B. thuringiensis infection leads to cellular energy imbalance in C. elegans
To investigate the intracellular physiological changes of C. elegans after pathogenic Bt infection, we conducted the C. elegans transcriptome analysis after infection by the nematicidal Bt strain BMB171/Cry5Ba, an acrystalliferous Bt mutant BMB171 transformed with toxin gene cry5Ba on the shuttle vector pHT304 30. As a control, we compared the transcriptome to a non-nematicidal Bt strain BMB171/pHT304, BMB171 transformed with the empty vector pHT304. Enrichment pathway analyses highlighted several pathways strongly affected by the infection of nematicidal Bt strain (Fig. 1A and Table S2). Interestingly, we found the energy metabolic related pathway was most strongly affected. This indicated that the regulators of the energy metabolic pathway may play important roles in host responses against Bt infection. To confirm this, we measured the concentrations of AMP and ATP by LC-MS 31 when wild-type C. elegans N2 fed with BMB171/Cry5Ba, the non-nematicidal control strain BMB171/pHT304 and the standard food strain E. coli OP50. The results showed that the AMP/ATP ratio had no significant difference after C. elegans N2 fed with BMB171/pHT304, but significantly increased after fed with BMB171/Cry5Ba, comparing with that of fed with E. coli OP50 (Fig. 1B). However, the AMP/ATP ratio showed no significant difference when the Cry5Ba-receptor mutant bre-5(ye17) worms were fed with either BMB171/Cry5Ba or BMB171/pHT304 (Fig. 1B). Next, we tested whether other nematicidal Bt can lead cell energy change, such as BMB171/Cry5Ca 30, BMB171/Cry6Aa 32, BMB171/Cry21Aa and non-nematicidal BMB171/Cry1Ac 33. We found that nematicidal Bt strains can cause significant energy imbalance of C. elegans comparing with non-nematicidal Bt (Fig. S1). Taking together, we demonstrated that nematicidal Bt infection triggers a cellular energy imbalance of C. elegans, which was mainly attributed to the nematicidal toxins.
B. thuringiensis infection leads to mitochondria damage
Mitochondria produces most of the cell's ATP through oxidative phosphorylation and the tricarboxylic acid cycle, and plays a vital influence on cell metabolism 34. Mitochondria are constantly in a state of fusion and division, which are essential for maintaining mitochondrial respiration and homeostasis, and even cell death 35,36. It has been proved that PFT from pathogens can caused serious mitochondrial disruption in C. elegans37. Also, toxins from Pseudomonas pathogens can target mitochondria and lead to mitochondria fragmentation (MF) and it dysfunctional 16,17. The MF phenomenon can result in bioenergetics defects, which cause cell energy imbalance 38. To assess how nematicidal Bt causes cellular energy imbalance. We tested several physiological and biochemical aspects of mitochondrial damage including MF, mitochondrial membrane potential (ΔΨm), and mitochondrial DNA (mtDNA) content. We used the transgene worms SJ4143(zcIs17[Pges−1::GFPmt]) as MF reporter which stably expressed GFP in mitochondria matrix of intestinal cells to detect the mitochondrial morphology 39. When worms fed with nematicidal Bt BMB171/Cry5Ba, 76.67% of worms showed MF (Fig. 2A and 2B). However, when worms were fed with non-nematicidal Bt strain BMB171/pHT304 or the standard food strain E. coli OP50, the mitochondria morphologies of most worms kept tubular, only less than 15% of worms showed MF (Fig. 2A and 2B). These results showed that nematicidal Bt infection leads to MF. Next, we tested whether BMB171/Cry5Ba infection can cause changes in mitochondrial membrane potential (ΔΨm) and mitochondrial DNA (mtDNA) content. Using wild type N2 worms, we found BMB171/Cry5Ba infection can lead to a significant reduction in mitochondrial membrane potential (ΔΨm) and mtDNA content comparing with non-nematicidal Bt BMB171/pHT304 (Fig. 2C, 2D and S2).
When the Cry5Ba receptor related gene bre-5 was silenced by RNA interference (RNAi) in transgene worms SJ4143(zcIs17 [Pges−1:: GFPmt]), only 12.22% of worms showed MF phenomenon after fed with BMB171/Cry5Ba (Fig. 2A and 2B), indicating that the Cry protein toxin Cry5Ba was the key factor that caused the MF phenomenon during Bt infection. However, when Cry5Ba-receptor null mutant bre-5(ye17) worms fed with BMB171/Cry5Ba in the same condition, the ΔΨm and mtDNA content were similar to bre-5(ye17) worms fed with BMB171/pHT304 (Fig. 2C-D). We conclude that Cry5Ba toxin is the key factor for Bt to manipulate host cell mitochondria and cause serious mitochondrial dysfunction.
Mdivi-1 is an efficient inhibitor to attenuates mitochondrial division by inhibiting the mitochondrial division dynamin, and it also suppresses mitochondrial outer membrane permeabilization 40. To assess the relationship between cell energy imbalance and mitochondrial dysfunction after Bt infection, we checked whether mitochondrial division inhibitor mdivi-1 can recover mitochondria damage. We observed that mdivi-1 can effectively reduce Cry5B-mediated MF (Fig. 2A-B). Besides, our results showed that when wild type N2 worms were fed with BMB171/Cry5Ba adding mdivi-1, the AMP/ATP ratio was significantly reduced, the ratio of mtDNA/nDNA returned to normal levels (Fig. 2D-E). However, we found mdivi-1 cannot recover the reduction of mitochondrial membrane potential (Fig. 2C). In general, these results indicate that mitochondrial damage is responsible for the increase in intracellular AMP/ATP.
To assess whether other nematicidal Bt strains can cause MF phenomenon, we fed transgene worms SJ4143(zcIs17 [Pges−1:: GFPmt]) with the other four nematicidal Bt BMB171/Cry5Ca, BMB171/Cry21Aa, BMB171/Cry6Aa, and non-nematicidal Bt BMB171/Cry1Ac. These former two strains produce Cry5-like three-domain (3D) group nematicidal Cry proteins Cry5Ca 30 and Cry21Aa 41, respectively. While BMB171/Cry6Aa produces a non-3D group nematicidal Cry toxin Cry6Aa 42. The transgene worms SJ4143 fed with nematicidal BMB171/Cry5Ca, BMB171/Cry6Aa and BMB171/Cry21Aa exhibited the fragmented mitochondrial morphology (Fig. S3) with a range from 65.42–86.73% (Fig. S4). However, the worms fed with the and BMB171/Cry1Ac showed no significant MF phenomenon (Fig. S3 and Fig. S4). The results showed that the toxins from nematicidal Bt are capable of causing mitochondrial morphology damage in C. elegans.
B. thuringiensis -induced mitochondrial damage is the result of intracellular potassium leakage
The Cry5Ba toxin in Bt is a typical pore-forming toxin (PFT) which can form pores in the cell membrane, causing the unbalance in selectively permeable of the plasma membrane 43,44. Mitochondria face various intracellular stressors, to explain the mechanism of Cry5Ba caused mitochondrial damage, we determine the direct stressors of mitochondria in response to Cry5Ba. We first check whether Cry5Ba can target mitochondrial directly, our results showed Cry5Ba can be translocated into epithelial cells, but can’t colocalize with mitochondria (Fig. 3A). In addition to target mitochondria directly, it had been proved PFTs can lead to severe ion dysregulation 45. The osmotic imbalance caused by ion dysregulation has been demonstrated important to mitochondria 46. Next, we assess the cellular Ca2+ and K+ levels after Bt infection. We found that Bt infection caused a significant decrease in potassium ion concentration, but not calcium ion. In a K+-free environment, the leakage of potassium is more serious. When the potassium concentration increased, the leakage of potassium would be alleviated. But in bre-5(ye17) worms, potassium content did not change at any condition (Fig. 3A-3B and S5-S6). So we can conclude that during the Bt infection, Cry5Ba caused intracellular potassium leakage.
To verify our hypothesis that potassium leakage is the direct reason for mitochondrial damage. We detect the MF phenomenon during different potassium concentrations, we found during the Bt infection, the restoration of intracellular potassium can also reduce the MF phenotype, but serious potassium leakage caused a greater proportion of MF phenotypes (Fig. 3C). At the same time, the mitochondrial membrane potential could also return to normal level with potassium addition (Fig. 3D). As we found, the MF phenotype caused by Cry5Ba toxin is responsible to energy imbalance. More importantly, our results showed the restoration of potassium concentration can restore the content of AMP/ATP (Fig. 3E). Therefore, we concluded the leakage of potassium ions caused by Bt infection directly causes mitochondrial stress,which subsequently leads to mitochondrial damage and energy imbalance.
Cell energy imbalance mediated by mitochondria damage activates the AMP-activated protein kinase
The AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains energy homeostasis and can be activated by a decrease in energy levels 47. The synthesis and catabolism of ATP are largely regulated by AMPK 47. AMPK is activated via phosphorylation of Thr172 on the α catalysis subunit (AAK-2 protein) 47. It is well known that increasing of AMP/ATP proportion is the classical way to activate the AMPK 47. We observed that aak-2 was significantly up-regulated and the AMP/ATP ratios were significantly increased when worms are infected by nematicidal Bt (Fig. 1B and S7). Therefore, we speculated that nematicidal Bt infection may activate AMPK. To confirm this hypothesis, we performed western blotting to analyze the Thr172 phosphorylation of AAK-2 protein in worms. The results showed that the Thr172 of AAK-2 was phosphorylated when C. elegans fed with BMB171/Cry5Ba but not in the control strain BMB171/pHT304 treatment (Fig. 4A). What’s more, the Thr172 of AAK-2 protein was not phosphorylated when Cry5Ba-receptor null mutant bre-5(ye17) worms were fed with BMB171/Cry5Ba under the same conditions (Fig. 4A). Inhibition of mitochondrial fragmentation using mdivi-1 could significantly suppress the Thr172 phosphorylation of AAK-2 (Fig. 4A). Besides, restore the leakage of potassium ions caused by Bt infection can also suppress the Thr172 phosphorylation of AAK-2 (Fig. 4B). These results demonstrated that the AMPK of worms is activated by nematicidal Bt infection via the phosphorylation of the core subunit AAK-2.
To assess the relationship between cell energy imbalance and the activating of AMPK after Bt infection, we knock down the aak-2 transcription levels by RNAi in the mitochondria reporter worms SJ4143(zcIs17[Pges−1:: GFPmt]). Then, the non-RNAi and aak-2 RNAi worms were fed with BMB171/Cry5Ba or BMB171/pHT304, respectively. The RNAi-aak-2 worms also showed a high level of MF when worms fed with BMB171/Cry5Ba, most mitochondria keep tubular when worms fed with control strain BMB171/pHT304 (Fig. 4C-4D).
We also measured the concentrations of AMP and ATP when wild-type C. elegans N2 and RNAi-aak-2 worms were fed with BMB171/Cry5Ba or BMB171/pHT304. The AMP/ATP ratio was significantly increased when RNAi-aak-2 worms were treated with BMB171/Cry5Ba compared to BMB171/pHT304 (Fig. 4E). However, the increasing of AMP/ATP ratio was no significant difference when RNAi-aak-2 and N2 worms were treated with BMB171/Cry5Ba (Fig. 4E). These results demonstrate that knockdown of the aak-2 does not affect the level of MF and energy imbalance during Bt infection, and suggest that AMPK activation is a result rather than a cause of the above MF phenomenon. Thus, we concluded that Bt-mediated cell energy imbalance activates the AMPK in C. elegans.
AMPK activation is involved in C. elegans defense responses against Bt infection
Several previous studies reported that AMPK defends against low glucose levels, dietary deprivation, paraquat, physical stress and pathogens 48–50. Therefore, we asked whether AMPK activation might be involved in defense against Bt infection. There are three subunits for the AMPK complexes, including a catalytic subunit (α), and two regulatory subunits (β and γ). To confirm this hypothesis, we tested the sensitivity of these four AMPK null alleles mutants and the wild type N2 worms exposed to BMB171/Cry5Ba, including aak-1(tm1944) (subunit α1 of AMPK), aak-2(ok524) (subunit α2 of AMPK), aakb-1(tm2658) (subunit β1 of AMPK), and aakg-4(tm5269) (subunit γ1 of AMPK). Compared to the wild type N2 worms, only aak-2(ok524) mutant worms showed more increased sensitivity to BMB171/Cry5Ba infection (Fig. 5A and Fig. 5B). Survival assays confirmed that the aak-2(ok524) mutant worms are more sensitive to BMB171/Cry5Ba infection (Fig. 5C).
To further confirm the importance of AMPK catalysis subunit α2 (AAK-2 protein) in defense to BMB171/Cry5Ba infection, we tested the sensitivity of null allele of aak-2 mutant worms, aak-2(gt33), feeding on BMB171/Cry5Ba. We found that aak-2(gt33) is also more sensitive to BMB171/Cry5Ba than wild type N2 (Fig. S8). A similar phenotype was observed in aak-2 RNAi worms (Fig. S9).
We next asked whether the AMPK subunit α2 knockout phenotype is specific for Bt infection. The sensitivity of the wild type N2 and aak-2(ok524) mutant worms were analyzed to the heavy metal copper sulfate and oxidative stress (hydrogen peroxide). Our results showed that it is no significant difference in these treatments between the wild type N2 and the mutant aak-2(ok524) worms (t-test, p > 0.05) (Fig. S10A and S10B).
In addition, we activated AMPK using 5-Aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR), a typical activator stimulating AAK-2 kinase activity of AMPK via phosphorylation of Thr172 on α catalysis subunit (AAK-2 protein) 51. Western blotting results showed that the AMPK is activated by 50 µM of AICAR via AAK-2 Thr172 phosphorylation (Fig. 3A). We then compared the sensitivity of the AICAR-treated with non-treated N2 worms to BMB171/Cry5Ba infection by survival assays and growth assay, respectively. The results showed that the AICAR-activating worms showed more significant resistance to BMB171/Cry5Ba infection comparing with the no-activating N2 worms (Fig. 4D and 4E). However, compared with the wild type N2 worms, the resistance to BMB171/Cry5Ba infection was not significantly different for aak-2 mutant either in AICAR-activating or no-activating conditions (Fig. 4D and 4E). Taking together, our results demonstrated that AMPK plays an important role in C. elegans defense against BMB171/Cry5Ba.
AMPK activity in the intestine is required for C. elegans resistance to Bt infection
To independently confirm the importance of AAK-2 in defense to BMB171/Cry5Ba infection, we constructed the transgenic strain aak-2(ok524) (Paak−2::aak-2), which expresses the aak-2 under the control of its native promoter Paak−2 to rescue the aak-2 function under the background of mutant aak-2(ok524) worms. The growth assay and survival assay results showed that aak-2 expression driven by its own promoter Paak−2 can completely alleviate the hypersensitivity of aak-2(ok524) mutant to BMB171/Cry5Ba infection (Fig. 6A and 6B), supporting that AAK-2 is independently important for worms to defense BMB171/Cry5Ba infection.
C. elegans apparently lacks professional immune cells, but can rely on epithelial cells for immune defenses 52. Cry5Ba can attack the intestine of worms and forming pores in the membrane of the intestine cell 53,54. We therefore hypothesized intestinal-specific activity of AMPK regulates immune responses to Bt infection immediately. We drove the aak-2 expression under different tissue-specific promoters, including the intestine-specific promoter Pvha−6 55, the muscle-specific promoter Pmyo−3 56, and the neuron-specific promoter Prab−3 57. We found that only aak-2 expression under the intestine-specific promoter Pvha−6 alleviated the hypersensitivity of aak-2(ok524) mutant to BMB171/Cry5Ba infection (Fig. 6C and 6D). In contrast, there were no significant difference in hypersensitivity to Bt infection among the aak-2(ok524) mutant and the muscle-specific Pmyo−3 or neuron-specific Prab−3 rescued worms (Fig. 6C and 6D). Also, we knocked down the aak-2 gene by RNAi in the intestine, muscle, and epidermis of worms, respectively. Only the intestine-specific knock-down of aak-2 made the worms more sensitive to BMB171/Cry5Ba infection (Fig. 6E). In contrast, epidermal-specific or muscular-specific aak-2 RNAi worms did not (Fig. S11A and S11B). These results suggest that the intestine serves as the first line of AMPK-mediated defense against BMB171/Cry5Ba attack.
AMPK activation triggers DAF-16 dependent innate immune singling pathway during Bt infection
AMPK pathway is evolutionarily conserved from C. elegans to mammals and regulates many downstream pathways 58. Here, AMPK has been shown to play a role in the defense of Bt infection in C. elegans. However, the AMPK downstream genes or pathways involved in the defense against Bt infection in C. elegans were not clear. It was reported that AAK-2 is capable of modulating the phosphorylation of the FOXO family transcription factor DAF-16, activating the DAF-16-dependent transcription when worms suffering from dietary restriction 59. DAF-16 can regulate many genes involved in metabolism, immune responses against several pathogens, and longevity of C. elegans 60,61. Moreover, the DAF-16 was also triggered by nematicidal Bt infection 62, and functioned as an important modulator in defense against nematicidal PFTs in C. elegans 63. Therefore, we speculated that AMPK may regulate the DAF-16-dependent signaling pathway in defense against Bt infection. To confirm this hypothesis, we compared previously identified DAF-16 target genes 60 with our RNA-Seq data. We found 60 of the genes up-regulated by Bt infection are also the targets of DAF-16 (Fig. 7A, Table S3 and S4) (t-test, p < 0.001). To verify this analysis, we selected 8 typical genes from these genes and determined their transcription by qPCR. The transcription of these 8 genes was significantly up-regulated after Bt infection in wild type N2 worms. Moreover, RNAi of daf-16 significantly suppressed the up-regulation of these genes induced by Bt infection (Fig. 7B), and RNAi of aak-2 also suppressed most gene upregulation, supported that these DAF-16-dependent genes are also regulated by AAK-2 during Bt infection.
Under standard growth conditions, DAF-16 is distributed predominately throughout the cytoplasm of all tissues. When activated, DAF-16 will be phosphorylated and translocated from cytoplasmic to the nucleus, then binds to the promoter region and activates the expression of target genes 60. Therefore, we monitored the cellular translocation of DAF-16 using transgenic worms TJ356(Isdaf-16:: gfp) as a reporter, which expresses functional DAF-16:: GFP fusion protein. Our results showed after BMB171/Cry5Ba infection, most of the DAF-16:: GFP was translocated from the cytoplasm to the nucleus in the intestine, especially the front and middle parts of the intestines (Fig. 7C and 7D). In contrast, the control strain BMB171/pHT304 failed to cause DAF-16 transfer to the nucleus at the same conditions (Fig. 7C, 7D and Fig. S12). These results demonstrated that nematicidal Bt infection can activate the DAF-16 nuclear translocation in wild-type worms.
To test whether the AMPK activity is required for the activation of DAF-16 during Bt infection, we monitored the cellular translocation of DAF-16 when the aak-2 gene was silenced by RNAi in the reporter worm TJ356 (Isdaf-16::gfp). The observations showed that the DAF-16 nuclear translocation induced by Bt is significantly diminished by RNAi aak-2 gene (Fig. 6C and 6D). To test whether DAF-16 activation can trigger the transcription of the downstream immune-related effectors, we tested the expression of sod-3, a well-known direct target of DAF-16 during Bt infection 64; sod-3 gene is also one of the most significant up-regulated genes in Bt-infected worms (Table S3 and S4). We observed the expression of sod-3 using transgenic worm CF1553(muIs84(sod-3::GFP)) 64. When the SOD-3::GFP reporter worms were exposed to BMB171/Cry5Ba, the expression of sod-3 was significantly up-regulated compared to BMB171/pHT-304. Meanwhile, the induction of sod-3 by BMB171/Cry5Ba infection was significantly inhibited when either daf-16 or aak-2 gene was silenced in the strain CF1553(muIs84(sod-3::GFP) (Fig. 7E and 7F). Additionally, the aak-2 deletion also significantly suppressed the up-regulation of the above selected 8 immune-related genes induced by Bt infection (Fig. 7B). Taking together, we concluded that the AMPK triggers the DAF-16-dependent innate immune pathway during Bt infection.