A novel gene-diet interaction promotes organismal lifespan and host protection during infection via the mitochondrial UPR

Abstract

Cells use a variety of mechanisms to maintain optimal mitochondrial function including the mitochondrial unfolded protein response (UPRmt). The UPRmt mitigates mitochondrial dysfunction by differentially regulating mitoprotective gene expression through the transcription factor ATFS-1. Since UPRmt activation is commensurate with organismal benefits such as extended lifespan and host protection during infection, we sought to identify pathways that promote its stimulation. Using unbiased forward genetics screening, we isolated novel mutant alleles that could activate the UPRmt. Interestingly, we identified one reduction of function mutant allele (osa3) in the mitochondrial ribosomal gene mrpl-2 that activated the UPRmt in a diet-dependent manner. We find that mrpl-2(osa3) mutants lived longer and survived better during pathogen infection depending on the diet they were fed. A diet containing low levels of vitamin B12 could activate the UPRmt in mrpl-2(osa3) animals. Also, we find that the vitamin B12-dependent enzyme methionine synthase intersects with mrpl-2(osa3) to activate the UPRmt and confer animal lifespan extension at the level of ATFS-1. Thus, we present a novel gene-diet pairing that promotes animal longevity that is mediated by the UPRmt.

Fig 1. Isolation of novel alleles that activate the UPR^mt in diet-dependent and–independent manners.

(A) Schematic of the UPR^mt response. (B) Schematic illustrating the strategy used to isolate mutants that activate the UPR^mt using forward genetics. (C, D) Photomicrographs and quantification of hsp-6p::GFP expression for the four isolated mutants fed different diets of E. coli. The alleles were named osa2, osa3, osa4 and osa5. Quantification of fluorescence intensities expressed as arbitrary units (A.U.); (mean ±SD; n ≥ 20 worms); ns denotes not significant, * denotes p<0.05, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001, **** denotes p ≤ 0.0001 (Student’s t test). (E) Schematics of gene structure and protein alignment of sucg-1(osa2), mrpl-2(osa3), and pdr-1(osa5) with the indicated mutations and amino acid changes.

Fig 2. Mitochondrial function is altered in mrpl-2(osa3) animals in a diet-dependent manner.

(A) Oxygen consumption rate determination for wild-type and mrpl-2(osa3) animals fed E. coli OP50, HT115, HB101, and BW25113. Oxygen consumption was normalized to total protein content (mean ±SD; n = 3); ns denotes not significant, ** denotes p ≤ 0.01, *** denotes p ≤ 0.001 (Student’s t test). (B) ATP production quantification for wild-type and mrpl-2(osa3) animals fed E. coli OP50, HT115, HB101, and BW25113. ATP levels are normalized to total protein content (mean ±SD; n = 3); ns denotes not significant, * denotes p ≤ 0.05 (Student’s t test). (C) Oxidative protein modification determination using the OxyBlot assay. OxyBlot values were normalized to actin for each sample and represented as arbitrary units (A.U.). (mean ± SD; n = 5); ns denotes not significant, * denotes p ≤ 0.05 (Student’s t test). (D) Mitochondrial membrane potential determination using TMRE and quantification for wild-type and mrpl-2(osa3) animals fed E. coli OP50, HT115, HB101, and BW25113 reflected as arbitrary units (A.U.). (mean ±SD; n ≥ 20 worms); ** denotes p ≤ 0.01, *** denotes p ≤ 0.001, **** denotes p ≤ 0.0001 (Student’s t test). (E) Animal size quantification of wild-type and mrpl-2(osa3) animals fed E. coli OP50, HT115, HB101, and BW25113. Animal size is expressed as the length and width of each animal and represented as arbitrary units (A.U.); (mean ±SD; n ≥ 20 worms); ns denotes not significant, **** denotes p ≤ 0.0001 (Student’s t test).

Fig 3. Diet-type determines lifespan in mrpl-2(osa3) animals.

(A-D) Lifespans of wild-type and mrpl-2(osa3) animals fed E. coli (A) OP50, (B) HT115, (C) HB101, and (D) BW25113. See S1 Table for all lifespan assay statistics. (E) Whole body thrashing rate quantification of wild-type and mrpl-2(osa3) animals fed E. coli OP50, HT115, HB101, and BW25113. (mean ±SD; n ≥ 10 worms); ns denotes not significant, *** denotes p ≤ 0.001, **** denotes p ≤ 0.0001 (Student’s t test). (F) Pharyngeal pumping rate quantification of wild-type and mrpl-2(osa3) animals fed E. coli OP50, HT115, HB101, and BW25113 (mean ±SD; n ≥ 10 worms). (G) Photomicrographs and quantification of gut autofluorescence in wild-type and mrpl-2(osa3) animals. Quantification of fluorescence intensities expressed as arbitrary units (A.U.); ns denotes not significant, ** denotes p ≤ 0.01, **** denotes p ≤ 0.0001 (Student’s t test).

Fig 4. mrpl-2(osa3) animals survive longer during infection depending on their prior diet.

(A, B) Photomicrographs and quantification of P. aeruginosa PA14-dsRed expression of infected wild-type or mrpl-2(osa3) animals previously fed different diets of E. coli. Quantification of fluorescence intensities expressed as arbitrary units (A.U.); (mean ±SD; n ≥ 20 worms); ns denotes not significant, **** denotes p ≤ 0.0001 (Student’s t test). (C-F) Survival analysis of wild-type and mrpl-2(osa3) animals infected with P. aeruginosa. E. coli diets prior to P. aeruginosa infection are (C) OP50, (D) HT115, (E) HB101, and (F) BW25113. See S1 Table for all survival assay statistics.

Fig 5. Vitamin B12 levels determine the activation of the UPR^mt in mrpl-2(osa3) animals under different diets.

(A, B) Photomicrographs and quantification of hsp-6p::GFP expression in wild-type and mrpl-2(osa3) animals fed E. coli OP50 in the presence or absence of 0.2 μg/ml methylcobalamin or adenosylcobalamin. Quantification of fluorescence intensities expressed as arbitrary units (A.U.); (mean ±SD; n ≥ 20 worms); **** denotes p ≤ 0.0001 (Student’s t test). (C) Lifespans of wild-type and mrpl-2(osa3) animals fed E. coli OP50 in the presence or absence of 0.2 μg/ml methylcobalamin. (D, E) Photomicrographs and quantification of hsp-6p::GFP expression in wild-type and mrpl-2(osa3) animals fed vitamin B12-restricted E. coli HT115 in the presence or absence of 0.2 μg/ml methylcobalamin. Quantification of fluorescence intensities expressed as arbitrary units (A.U.); (mean ±SD; n ≥ 20); ns denotes not significant, **** denotes p ≤ 0.0001 (Student’s t test). (F) Lifespans of wild-type and mrpl-2(osa3) animals fed vitamin B12-restricted E. coli HT115 in the presence or absence of 0.2 μg/ml methylcobalamin.

Fig 6. Methionine supplementation suppresses UPR^mt activation in mrpl-2(osa3) animals fed a vitamin B12 deficient diet.

(A) Schematic illustration of vitamin B12-dependent metabolic pathways. (B, C) Photomicrographs and quantification of hsp-6p::GFP expression of wild-type, mrpl-2(osa3), metr-1(ok521), mmcm-1(ok1637), metr-1(ok521); mrpl-2(osa3), and mmcm-1(ok1637); mrpl-2(osa3) fed an E. coli HT115 diet. Quantification of fluorescence intensities expressed as arbitrary units (A.U.); (mean ±SD; n ≥ 20); ns denotes not significant, **** denotes p ≤ 0.0001 (Student’s t test). (D) Lifespans of wild-type, mrpl-2(osa3), metr-1(ok521), metr-1(ok521); mrpl-2(osa3) fed an E. coli HT115 diet. (E) Lifespans of wild-type, mrpl-2(osa3), mmcm-1(ok1637), mmcm-1(ok1637); mrpl-2(osa3) fed an E. coli HT115 diet. (F) Lifespans of wild-type, mrpl-2(osa3), metr-1(ok521), metr-1(ok521); mrpl-2(osa3) fed an E. coli OP50 diet. (G) Lifespans of wild-type, mrpl-2(osa3), mmcm-1(ok1637), mmcm-1(ok1637); mrpl-2(osa3) fed an E. coli OP50 diet. (H, I) Photomicrographs and quantification of hsp-6p::GFP expression of wild-type and mrpl-2(osa3) fed an E. coli OP50 diet in the presence or absence of 10 mM methionine. Quantification of fluorescence intensities expressed as arbitrary units (A.U.); (mean ±SD; n 20); denotes p 0.0001 (Student’s t test). (J) Lifespans of wild-type and mrpl-2(osa3) fed an E. coli OP50 diet in the presence or absence of 10 mM methionine.

Fig 7. ATFS-1/UPR^mt mediate the lifespan extension of mitonuclear imbalance and methionine restriction.

(A) Heat maps representing gene expression changes in wild-type, mrpl-2(osa3), and metr-1(ok521) fed an E. coli OP50 diet. Genes were considered as differentially expressed if there was a significance difference of p ≤ 0.05 (after Benjamini-Hochberg correction). (B) Functional categories of differentially expressed genes. (C) Venn diagram of differentially expressed genes shared between mrpl-2(osa3) and metr-1(ok521). (D, E) Photomicrographs and quantification of hsp-6p::GFP expression for mrpl-2(osa3), mrpl-2(osa3); atfs-1(tm4525), metr-1(ok521), and metr-1(ok521); atfs-1(tm4525) fed a diet of E. coli OP50. Quantification of fluorescence intensities expressed as arbitrary units (A.U.); (mean ±SD; n ≥ 20); **** denotes p ≤ 0.0001 (Student’s t test). (F) Lifespans of wild-type, mrpl-2(osa3), atfs-1(tm4525), and mrpl-2(osa3); atfs-1(tm4525) fed a diet of E. coli OP50. (G) Lifespans of wild-type, metr-1(ok521), atfs-1(tm4525), and metr-1(ok521); atfs-1(tm4525) fed a diet of E. coli OP50. (H) Model. Wild-type animals fed a diet of E. coli OP50 experience a mild vitamin B12 restriction which reduces methionine synthase activity resulting in a subtle methionine restriction that is insufficient to activate the UPR^mt. However, the UPR^mt is activated in combination with the mild mitonuclear imbalance of the mrpl-2(osa3) reduction of function mutant which results in extended lifespan. In contrast, methionine supply is higher when fed a vitamin B12 replete diet of E. coli HT115, and therefore the mild mitonuclear imbalance of mrpl-2(osa3) mutant alone is incapable of inducing the UPR^mt in this scenario resulting in animals that display normal aging rates.