Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection (original) (raw)

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Acknowledgements

We thank E. Troemel, M. Pilon, S. Mitani of the National Bioresource Project, and the Caenorhabditis Genetics Center for providing C. elegans strains. We thank D. Kim and J. Xavier for providing P. aeruginosa strains, and T. Langer and S. Troeder for mammalian reagents. This work was supported by the Ellison Medical Foundation, the Lucille Castori Center for Microbes, Inflammation and Cancer at MSKCC and the National Institutes of Health (R01AG040061) to C.M.H., (F32AI100501) to N.V.K. and (R01AI085581) to F. Ausubel.

Author information

Authors and Affiliations

  1. Cell Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA,
    Mark W. Pellegrino, Amrita M. Nargund, Reba Gillis & Cole M. Haynes
  2. Department of Molecular Biology, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA
    Natalia V. Kirienko
  3. Department of Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USA
    Natalia V. Kirienko
  4. BCMB Allied Program, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA,
    Christopher J. Fiorese & Cole M. Haynes

Authors

  1. Mark W. Pellegrino
  2. Amrita M. Nargund
  3. Natalia V. Kirienko
  4. Reba Gillis
  5. Christopher J. Fiorese
  6. Cole M. Haynes

Contributions

M.W.P., A.M.N., N.V.K., R.G. and C.J.F. performed experiments. M.W.P. and C.M.H. conceived of and planned the experiments and wrote the paper.

Corresponding author

Correspondence toCole M. Haynes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Nuclear accumulation of ATFS-1 is required for UPRmt activation during P. aeruginosa exposure.

a, Representative photomicrographs of F35E12.5_pr::gfp transgenic worms raised on control or spg-7(RNAi). No detectable increase in expression was observed following spg-7(RNAi) treatment. In contrast, strong expression of F35E12.5_pr::gfp was observed following exposure to P. aeruginosa compared to E. coli controls. Scale bar, 0.5 mm. b, Wild-type or atfs-1(tm4525);hsp-60_pr::gfp worms on E. coli or P. aeruginosa. Lower panels are magnified views of the intestine showing enhanced expression of hsp-60_pr::gfp (asterisks). Scale bars, 0.05 mm. c, Diagrams of wild-type ATFS-1 (ATFS-1FL) and ATFS-1 with a mutated nuclear localization signal (ATFS-1ΔNLS). d, Photomicrographs of _atfs-1(tm4525);hsp-60_pr::gfp worms expressing ATFS-1FL or ATFS-1ΔNLS via the hsp-16 promoter exposed to E. coli or P. aeruginosa. Scale bar, 0.1 mm.

Extended Data Figure 2 Multiple P. aeruginosa virulence genes contribute to UPRmt activation.

a, b, Expression of hsp-60 and hsp-6 mRNA for glp-4(bn2) worms exposed to E. coli or P. aeruginosa liquid-killing using qRT–PCR (n = 3, ± s.d.). Fold inductions are normalized to wild-type E. coli test group, *P < 0.05 (Student’s t_-test). c, Quantification of survival for glp-4(bn2) worms raised on control or atfs-1(RNAi) and exposed to P. aeruginosa liquid-killing, *P < 0.0001 (Student’s t_-test). d, List of P. aeruginosa toxin mutants. e, Quantification of the proportion of worms showing increased hsp-6_pr::gfp expression in the intestine under slow-killing conditions. Exposure to P. aeruginosa caused hsp-6_pr::gfp induction (n = 3, ± s.e.m.), *P < 0.05 (Student’s _t_-test). However, exposure to P. aeruginosa with mutations in the pvdA, pvdD, pvdF, phzM, hcnB, or hcnC toxin genes resulted in relatively less UPRmt activation (n = 3, ± s.e.m.), **P < 0.05 (Student _t_-test).

Extended Data Figure 3 Intestinal accumulation of lys-2 during mitochondrial stress and P. aeruginosa exposure requires ATFS-1.

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.

Extended Data 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, ± s.d.), * P < 0.05 (Student’s t test). b, Schematic diagram of the atfs-1 genomic open reading frame showing positions of exons 1–8 (boxes) and locations of the tm4525 (ref. 5) and tm4919 deletions in red. The tm4919 allele is a 334 base pair deletion beginning 107 base pairs upstream of the atfs-1 start codon and ends within the second intron of the atfs-1 genomic open reading frame. c, Representative photomicrographs of a germline in wild-type and atfs-1(tm4919) worms. Scale bar, 0.02 mm.

Extended Data Figure 5 ATFS-1 is not required for pathogen avoidance during P. aeruginosa exposure.

a, Quantification of avoidance behaviour 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 of worms (n = 4, ± s.d.). *P < 0.0001, **P = 0.1914 (Student’s _t_-test). b, Quantification of avoidance behaviour 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 of worms (n = 3, ± s.d.). *P < 0.0001, **P = 0.8706 (Student’s _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 h. Images are overlays of DIC and GFP. Scale bar, 0.1 mm. e, Quantification 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 analysed 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, Quantification of pharyngeal pumping rate per minute for wild-type worms raised on control or spg-7(RNAi) (n = 10, + s.d.). n.s., no significant difference (P = 0.10; Student’s _t_-test).

Extended Data Figure 6 atfs-1(et18) gain of function mutant worms induce innate immune gene expression in the absence of mitochondrial stress.

a–d, Expression levels of abf-2, lys-2, clec-4 and clec-65 mRNA in wild-type or atfs-1(et18) worms using qRT–PCR (n = 3, ± s.d.), *P < 0.05 (Student’s t test). e, Representative photomicrographs of wild-type and atfs-1(et18) worms carrying the _irg-1_pr::gfp transgene raised on control or zip-2(RNAi). Scale bar, 0.10 mm. f, Survival analysis of wild-type and atfs-1(et18) worms raised on control or lys-2(RNAi) and exposed to P. aeruginosa. Statistics for each survival analysis are presented in Extended Data Table 3.

Extended Data Figure 7 Mitochondrial protective and innate immune gene induction contributes to ATFS-1-mediated resistance to P. aeruginosa infection.

a, Representative photomicrographs of wild-type hsp-60_pr::gfp_ worms raised on control, atp-2(RNAi), spg-7(RNAi), eft-2(RNAi), sca-1(RNAi), T25B9.9(RNAi) or T08A11.2(RNAi). Scale bar is 0.1 mm. b, Representative photomicrographs of wild-type irg-1_pr::gfp_ worms raised on control, atp-2(RNAi), eft-2(RNAi), sca-1(RNAi), T25B9.9(RNAi) or T08A11.2(RNAi). Scale bar is 0.1 mm. c, Survival analysis of wild-type worms raised on control, atp-2(RNAi), eft-2(RNAi), sca-1(RNAi), T25B9.9(RNAi) or T08A11.2(RNAi) and exposed to P. aeruginosa. Statistics for each survival analysis are presented in Extended Data Table 3. d, Survival analysis of wild-type worms raised on control, atp-2(RNAi), eft-2(RNAi), sca-1(RNAi), T25B9.9(RNAi) or T08A11.2(RNAi) and exposed to E. coli. Statistics for each survival analysis are presented in Extended Data Table 3. e, Representative photomicrographs of wild-type or kgb-1(km21);hsp-60_pr::gfp_ worms raised on E. coli plates with or without 30 μg ml−1 ethidium bromide, suggesting that the KGB-1 Jun kinase pathway negatively regulates the UPRmt during mitochondrial stress29. Scale bar, 0.5 mm.

Extended Data Table 1 ATFS-1-dependent innate immune genes upregulated when raised on spg-7(RNAi)5

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Extended Data Table 2 ATFS-1-dependent UPRmt genes in common with genes induced following P. aeruginosa exposure (ref.10)

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Extended Data Table 3 Statistics for survival analysis

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Pellegrino, M., Nargund, A., Kirienko, N. et al. Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection.Nature 516, 414–417 (2014). https://doi.org/10.1038/nature13818

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