Interspecies systems biology uncovers metabolites affecting C. elegans gene expression and life history traits

Abstract

Diet greatly influences gene expression and physiology. In mammals, elucidating the effects and mechanisms of individual nutrients is challenging due to the complexity of both the animal and its diet. Here, we used an interspecies systems biology approach with Caenorhabditis elegans and two of its bacterial diets, Escherichia coli and Comamonas aquatica, to identify metabolites that affect the animal's gene expression and physiology. We identify vitamin B12 as the major dilutable metabolite provided by Comamonas aq. that regulates gene expression, accelerates development, and reduces fertility but does not affect lifespan. We find that vitamin B12 has a dual role in the animal: it affects development and fertility via the methionine/S-Adenosylmethionine (SAM) cycle and breaks down the short-chain fatty acid propionic acid, preventing its toxic buildup. Our interspecies systems biology approach provides a paradigm for understanding complex interactions between diet and physiology.

Figure 1. Bacterial Screens

(A) Diagram of the Keio E. coli deletion collection screen. (B) Diagram of Comamonas DA1877 transposon mutagenesis screen. (C) Network of metabolites implicated by bacterial screens. SAICAR = 5-Amino-4-imidazole-N-succinocarboxamide ribonucleotide; [acp] = acyl carrier protein. See also Figure S1, Tables S1 and S2.

Figure 2. Metabolite Screen

(A) Summary of the observed effects of supplementing metabolites to Pacdh-1::GFP animals fed either Comamonas aq. DA1877 (left column) or E. coli OP50 (right column). Shades of green represent relative GFP expression. Numbers indicate metabolite concentrations (in mM). (B) Pacdh-1::GFP animals fed Comamonas aq. DA1877 bacteria alone or supplemented with the indicated compounds that activate GFP expression. Exposure time is indicated in yellow. Exposure time for inset images is 400 ms. (C) Pacdh-1::GFP animals fed E. coli OP50 bacteria supplemented with the indicated compounds that repress GFP expression. Exposure time is indicated in yellow. Exposure time for inset images is 400 ms. See also Table S3.

Figure 3. Correlation Between Vitamin B12 Biosynthesis and Repression of the Dietary Sensor

(A) Ado-Cbl content measured by mass spectrometry in indicated bacterial strains. The red line indicates background levels of Ado-Cbl in the bacteria-free control. (B) Correlation between vitamin B12 biosynthesis pathway presence and dietary sensor repression. Images of Pacdh-1::GFP animals fed various bacterial strains are located below a cartoon that indicates pathway status. Differences in exposure times for the inset images are indicated in yellow. See also Figure S2.

Figure 4. Chemical Epistasis with Vitamin B12 and Propionic Acid on C. elegans Gene Expression

(A) Network of the two vitamin B12-dependent pathways. Metabolites are indicated in rectangles. Green metabolites activate the dietary sensor when supplemented to the bacterial diet. Genes encoding the metabolic enzymes involved are indicated with arrows between metabolites. Genes that, when mutated, activate the dietary sensor are indicated in red. Red hexagons – vitamin B12; green hexagons – vitamin B6; blue indicates folate metabolism. 5-Me-THF = 5-methyltetrahydrofolate; THF = tetrahydrofolate, the biologically active form of folate. (B) Chemical epistasis of combined supplementation of Ado-Cbl and propionic acid on Pacdh-1::GFP dietary sensor animals fed the E. coli OP50 diet. (C) qRT-PCR of 14 Comamonas-upregulated and 14 Comamonas-downregulated genes in wild type animals fed indicated diets and supplemented metabolites. PA = propionic acid. N2 = wild type animals. Changes in expression for each gene are plotted as log₂ fold change compared to respective mRNA levels in wild type animals fed E. coli OP50 (first and third columns) or Comamonas aq. DA1877 (middle column). See also Table S4.

Figure 5. Effects of Metabolite Supplementation on C. elegans Development

(A) Developmental progression of synchronized wild type populations of animals fed indicated diets and supplemented metabolites. The parental generation was cultivated on E. coli OP50. (B) Developmental progression of synchronized wild type populations of animals fed indicated diets and supplemented metabolites. PA = propionic acid. Vitamin B12 = 64 nM Ado-Cbl. The parental generation was cultivated on E. coli OP50. (C) Developmental progression of synchronized wild type and mutant populations of animals fed indicated diets and supplemented metabolites. 64 nM of each variant of vitamin B12 was used. (D) Developmental progression of synchronized wild type and mutant populations of animals fed indicated diets and supplemented metabolites. L-Met = 4 mM methionine. 56 μM folate was used. See also Figure S3.

Figure 6. Effects of Vitamin B12 on Propionic Acid Toxicity

(A) Toxicity analysis of incremental doses of propionic acid on wild type and Δpcca-1 mutant C. elegans in the absence and presence of 64 nM Ado-Cbl (B12). Mean +/− SEM is plotted from three combined experiments. (B) Percent survival of different C. elegans strains fed E. coli OP50 at two concentrations of propionic acid (PA) in the absence or presence of 64 nM Ado-Cbl. Mean +/− SEM is plotted from four combined experiments.

Figure 7. Model

Our findings demonstrate that the Comamonas aq. DA1877 diet provides high levels of vitamin B12, whereas the E. coli OP50 diet is low in this cofactor. Vitamin B12 acts through two different pathways: it lowers levels of propionyl-CoA, thereby preventing propionic acid toxicity, and enables the synthesis of methionine and SAM, thereby accelerating development and reducing fertility. Methionine synthesis reduces levels of homocysteine and likely propionyl-CoA levels. Our data suggest that vitamin B12 may affect these processes by regulating the expression of different sets of genes (type 1 and type 2); some that respond oppositely to propionyl-CoA and others that are insensitive to this metabolite.