; (B) Volcano plot showing differential genes selected by p-value (<0.05) (Green: down-regulated; Red: up-regulated); (C) KEGG pathway enrichment analysis conducted on DEGs; (D) A schematic of the interconnected central carbon metabolic pathways. (E) Transcription profiles of genes involved in the TCA cycle, glycolysis and carbon metabolism. Values reported as log₂(foldchange) over the control group.

Carbon metabolism is critical for providing energy and developing antibiotic resistance.³⁷ Generally, carbon flux from glucose, glutamine and fatty acids flows through the interlinked central carbon metabolic pathways (Fig. 12D). We therefore focused on transcript levels of key genes in the TCA cycle, glycolysis and carbon metabolism (Fig. 12E). Notably, the transcription levels of most genes encoding key enzymes in the TCA cycle of S. aureus were significantly downregulated by auranofin, e.g., isocitrate dehydrogenase (icd), succinate dehydrogenase (sdhA/B), and succinate-CoA ligase (sucC/D). As a major metabolic process of the central carbon metabolism, S. aureus used glycolysis to generate ATP via substrate-level phosphorylation. Key enzymes in glycolysis were found to be predominantly upregulated in S. aureus treated with auranofin, such as the PTS system glucose-specific EIICBA component (ptsG), glyceraldehyde-3-phosphate dehydrogenase (gapA), phosphoglycerate kinase (pgk), and enolase (eno). Together, these results confirmed that auranofin induced metabolic reprogramming in S. aureus, leading to suppression of the TCA cycle activity and enhancement of glycolytic metabolic flow. In addition to glucose utilization, other carbon metabolic pathways like the folate cycle and arginine metabolism were involved in the regulation of carbon flux in S. aureus. Among them, there was a significant increase in the gene encoding phosphate acetyltransferase (pta) and a significant decrease in that encoding carbamate kinase (arcC), both of which could be regulated by catabolite control protein A (ccpA).^38,39 Meanwhile, CcpA regulated the pyruvate formate-lyase (pflB)-dependent conversion of pyruvate to acetyl coenzyme A and acetate.⁴⁰ It was confirmed that the deletion of ccpA derepressed the TCA cycle and inhibited biofilm formation.⁴¹ Thus, there was an imbalance of the overall carbon metabolism in S. aureus after treatment with auranofin, indicating that the function of CcpA was interfered.

3 Conclusion

The issue of drug-resistant S. aureus is a global concern, which poses a great threat to human health. Therefore, novel antibiotics are urgently needed to cope with antibiotic resistance. However, the combination of high cost, long cycle and low benefit has slowed the discovery and development of these life-saving drugs. Identifying new applications of old approved drugs is an effective method for the development of new antibiotics, which can greatly reduce costs and improve the success rate. Auranofin is a typical gold-based organogold drug, which was approved by the Food and Drug Administration (FDA) for rheumatoid arthritis. Notably, it also exhibited multiple biological activity, such as anticancer, antiparasitic and antibacterial efficacy. Especially in the aspect of anti-infection, auranofin has robust bactericidal potency and effectively overcomes bacterial resistance. Thus, it is a very promising metalloantibiotic. However, up to now, the underlying mechanism of the antimicrobial activity of auranofin is not fully understood. Herein, we show that auranofin can directly bind to CcpA in vitro and in vivo via two cysteine residues. In addition, both the β-galactosidase (β-Gal) assay and toxin secretion experiment validated that auranofin can disrupt the biological activity of CcpA in living bacterial cells. Importantly, we also confirmed that CcpA is an important target of auranofin against S. aureus infection. S. aureus was more sensitive to auranofin treatment than S. aureus ccpA::ccpA^2CS mutant in vitro and in the G. mellonella infection model. More importantly, at the cellular and animal levels, we demonstrated that auranofin can efficiently enhance the efficacy of antibiotics by targeting CcpA. Lastly, the analysis of the overall genetic changes in S. aureus also indicated that the function of CcpA was interrupted by auranofin. In summary, the results herein confirmed that CcpA is one of the potential targets of auranofin against S. aureus, further revealing the antibacterial mechanism of auranofin.

4 Experimental methods

4.1 Combined time-killing experiment

Bacteria were diluted with fresh TSB medium and then transferred into a bacterial culture tube. Then, either auranofin, antibiotics or the combination of auranofin and antibiotics was added into a tube. After incubation at 37 °C on a shaking incubator, 50 μL bacteria solution was collected at regular intervals and spread on a solid culture dish for counting. Lastly, time-killing curves were plotted.

4.2 Checkerboard assay

In a 96-well plate, 25 μL antibiotics was added into the well in each row, and 25 μL auranofin was added into the well in each column. Then, gradient dilutions were performed with distilled water. Afterwards, 200 μL diluted S. aureus or S. aureus ccpA::ccpA^2CS was added into each well. After incubation at 37 °C on a shaker for 20 hours, the absorbance of each well was measured using a microplate reader, and the FICI value was calculated.

4.3 The G. mellonella infection model

Bacteria in the log-phase stage were collected, washed with PBS, and then used to prepare the bacterial suspension (OD₆₀₀ = 0.3). The hind legs of G. mellonella were disinfected with alcohol, and then a 5 μL bacterial suspension was injected into G. mellonella from its left hind leg. One hour later, 5 μL aliquots of drugs were further injected (auranofin, 10 mg kg⁻¹; kanamycin, 10 mg kg⁻¹; gentamicin, 10 mg kg⁻¹; auranofin + antibiotics, 10 mg kg⁻¹) into G. mellonella from its right hind leg. Each group consisted of 10 G. mellonella. Lastly, the survival rate of the larvae was recorded every 24 hours.

4.4 Mouse wound infection model

Bacteria in the log-phase stage were collected, washed with PBS, and then used for the preparation of the bacterial suspension (OD₆₀₀ = 1). The backs of mice were shaved, and the mice were then anesthetized. Afterwards, a circular wound was cut on the mouse skin, and 100 μL of bacterial suspension was applied onto the area. After 24 hours, all mice were treated with drugs twice daily, once with 50 μL each of the following: auranofin, kanamycin, gentamicin, auranofin combined with the antibiotic (50 μg mL⁻¹). On the fifth day, the mice were euthanized and the skin tissue was obtained for grinding and spreading to monitor the number of viable bacteria in the infected tissue.

Ethics statement

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Jiangxi Science& Technology Normal University (No. 3601020137931).

Author contributions

Wenjing Lin: data curation, investigation, writing – original draft; Jingjing Chen: data curation, investigation; Ziying Huang: investigation; Haijun Li: investigation; Yushou Chen: investigation; Xuemin Duan: supervision; Yanshi Xiong: formal analysis, methodology; Bingjie Han: methodology, formal analysis; Guijuan Jiang: methodology, writing; Jintao Wang: funding acquisition, project administration; Xiangwen Liao: funding acquisition, project administration, supervision, review & editing.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors have declared that there is no conflict of interest.

Acknowledgements

We gratefully acknowledge the generous supported by the National Natural Science Foundation of China (22067006); supported by Jiangxi Provincial Natural Science Foundation, China (20232ACB213010, 20212BAB203007); and the Department of Education Science and Technology Research Project of Jiangxi, China (GJJ211106, GJJ201139, GJJ2201313).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5qi00073d

‡ These authors contributed equally to this work.

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