Ginkgolide a enhances the resistance to pathogen infection through mitochondrial unfolded protein response

Abstract

The normal function of mitochondria plays a key role in innate immunity. Normally, changes in the internal and external environment will lead to mitochondrial stress, and then the body will produce mitochondrial unfolded protein response (UPR^mt) to maintain mitochondrial homeostasis. Ginkgolide A (GA) is a diterpenoid isolated from Ginkgo, which has many important biological activities such as anti-inflammatory, anticancer, anxiolytic-like, anti-antherosclerosis and anti-atherombosis. However, whether GA affects innate immune responses and the underlying molecular mechanisms are still unknown. In the present study, we show that 100 µM GA enhances the resistance to Gram-negative pathogens Pseudomonas aeruginosa, Salmonella enterica and Gram-positive pathogens Staphylococcus aureus, Enterococcus faecalis in Caenorhabditis elegans by clearance intestinal bacterial loads. We also find that GA enhances innate immunity through a homeodomain transcriptional regulator DVE-1, which activates the UPR^mt. Because DVE-1 encodes a homeodomain transcription regulator that is homologous to the mammalian SATB2 transcription factor. Furthermore, we demonstrate that this function was conserved, because GA also manifested protective function in lung epithelial cell and mice during P. aeruginosa infection via the homeodomain transcription factor SATB2. Hence, our research suggests that GA has the potential therapeutic compound to protect patients from pathogen infection.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-025-05869-5.

Introduction

The innate immune system serves as the host’s first line of defense against invading pathogens. Its rapid activation not only facilitates the effective elimination of pathogens but also minimizes excessive inflammation and tissue damage [1, 2]. Upon triggering, the innate immune system promptly activates downstream signaling pathways, enhances immune cell activity, and initiates a robust response to combat pathogen infection [3, 4]. Caenorhabditis elegans has emerged as a valuable model organism for studying host-pathogen interactions due to its ease of cultivation, short life cycle, well-defined genetic background, and strong experimental flexibility [5]. Using this model, researchers have identified several classical signaling pathways that regulate innate immunity, including: MAPK Signaling Pathway, conserved pathway includes p38 MAPK, ERK MAPK, and c-JNK MAPK and plays a critical role in host defense [6, 7]. Insulin-like Receptor DAF-2/DAF-16 Signaling Pathway, DAF-2, similar to the insulin receptor, regulates innate immunity, lifespan, stress responses, and dauer formation by controlling the nuclear localization of the FOXO transcription factor DAF-16 [8]. Mitochondrial Unfolded Protein Response (UPR^mt), accumulation of misfolded or unfolded proteins in the mitochondrial matrix triggers the UPR^mt to maintain mitochondrial protein homeostasis [9], primarily mediated by transcription factors ATFS-1 [10] and DVE-1 [11]. Endoplasmic Reticulum Unfolded Protein Response (UPR^er), Similar to UPR^mt, UPR^er is involved in nematode innate immunity and is mediated by atf-6, pek-1, and ire-1α [12, 13]. Other known signal pathways involved in innate immunity of C. elegans, Wnt Signaling Pathway [14] and TGF-β signaling pathway which can regulate the expression of antimicrobial peptides and activate antimicrobial responses [15].

Natural products derived from plants have consistently demonstrated their therapeutic potential and remain a vital source for identifying novel drug candidates [16, 17]. In recent years, increased attention has been paid to the pharmacological properties of ginkgolide A, a diterpenoid isolated from Ginkgo, exhibits various biological activities, including anti-cancer [18], anxiolytic [19], anti-atherosclerotic [20] and liver-protective effects [21]. Notably, several studies have demonstrated its potent anti-inflammatory effects [22], yet its role in innate immunity and the underlying mechanisms remain unexplored.

Mitochondria, double-membraned organelles, regulate critical cellular activities such as metabolic reprogramming and immune homeostasis, serving as central hubs for signal transduction [23]. The stability of mitochondrial function determines the host’s immune resilience under pathogenic stress [24, 25]. The accumulation of misfolded proteins in mitochondria induces the expression of mitochondrial chaperones and activates the UPR^mt [26]. Chaperone proteins like hsp-60 and hsp-6 assist in folding newly imported proteins or refolding damaged ones [27]. Recent research has shown that the homeodomain-containing transcription factor DVE-1 binds to the hsp-60 promoter and drives chromatin remodeling in response to mitochondrial dysfunction, which is a critical precursor to the transcriptional response to mitochondrial stress [26]. Furthermore, increased nuclear localization of DVE-1 initiates downstream immune responses by activating UPR^mt, thereby enhancing innate immunity in C. elegans [11].

In our study, we explored the mechanism by which GA protects C. elegans from pathogenic bacterial infections. We confirmed that GA enhances host resistance to pathogens through the transcription factor DVE-1. Additionally, we demonstrated that GA activates UPR^mt and immune response genes in a DVE-1-dependent manner to bolster innate immunity in C. elegans. Importantly, this mechanism was shown to be highly conserved across species, from nematodes to mammals.

Materials and methods

Worm strains and cultivation

Worms were cultured and maintained under standard conditions as described previously [28–30]. The following nematode strains were acquired from the Caenorhabditis Genetics Center: N2 Bristol wild-type, VC3201 (atfs-1(gk3094)), SJ4058 (HSP-60::GFP), RB925 ire-1(ok799), KU25 pmk-1(km25), SD184 mpk-1(n2521), VC8 jnk-1(gk7), CB1370 daf-2(e1370), and SJ4197 (dve-1p::dve-1::GFP). The dve-1(tm4803) strain was obtained from the National BioResource Project. All C. elegans mutants were backcrossed three times with the wild-type N2 strain before use in laboratory experiments.

Infection assay

Escherichia coli OP50, Salmonella enterica SL1344, Enterococcus faecalis ATCC 29,212, and Pseudomonas aeruginosa PA14 were cultured overnight in LB broth at 37 °C. Similarly, Staphylococcus aureus NCTC8325 was grown overnight in tryptic soy broth (TSB, BD, Sparks, MD) at 37 °C with shaking at 180 rpm. The cultures were then spread onto NGM plates. All infection assays were conducted on NGM agar plates, either with or without the addition of GA. Synchronized worm populations were first cultured on NGM plates seeded with E. coli OP50 at 20 °C until they reached the young adult stage. Subsequently, 50 nematodes were transferred to individual NGM plates containing P. aeruginosa PA14, S. enterica SL1344, S. aureus NCTC8325, or E. faecalis ATCC 29,212. The plates were incubated at 25 °C, and nematode survival was monitored every 12 h. Worms that were immobile and unresponsive to touch were considered dead [31, 32]. All experiments were performed independently in triplicate, with three plates included in each assay.

Fluorescence microscopy

HSP-60::GFP and DVE-1::GFP nematode strains were synchronized and cultured to the L1 stage before being transferred to NGM plates supplemented with or without 100 µM GA. Fluorescent images were captured using a Zeiss Axioskop 2 Plus fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with a digital camera. Fluorescence intensity was quantified using ImageJ software (NIH). Each experiment included three plates, with approximately 30 worms per plate, and all trials were conducted independently three times.

Quantification of intestinal bacterial loads

P.aeruginosa/GFP was cultured overnight at 37 °C in LB liquid medium containing 100 µg/ml ampicillin, after which the bacterial solution was evenly spread onto NGM agar plates with or without 100 µM GA. Worms were then transferred to these NGM plates containing P. aeruginosa/GFP and incubated at 25 °C for 48 h [31]. To remove P. aeruginosa/GFP from the worm surface, worms were sequentially transferred to NGM agar plates seeded with E. coli OP50 for 30 min, repeating this process three times. Subsequently, 10 worms were collected and placed into 50 µL PBS containing 0.1% Triton solution for homogenization [33]. The resulting lysate was serially diluted with sterilized water and plated onto LB agar containing ampicillin. Plates were incubated at 37 °C for 24 h, and the number of P. aeruginosa/GFP colonies was counted. Six plates were analyzed per trial, and all experiments were conducted independently three times.

Quantitative real-time PCR

Nematodes treated with 100 µM GA and lung tissues from mice administered 20 mg/kg GA were collected for analysis. Total RNA was extracted from the worm and mouse tissues using TRIzol reagent (Invitrogen). Reverse transcription of the RNA into random-primed cDNA was performed with SuperScript III (Invitrogen). The resulting cDNA was analyzed using an Applied Biosystems Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Quantitative polymerase chain reaction (qPCR) was conducted with SYBR Premix-Ex Tag™ (Takara, Dalian, China). For normalization, pmp-3 was used as the internal control in nematodes [34, 35], while actin served as the internal control in cells and mice. The primers utilized in this study are listed in Table S2.

Cell culture and cell SiRNA transfection

A549 human lung epithelial cells (American Type Culture Collection, Manassas, VA) were removed from liquid nitrogen and rapidly thawed in a 37 °C water bath. Once thawed, the cells were transferred to a medium containing 10% FBS and incubated at 37 °C in a 5% CO₂ incubator, with the medium replaced every 8 to 12 h. Cells in optimal growth condition were seeded into six-well plates and transfected with SATB2 siRNA. The siRNA and corresponding negative control (siNC) targeting SATB2 were procured from Genechem (Shanghai, China). After 48 h of siRNA treatment, the cells were harvested, and the knockdown efficiency was evaluated using real-time quantitative PCR.

Cell cytotoxicity assays

A549 human lung cancer epithelial cells (American Type Culture Collection, Manassas, VA, USA) were rinsed and incubated in a serum-free medium for 1 h. Subsequently, the cells were infected with P. aeruginosa PA14, prepared in PBS from the logarithmic growth phase, at a multiplicity of infection of 100:1 [36]. Cytotoxic effects were assessed 4 h post-infection using Trypan blue (Sigma-Aldrich, St. Louis, MO), which stained non-viable cells blue.

Statistics

The data are expressed as the mean ± SEM. Statistical analyses were performed using an unpaired, two-tailed t-test or analysis of variance (ANOVA). Survival analysis was conducted using the log-rank (Mantel-Cox) test. A P-value of less than 0.05 was considered statistically significant. All visualizations were generated using GraphPad Prism 9.0 software (GraphPad, San Diego, CA, USA).

Results

GA increases the resistance to pathogen infection in C. elegans

Figure 1A represents the chemical structure of GA. To test whether GA enhances innate immunity in C. elegans, we utilized wild-type animals treated with GA (0, 1, 10, and 100 µM) and then exposed them to the human opportunistic pathogen P. aeruginosa PA14. We discovered that wild-type (WT) animals treated with GA were resistant to P. aeruginosa PA14, and this effect might have been dose-dependent (Fig. 1B, Table S1). Since intestinal bacterial colonization is one of the manifestations of host pathogen infection, we explored whether GA has an impact on the accumulation of intestinal bacteria [37, 38]. Worms treated with GA had a reduced number of bacteria in the intestine compared to control animals (Fig. 1C). These results indicate that GA enhances resistance to pathogen infection. Additionally, we examined whether GA has a broad-spectrum effect on pathogen resistance. We exposed both GA-treated and untreated wild-type worms to the gram-negative pathogen Salmonella enterica and the gram-positive pathogens Staphylococcus aureus and Enterobacter faecalis. The results demonstrated that GA enhanced the resistance to both Gram-positive and Gram-negative bacteria in C. elegans (Fig. 1D-F, Table S1). Overall, these results indicated that GA promoted innate immunity in C. elegans.

Fig. 1.

GA increases innate immunity in C. elegans (A) The chemical structure of ginkgolide A (GA). (B) The survival of N2 worms that were exposed to various concentrations of GA and infected with P. aeruginosa PA14. (*P < 0.05; log-rank test). (n ≥ 40). (C) With the enhancement of GA concentration, the CFUs of P. aeruginosa PA14 in the intestinal tract of nematodes declined. (*P < 0.05, unpaired t-test). (n ≥ 10). (D-F) GA (100µM) enhanced the resistance of C. elegans to S. enterica (D), S. aureus (E) and E. faecalis (F). (*P < 0.05, log-rank test). (n ≥ 40). The error bars indicate the mean ± SEM of three independent biological replicates. NS means (no significance)

GA activates the UPR^mt to promote innate immunity in C. elegans via DVE-1

Mitochondria possess a variety of biological functions and play a crucial role in regulating the pathogenesis of bacterial infections [39]. Therefore, we hypothesize whether GA promotes innate immunity by activating the UPR^mt. We first examined the expression of the hsp-60 chaperone protein after GA treatment, because the chaperone protein hsp-60 is one of the important markers for the activation of UPR^mt. The results indicated that GA increased the fluorescence intensity of the HSP-60::GFP (Fig. 2A). This implicated that GA promoted innate immunity by activating the UPR^mt. We did not observe the mRNA levels of dve-1 in dve-1(tm4803) mutant worms and the mRNA levels of atfs-1 in atfs-1(gk3094) mutant worms (Figure S1A and B). To further explore this mechanism, we examined the survival of atfs-1(gk3094) and dve-1(tm4803) mutant worms to the pathogen P. aeruginosa PA14 after treatment GA. We found that GA did not enhance innate immunity in dve-1(tm4803) mutant worms. However, GA increased innate immunity in atfs-1(gk3094) mutant worms (Fig. 2B, Table S1). To perform the rescue experiments, we used the [dve-1(tm4803) + lfEX1(dve-1p::dve-1)] transgenic worms, which carried dve-1 under the control of endogenous dve-1 promoter in dve-1(tm4803) worms. Expectedly, GA enhanced innate immunity in [dve-1(tm4803) + lfEX1(dve-1p::dve-1)] transgenic worms (Figure S2). In addition, we found that GA did not change CFUs of P. aeruginosa in dve-1(tm4803) mutant worms. However, GA decreased CFUs of P. aeruginosa in atfs-1(gk3094) mutant worms (Fig. 2C). These results suggested that GA enhanced innate immunity through DVE-1. Subsequently, quantitative real-time PCR analysis showed that GA increased the mRNA levels of dve-1 rather than atfs-1 (Fig. 2D). Furthermore, we found that GA significantly increased the expression level of DVE-1::GFP fusion protein (Fig. 2E). Next, the dve-1 target genes, hsp-60, hsp-6, irg-6, mul-1 and lys-2 were detected [39]. Quantitative real-time PCR analysis demonstrated that these genes were increased in animals treated with GA compared to the control. However, GA did not change their expression in dve-1 mutant worms (Fig. 2F). Meanwhile, we found that GA increased the mRNA levels of hsp-60, hsp-6, irg-6, mul-1 and lys-2 in Control animals. However, knockout atfs-1 did not decrease the mRNA levels of these genes after GA treatment (Figure S3). These results suggested that GA activated the UPR^mt to promote innate immunity via DVE-1.

Fig. 2.

GA activates UPR^mt to promote innate immunity through DVE-1 (A) The expression of HSP-60::GFP was elevated in worms that were treated with GA. The quantification of fluorescence intensity (n ≥ 20) is presented in the right panel. Scale bars: 100 μm. These results are presented as mean ± SEM from three independent experiments, each performed in triplicate. (**P < 0.01, unpaired t-test). (B) The survival curves regarding the GA treatment of wild-type, ATFS-1, and DVE-1 in P. aeruginosa PA14 (*P < 0.05; log-rank test). (C) The CFUs of P. aeruginosa PA14 in N2, atfs-1(gk3094), dve-1(tm4803) worms. (*P < 0.05, unpaired t-test). The error bars indicate the mean ± SEM of three independent biological replicates. (D) The levels of mRNA expression of atfs-1 and dve-1 in worms that were subjected to the treatment with GA. These findings are presented as the mean ± SEM based on three independent experiments, with each experiment being carried out in triplicate. (**P < 0.01, unpaired t-test). (E) The expression of DVE-1::GFP was increased in worms that were treated with GA when compared with the controls. The quantification of DVE-1::GFP (n ≥ 20) is displayed in the right panel. These outcomes are expressed as the mean ± SEM from three independent experiments (**P < 0.01, unpaired t-test). Scale bars: 100 μm. (F) The mRNA levels of dve-1 target genes in 100 µM GA-treated worms or in dve-1 mutant worms. (*P < 0.05, unpaired t-test). The error bars indicate the mean ± SEM of three independent biological replicates

GA enhances innate immunity independently of other known innate immune pathways

In addition, we examined whether the regulation of innate immunity by GA depended on other evolutionarily highly conserved innate immune pathways, such as PMK-1/P38 MAPK pathway, MPK-1/ERK MAPK pathway, JNK-1/JNK MAPK pathway, DAF-2/insulin pathway, and IRE-1/endoplasmic reticulum UPR pathway. We found that GA also enhanced innate immunity in pmk-1(km25), mpk-1(n2521), jnk-1(gk7), daf-2(e1370) and ire-1(ok799) mutant worms (Fig. 3A-F). Furthermore, we also found that GA reduced CFUs of P. aeruginosa in pmk-1(km25), mpk-1(n2521), jnk-1(gk7), daf-2(e1370) and ire-1(ok799) mutant worms (Fig. 3G). These results suggested that GA promoted innate immunity in C. elegans independently of other known innate immune pathways.

Fig. 3.

GA extends innate immunity independently of other known innate immune pathways. (A-F) Survival of (N2) (A), pmk-1(km25) (B), mpk-1(n2521) (C), jnk-1(gk7) (D), daf-2(e1370) (E), and ire-1(ok799) (F) mutant worms exposed to P. aeruginosa PA14 after 100 µM GA treatment. (*P < 0.05, log-rank test). (G) The CFUs of P. aeruginosa PA14 in N2, (N2), pmk-1(km25), mpk-1(n2521), jnk-1(gk7), daf-2(e1370), and ire-1(ok799) worms. (*P < 0.05, unpaired t-test). The error bars indicate the mean ± SEM of three independent biological replicates

GA protects A549 lung epithelial cells from infection by activating the SATB2-mediated UPR^mt

The identification of GA promoting innate immune responses in C. elegans via the UPR^mt led us to wonder whether GA exerted similar effects on cells. To determine whether GA was involved in the local infection response in human cells, we used A549 lung epithelial cells infected with P. aeruginosa PA14 [36]. We discovered that treatment with different concentrations of GA significantly reduced P. aeruginosa PA14-induced cytotoxicity in A549 lung epithelial cells and this effect might be dose-dependent (Fig. 4A). DVE-1, a homeodomain transcription factor, is homologous to the mammalian SATB2 [40, 41]. Subsequently, we examined the expression of SATB2. We found that the expression level of SATB2 was increased in A549 lung epithelial cells treated with GA (Fig. 4B). Additionally, knockdown SATB2 decreased the mRNA level of SATB2 (Fig. 4C). Meanwhile, we found that GA did not significantly reduce the cytotoxicity of P. aeruginosa PA14 in SATB2 knockdown A549 lung epithelial cells (Fig. 4D). Finally, we examined the target genes of the UPR^mt, such as HSPA1, HSPA9, LONP1, and YME1L1 [42–44]. Quantitative real-time PCR analysis demonstrated that these UPR^mt genes were increased in the GA-treated group compared with the control group. However, this phenomenon vanished in SATB2 knockdown A549 lung epithelial cells (Fig. 4E). These results indicated that GA protected A549 lung epithelial cells from pathogen infection through activation of the SATB2 mediated UPR^mt.

Fig. 4.

GA protects lung epithelial cells from infection through activation of SATB2 mediated UPR^mt (A) The cytotoxicity of cells exposed to P. aeruginosa PA14 for 4 h was significantly reduced in A549 lung epithelial cells after GA treatment. (**P < 0.01, unpaired t-test). (B) The mRNA level of SATB2, after GA treatment. These results are mean ± SEM of three independent experiments performed in triplicate. (**P < 0.01, unpaired t-test). (C) Quantitative real-time PCR was used to measure the knockdown efficiency of SATB2. (**P < 0.01, unpaired t-test). (D) The cytotoxicity of cells exposed to P. aeruginosa PA14 for 4 h in SATB2 knockdown and Control A549 lung epithelial cells after GA treatment. (**P < 0.01, ANOVA). (E) The mRNA levels of the mitochondrial unfolded protein response genes in SATB2 knockdown and Control A549 lung epithelial cells after GA treatment. These results are mean ± SEM of three independent experiments performed in triplicate (*P < 0.05, unpaired t-test)

GA protects mice from P. aeruginosa infection by activating the SATB2-mediated UPR^mt

Next, we investigated whether ginkgolide A enhanced innate immunity in mice. Mice in the GA treatment groups (with doses of 5 mg/kg body weight, 10 mg/kg body weight, and 20 mg/kg body weight) and the control mice were infected with P. aeruginosa PA14 (1.0 × 10⁶ colony-forming units per mouse). We found that GA treatment mice had significantly longer survival than the controls (Fig. 5A). Moreover, we tested the bacterial load in the lung by counting the viable colony-forming units (CFUs). We found that GA treatment mice significantly reduced bacterial load in the lung compared to the control group (Fig. 5B). The UPR^mt is a protective reaction that is highly conserved from worms to mammals [45, 46]. To determine whether GA boosted innate immunity in mice through the UPR^mt, we tested the mRNA expression level of SATB2 in mice. Quantitative real-time PCR analysis indicated that the mRNA level of SATB2 was significantly elevated in the group treated with GA compared with the control group (Fig. 5C). Subsequently, we examined the expression levels of the UPR^mt genes, HSPD1, HSPA9, LONP1, and YMME1L1, after treatment GA [42–44]. We found that the expression levels of HSPD1, HSPA9, LONP1, and YMME1L1 were upregulated in the GA treatment group compared to the control group (Fig. 5D). These results indicated that GA increased the resistance to pathogen infection via the SATB2-mediated UPR^mt.

Fig. 5.

GA protects mice from P. aeruginosa infection through activation of SATB2 mediated UPR^mt (A) Mice treated with GA (5, 10, 20 mg/kg) showed increased resistance to P. aeruginosa PA14 compared with the control group (*P < 0.05, log-rank test). (B) Lung homogenates were collected and plated on LB agar plates for P. aeruginosa PA14 counts. (*P < 0.05, unpaired t-test). (C) The mRNA level of SATB2 in mice with GA treatment. (*P < 0.05, unpaired t-test). (D) The mRNA levels of HSPD1, HSPA9, LONP1, and YME1L1 genes after GA treatment. These results are mean ± SEM of three independent experiments performed in triplicate (*P < 0.05, unpaired t-test)

Discussion

The widespread use of antibiotics has proven to be a double-edged sword, contributing to the emergence of multidrug-resistant microorganisms. Antibiotic resistance has recently become a significant global public health challenge for both clinicians and patients [47]. GA, a diterpenoid extracted from Ginkgo biloba leaves, exhibits a range of biological activities, including antitumor, antianxiety, anti-atherosclerosis, and hepatoprotective effects [48]. Extracts of ginkgo leaves are widely used as herbal medicine in Europe and as dietary supplements in the United States [49]. Several studies have demonstrated its potent anti-inflammatory properties [22, 50]. However, its role in innate immunity and the underlying mechanisms remain unexplored. In this study, we reveal that therapeutic application of GA protects the host against infections caused by Gram-negative pathogens such as P. aeruginosa and S. enterica, as well as Gram-positive pathogens like S. aureus and E. faecalis. This highlights its potential as a lead compound for developing broad-spectrum antibacterial drugs. Furthermore, our findings suggest that GA enhances innate immune responses by reducing intestinal bacterial loads in a dose-dependent manner.

Mitochondria are essential organelles that play critical roles in cellular functions, including energy production, metabolic regulation, and intracellular signaling [9]. Recent studies have emphasized the role of metabolic changes in regulating immune function and identified mitochondria as key elements in immune responses [51, 52]. Under mitochondrial stress, cells activate a protective mechanism known as the UPR^mt [26]. Key regulators of UPR^mt include ATFS-1, DVE-1, and UBL-5, with studies suggesting a cooperative interaction between DVE-1 and UBL-5 for coactivating UPR^mt alongside ATFS-1 [9]. However, the precise molecular mechanisms of this interaction remain unclear. Mark W. Pellegrino et al. reported that hyperactivation of ATFS-1 and UPR^mt enhanced the clearance of P. aeruginosa from the gut and prolonged the survival of C. elegans, independent of traditional innate immune pathways [53]. Similarly, Xiao et al. demonstrated that caffeic acid protects the host from pathogen infection by activating UPR^mt and immune response genes via ATFS-1 [54]. Furthermore, Chlorogenic acid activates UPR^mt to enhance innate immunity through ATFS-1 [55]. Interestingly, we found that GA enhances pathogen resistance by modulating UPR^mt through the transcription factor DVE-1. This effect was dose-dependent (Fig. 1B). In our experiments, treatment with GA did not improve resistance to P. aeruginosa in dve-1 mutant worms. However, GA increased innate immunity in atfs-1 mutant worms (Fig. 2B). These findings suggest an alternative mechanism by which natural products like GA protect hosts from diverse pathogens. To assess whether this mechanism is conserved, we evaluated the expression of SATB2, the human homolog of DVE-1, in both a lung epithelial cell model and a pathogen-infected animal model treated with GA. Our results demonstrated that GA enhanced the resistance of A549 lung epithelial cells and mice to pathogenic bacteria by activating the SATB2-mediated UPR^mt. Notably, GA upregulated SATB2 gene expression in A549 cells (Fig. 4B) and in the mouse model, leading to changes in target genes associated with UPR^mt (Fig. 5C and D). In conclusion, this study identifies DVE-1/SATB2 as an evolutionarily conserved transcription factor involved in pathogen resistance across species, from worms to mammals (Fig. 6). These findings provide foundational data for future investigations.

Fig. 6.

The mechanisms of GA-mediated innate immunity

Our research explores the role of GA in host innate immunity and demonstrates its capacity to augment innate defenses against both Gram-negative and Gram-positive bacteria. This highlights its potential as a broad-spectrum antibacterial agent. Importantly, our study also uncovers the role of the DVE-1-mediated mitochondrial unfolded protein response in host immunity, expanding the understanding of UPR^mt beyond the ATFS-1-mediated pathway. The conservation of the DVE-1-mediated UPR^mt mechanism from C. elegans to mice offers novel insights into the role of mitochondrial stress responses in immune regulation.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Y.X., and G.B. conceptualized and designed the study, aided in acquiring and analyzing data, drafted and critically revised the manuscript. Y.C., R.W., X.L., participated in experiments and the data analysis. Y.C. wrote the paper. All authors read and approved the final manuscript.

Funding

This work was supported by The Science and Technology Plan Project of Guizhou (QKHJC-ZK[2024]ZD071), Science & Technology Plan of Zunyi (2024[305]), The Xin miao Funding of Zunyi Medical University ([2021]1350-040).

Data availability

All datasets generated for this study are included in the article/Supplementary Material.

Declarations

Ethics

All animals were obtained from Zunyi Medical University. All mouse studies were carried out under standard conditions and in accordance with Zunyi Medical University Animal Care Committee (ZMU21-2305-003) guidelines.

Consent for publication

All authors read and approved the final manuscript.

Competing interest

The authors declared that they had no known competing interests.