Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection

Metazoans identify and eliminate bacterial pathogens in microbe-rich environments such as the intestinal lumen, however the mechanisms are unclear. Potentially, host cells employ intracellular surveillance or stress response programs to detect pathogens that target monitored cellular activities to initiate innate immune responses1–3. Mitochondrial function is evaluated by monitoring mitochondrial protein import efficiency of the transcription factor ATFS-1, which mediates the mitochondrial unfolded protein response (UPRmt). During mitochondrial stress, import is impaired4 allowing ATFS-1 to traffic to the nucleus where it mediates a transcriptional response to re-establish mitochondrial homeostasis5. Here, we examined the role of ATFS-1 during pathogen exposure because in addition to mitochondrial protective genes, ATFS-1 induced innate immune genes during mitochondrial stress that included a secreted lysozyme and anti-microbial peptides. Exposure to the pathogen Pseudomonas aeruginosa caused mitochondrial dysfunction and activation of the UPRmt. Animals lacking atfs-1 were susceptible to P. aeruginosa, while hyper-activation of ATFS-1 and the UPRmt improved clearance of P. aeruginosa from the intestine and prolonged C. elegans survival largely independent of known innate immune pathways6,7. We propose that ATFS-1 import efficiency and the UPRmt is a means to detect pathogens that target mitochondria and initiate a protective innate immune response.

activity are sufficient to activate innate immune responses suggesting the elegant hypothesis that host cells utilize intracellular stress responses to initiate innate immunity programs when pathogens perturb monitored cellular processes [1][2][3] .
Cells respond to mitochondrial dysfunction by activating the UPR mt , which is regulated by the transcription factor ATFS-1. In healthy cells, ATFS-1 is efficiently imported into mitochondria and degraded. However, during mitochondrial stress, import efficiency is reduced 4,5 , allowing a small percentage of ATFS-1 to accumulate in the cytosol 5 . Because ATFS-1 has a nuclear localization sequence (NLS), it then traffics to the nucleus where it activates a protective transcriptional response (Fig. 1a). Our expression profiling studies indicated that ATFS-1 induces genes that promote mitochondrial protein folding, ROS detoxification and mitochondrial protein import, suggesting the UPR mt stabilizes the mitochondrial protein folding environment to promote organelle homeostasis 5 .
Intriguingly, a number of transcripts induced during mitochondrial stress caused by inhibition of the mitochondrial protease SPG-7 encode innate immunity proteins 5 (Extended Data Table 1), some of which were also found to be induced following exposure to the pathogen P. aeruginosa 10 (Fig. 1b and Extended Data Table 2). The antimicrobial peptide abf-2 and the secreted lysozyme lys-2, both of which are required for resistance to pathogen infection 11,12 , were induced during mitochondrial stress ( Fig. 1c-d), as were two C-type lectins, which are involved in pathogen recognition 13 (Fig. 1e-f). Of note, mitochondrialspecific stress also caused induction of antimicrobial peptides 14 in mammalian cells ( Fig.  1g-j), suggesting the response is conserved. In C. elegans, induction of innate immune genes by spg-7(RNAi) required ATFS-1 ( Fig. 1c-f). Thus, in addition to inducing mitochondrial-protective genes, ATFS-1 also transcriptionally up-regulated innate immune genes during mitochondrial stress. Therefore, we hypothesized that ATFS-1 and the UPR mt are involved in regulating innate immunity during exposure to pathogens that perturb mitochondrial function.
P. aeruginosa produces virulence factors that target many cellular functions including the mitochondrial toxins cyanide and pyocyanin 15,16 . P. aeruginosa also produces exotoxin A, which impairs protein synthesis and leads to the induction of the innate immune gene irg-1 via the transcription factor ZIP-2 2,3,17 . Mitochondrial stress also caused irg-1 pr ::gfp induction, which was blocked in atfs-1(tm4919) and partially so in zip-2(tm4248) worms (Fig. 1k), suggesting that multiple transcription factors and stressors influence innate immune gene expression. Additionally, zip-2 mRNA was induced during mitochondrial stress, which also required atfs-1 (Fig. 1l). Of note, F35E12.5, which is induced by the MAP kinase PMK-1 and the transcription factor ATF-7 during P. aeruginosa exposure 10,18 , was not induced during mitochondrial stress (Extended Data Fig. 1a). Thus, ATFS-1 regulates a subset of innate immune genes during mitochondrial stress in addition to its cytoprotective role in promoting mitochondrial homeostasis.
We next examined if P. aeruginosa exposure caused mitochondrial stress capable of activating the UPR mt . Slow-killing conditions were used in which the pathogen accumulates in the intestine leading to infection 19 . Interestingly, P. aeruginosa exposure caused intestinal cell mitochondria to elongate similar to spg-7(RNAi) (Fig. 2a), consistent with the pathogen causing mitochondria stress, and mitochondrial fusion providing protection 20 . Additionally, exposure to P. aeruginosa caused dramatic developmental delays in combination with mild mitochondrial stresses such as ethidium bromide 5 , paraquat 5 , or the clk-1(qm30) allele 21 (Fig. 2b), consistent with the pathogen causing modest mitochondrial stress. Importantly, P. aeruginosa exposure caused increased mitochondrial chaperone reporter (hsp-6 and hsp-60 pr ::gfp) activation in the intestine that required atfs-1 ( Fig. 2c and Extended Data Fig.  1b), which correlated with increased nuclear accumulation of ATFS-1::GFP and required the NLS in ATFS-1 ( Fig. 2d and Extended Data Fig. 1c, d). Exposure to P. aeruginosa liquidkilling conditions, which requires pathogen expressed iron chelating siderophores 22 , also induced mitochondrial chaperone genes, suggesting multiple P. aeruginosa virulence factors can activate the UPR mt (Extended Data Fig. 2a, b). Interestingly, both synthetic growth arrest and UPR mt activation by P. aeruginosa required the global virulence activator gene gacA 23 (Fig. 2b, c). Furthermore, exposure to P. aeruginosa strains lacking individual siderophore, pyocyanin or cyanide toxin genes resulted in less UPR mt activation than wildtype P. aeruginosa (Extended Data Fig. 2d, e), suggesting that multiple pathogen toxins target mitochondrial function resulting in UPR mt activation. However, UPR mt activation may also be due to indirect damage associated with activation of a separate immune response 24 .
Consistent with a role for ATFS-1 in inducing innate immune and mitochondrial protective genes 5 , the survival of worms raised on atfs-1(RNAi) was significantly reduced when exposed to P. aeruginosa, but not E. coli ( Fig. 3a-b). atfs-1(RNAi) treated worms were also susceptible to P. aeruginosa liquid-killing (Extended Data Fig. 2c), supporting a role for ATFS-1 in activating a protective transcriptional response to pathogen exposure. Of note, RNAi was used to reduce atfs-1 activity for the survival studies rather than atfs-1(tm4919) because of germline defects that complicate the analysis (Extended Data Fig. 4b, c).
We next examined if UPR mt activation is sufficient to protect against P. aeruginosa. The UPR mt was induced by allowing worms to develop on spg-7(RNAi) for two days 5 prior to pathogen exposure. UPR mt pre-activation dramatically reduced the intestinal accumulation of P. aeruginosa expressing GFP (P. aeruginosa-GFP 19 (Fig. 3c, d)). Importantly, P. aeruginosa-GFP accumulated in the intestine of atfs-1(tm4919) worms following spg-7(RNAi) treatment indicating that UPR mt activation promotes pathogen clearance. In addition to adapting transcription, worms are also able to avoid P. aeruginosa, which was unaffected by atfs-1(tm4919) or pretreatment with spg-7(RNAi) (Extended Data Fig. 5a-e). Consistent with increased pathogen clearance via anti-microbial gene induction, UPR mt pre-activation prolonged the survival of animals challenged with P. aeruginosa, which required atfs-1 (Fig. 3e) and was independent of germline defects or feeding behavior (Extended Data Fig. 5f-g).
Because mitochondrial stress can activate multiple stress response pathways in addition to the UPR mt 25,26 , we examined an atfs-1 gain-of-function mutant, which constitutively activates the UPR mt independent of mitochondrial dysfunction. atfs-1(et18) worms express ATFS-1 with an amino acid substitution in the mitochondria targeting sequence that reduces mitochondrial import efficiency causing constitutive UPR mt activation 27 and innate immune gene induction (Extended Data Fig. 6a-e). Impressively, atfs-1(et18) worms accumulated less P. aeruginosa-GFP in the intestine (Fig. 3f, g) and survived longer than wild-type worms (Fig. 3h) indicating that UPR mt activation is sufficient to provide resistance to P. aeruginosa. Importantly, atfs-1(RNAi) and lys-2(RNAi) reduced atfs-1(et18) worm survival ( Fig. 3h and Extended Data Fig. 6f), suggesting that ATFS-1-mediated innate immune gene induction provides resistance to P. aeruginosa.
Inhibition of additional cellular activities including translation (eft-2), mRNA splicing (T08A11.2), calcium transport (sca-1) and the pentose phosphate pathway (T25B9.9) also induce innate immune gene expression 1-3 but do not induce the UPR mt (Extended Data Fig.  7a, b). Thus, we examined if other stress-activated innate immune responses are also protective against P. aeruginosa. Knockdown of eft-2, T25B9.9, sca-1 or T08A11.2 did not increase survival on P. aeruginosa (Extended Data Fig. 7c) however, sca-1(RNAi) and T08A11.2(RNAi) decreased lifespan on E. coli, indicating a reduction in general fitness (Extended Data Fig. 7d). In contrast, knockdown of the mitochondrial ATP synthase subunit atp-2, which activates mitochondrial protective and innate immune gene expression (Extended Data Fig. 7a, b), prolonged survival during P. aeruginosa exposure (Extended Data Fig. 7c). Our data suggest the UPR mt provides protection from P. aeruginosa by coupling mitochondrial-protective and antimicrobial gene expression.
Lastly, we determined if ATFS-1 and the UPR mt interacted with established C. elegans innate immune pathways, which include a MAP kinase pathway mediated by NSY-1/ SEK-1/PMK-1 6,7,10 , the MLK-1/MEK-1/KGB-1 c-Jun kinase pathway 7,28 , as well as that mediated by ZIP-2 17 . Interestingly, pre-activation of the UPR mt enhanced the survival of the pmk-1 and sek-1 mutants (Fig. 4a, b), as well as the kgb-1 and mlk-1 mutants (Fig. 4c, d). Of note, increased survival by spg-7(RNAi) was further enhanced in kgb-1(km21) worms, consistent with kgb-1 being a negative regulator of the UPR mt 29 (Extended Data Fig. 7e). Alternatively, zip-2(tm4248) modestly reduced the enhanced resistance conferred by spg-7(RNAi) (Fig. 4e), consistent with atfs-1 functioning in the same pathway as zip-2 during mitochondrial stress. In sum, our data suggest that the UPR mt can function independent of the MAP and c-Jun kinase-regulated innate immune pathways.
Our studies indicate that the UPR mt is activated by and protects against P. aeruginosa, and thus support a mechanistic means 5 by which host cells can detect pathogens that target mitochondrial function (Fig. 4f), which is consistent with only a subset of bacterial species inducing the UPR mt 30 . Because ATFS-1 responds directly to mitochondrial dysfunction and induces a transcriptional response that is both mitochondrial protective 5 and antimicrobial, the UPR mt is a uniquely comprised pathway to mitigate mitochondrial damage stemming from genetic defects or pathogen exposure (Fig. 4f).

Worm and bacterial strains
The atfs-1(tm4919) mutant strain was a gift from the National BioResource Project and backcrossed to wild-type N2 twice. Worm strains were provided by the Caenorhabditis Genetics Center unless otherwise noted. Hermaphrodite worms were raised on the OP50 strain of E. coli unless they were treated with RNAi, in which the HT115 E. coli strain expressing the described RNAi plasmid was used 31,32 . Where indicated, worms were exposed to the pathogenic strain of P. aeruginosa, PA14.

Cell culture
Expression of dominant-negative AFG3L2 was induced in stable HEK293 cells by the addition of 1µg/ml tetracycline 33 and the cells were harvested 48 hours later. The ΔOTC expression plasmid 34 was transfected into Hela cells via Lipofectamine and the cells were harvested after 72 hours.

C. elegans slow-killing assay
Slow-killing experiments were performed as previously described 35,36 with minor modifications. E. coli or P. aeruginosa overnight cultures were used to seed slow-killing nematode growth medium (NGM) agar plates (with 0.35% peptone). Plates were allowed to dry overnight at room temperature, incubated at 37°C for 24 hours and allowed to equilibrate at room temperature. Synchronized L1 worms were allowed to develop on E. coli until the L4 stage and then transferred to P. aeruginosa slow-killing plates and incubated at 25°C. RNAi was performed as described previously 37 . For atfs-1(RNAi) (Fig. 3a-b), eri-1(mg366) (enhanced RNAi) worms 38 were raised on control or atfs-1(RNAi) bacteria at 16°C until the L4 stage. All animals were transferred to fresh P. aeruginosa slow-killing plates in a randomized fashion. Animals were counted at the described times and were scored as dead if they failed to respond when touched. Fifty worms were used per experiment and those that had crawled off the plate or exploded at the vulva were censored. All data related to the survival analysis is presented in Extended Data Table 3. Each experiment was performed in triplicate and the log rank (Mantel-Cox) statistical test was used to evaluate p values.
Intestinal mitochondrial morphology was visualized using ges-1 pr ::gfp mt worms 39 . The worms were synchronized by bleaching and allowed to hatch on plates containing P. aeruginosa and raised for 48 hours at 25°C. Visualization of hsp-6 pr ::gfp, hsp-60 pr ::gfp and atfs-1 pr ::atfs-1::gfp was performed essentially as described 36,37 . P. aeruginosa was grown at 16°C for 24 hours and seeded onto slow-killing plates. Plates were incubated overnight at room temperature. Synchronized L1s were transferred to P. aeruginosa plates and incubated at 20°C for 24 hours before imaging.
To examine growth rates, eggs were allowed to hatch on plates containing P. aeruginosa and raised for 3 days at 25°C. 30 µg/ml ethidium bromide or 0.2 mM paraquat was added to E. coli or P. aeruginosa slow-killing plates. For clk-1(qm30) growth rates, worms were raised for 4 days at 25°C.

Statistics
All experiments were performed three times yielding similar results and comprised of biological replicates. The sample size and statistical tests were chosen based on previous studies with similar methodologies and the data met the assumptions for each statistical test performed. No statistical methods were used in deciding sample sizes, nor were any blinded experiment performed. For all figures, the mean ± standard deviation (SD) is represented unless otherwise noted.

C. elegans liquid-killing assay
glp-4(bn2) worms were raised at 25°C to sterilize them while being fed atfs-1(RNAi). At the L4-early adult stage, the described worms were exposed to P. aeruginosa under conditions used for the liquid-killing assay 40 .

RNA isolation and quantitative real time PCR (qRT-PCR)
Total RNA was obtained using the RNA STAT reagent (Tel-Test Inc.) and used for cDNA synthesis via the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories). qRT-PCR was performed using Thermo-Scientific™; SyBr Green Maxima Mix. For Figures 1c-f and 1l, worms were hatched onto RNAi-expressing plates and harvested after 48 hours. For Figures 2e-h, synchronized L4 worms were fed on E. coli or P. aeruginosa for 8 hours using the slow-killing method prior to harvesting. For Extended Data Figures 2a-b, synchronized glp-4(bn2) L4 worms were raised in liquid culture using E. coli or P. aeruginosa for 16 hours. All values were normalized to wild-type worms grown on control bacteria for RNAi experiments (Fig. 1c-f, l) or wild-type worms grown on E. coli for P. aeruginosa experiments (Fig. 2e-h). act-3 and snb-1 mRNA were used as controls for slow-killing and liquid-killing experiments, respectively. HPRT mRNA was used as a control for dominantnegative AFG3L2 and ΔOTC experiments.

P. aeruginosa intestinal accumulation assay
To examine bacterial accumulation in the worm intestine, wild-type or atfs-1(tm4919) worms were synchronized and raised on control or spg-7(RNAi) plates for 48 hours. Overnight cultures of P. aeruginosa expressing GFP (P. aeruginosa-GFP) were seeded onto slow-killing NGM plates, allowed to dry overnight at room temperature and then incubated at 37°C for 24 hours. To exclude pathogen avoidance as a means of decreased intestinal colonization, where indicated P. aeruginosa-GFP was also spread across the entire surface of the slow-killing plate (Extended Data Fig. 5d, e). Worms at the L4 stage were transferred to P. aeruginosa-GFP plates and allowed to feed for 24-48 hours prior to examination. The extent of bacterial accumulation was scored as either "none/mild", "moderate" or "strong" as indicated (Extended Data Fig. 5c).

Plasmid construction
The hsp-16 pr ::atfs-1 FL and hsp-16 pr ::atfs-1 ΔNLS plasmids were described previously 37 . To construct the lys-2 pr ::gfp plasmid, a 803 base pair fragment of the lys-2 promoter sequence upstream of the start codon was amplified using PCR and cloned into the HindIII and PstI sites of pPD95.75. lys-2 pr ::gfp was microinjected into wild-type worms at a concentration of 20 ng/µl along with myo-3 pr ::mCherry at a concentration of 60 ng/µl.

P. aeruginosa avoidance assay
Synchronized L1 wild-type and atfs-1(tm4919) worms were allowed to develop on control or spg-7(RNAi) plates to the L4 stage and then transferred to E. coli or P. aeruginosa slowkilling plates for 17 hours when the worms were scored. The extent of avoidance was expressed as the percent of animals off of the bacterial lawn over the total the number of animals on the plate (Extended Data Fig. 5a, b).

Microscopy
C. elegans were imaged using a Zeiss AxioCam MRm mounted on a Zeiss Imager.Z2 microscope. Exposure times were the same in each experiment. a, Representative photomicrographs of wild-type and atfs-1(tm4919) worms carrying the lys-2 pr ::gfp transgene raised on control or spg-7(RNAi). Scale bar, 0.1 mm. b, Representative photomicrographs of wild-type and atfs-1(tm4919) worms carrying the lys-2 pr ::gfp transgene exposed to E. coli or P. aeruginosa. Scale bar, 0.1 mm. Figure 4. ATFS-1 partially regulates zip-2 expression during P. aeruginosa exposure a, Expression levels of zip-2 mRNA in wild-type or atfs-1(tm4525) worms raised on E. coli or P. aeruginosa using qRT-PCR (N=3, ± SD), * p<0.05 (Student's t test). Extended Data Figure 5. ATFS-1 is not required for pathogen avoidance during P. aeruginosa exposure a, Quantitation of avoidance behavior for wild-type and atfs-1(tm4919) worms raised on E. coli or P. aeruginosa expressed as a percentage of the number of animals off the bacterial lawn relative to the total number worms (N=4, ± SD). *p<0.0001, **p=0.1914 (Student ttest). b, Quantitation of avoidance behavior for wild-type worms raised on control or spg-7(RNAi) and exposed to E. coli or P. aeruginosa expressed as a percentage of the number of animals off the bacterial lawn relative to the total number worms (N=3, ± SD). *p<0.0001, **p=0.8706 (Student t-test). c, Representative photomicrographs illustrating the scored level of infection for P. aeruginosa colonization assay using P. aeruginosa-GFP. Three categories of P. aeruginosa-GFP infection were used: none/mild, moderate and strong. Scale bar, 0.1 mm. d, Representative photomicrographs of wild-type and atfs-1(tm4919) worms raised on spg-7(RNAi) and exposed to a lawn of P. aeruginosa-GFP that completely covered the surface of the slow-killing plate for 24 hours. Images are overlays of DIC and GFP. Scale bar, 0.1 mm. e, Quantitation of P. aeruginosa intestinal colonization as shown in Extended Data Fig. 5d. White, grey and black bars denote no/mild infection, moderate infection and strong infection, respectively. Forty worms were analyzed per treatment. f, Survival analysis of glp-4(bn2) and atfs-1(tm4919); glp-4(bn2) worms raised on control or spg-7(RNAi) and exposed to P. aeruginosa. Statistics for each survival analysis are presented in Extended Data Table 3. g, Quantitation of pharyngeal pumping rate per minute for wild-type worms raised on control or spg-7(RNAi) (N=10, + SD). n.s., no significant difference (p=0.10; Student ttest).

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Extended Data Table 2 ATFS-1 dependent UPRmt genes in common with genes induced following P. aeruginosa exposure.  Table 3 Statistics for survival analysis.
h, Survival of wild-type and atfs-1(et18) worms on control or atfs-1(RNAi) exposed to P. aeruginosa. Statistics are in Extended Data Table 3. worms on control or spg-7(RNAi) exposed to P. aeruginosa. Statistics are in Extended Data Table 3. e, Survival of wild-type, zip-2(tm4248) and atfs-1(tm4919) worms raised on control or spg-7(RNAi) and exposed to P. aeruginosa. Statistics are in Extended Data Table 3. f, ATFS-1 signaling schematic.