DDS promotes longevity through a microbiome-mediated starvation signal

Abstract

The antibiotic diaminodiphenyl sulfone (DDS) is used in combination with other antibiotics as a first line treatment for leprosy. DDS has been previously reported to extend lifespan in Caenorhabditis elegans through inhibition of pyruvate kinase and decreased mitochondrial function. Here we report an alternative mechanism of action by which DDS promotes longevity in C. elegans by reducing folate production by the microbiome. This results in altered methionine cycle metabolite levels mimicking the effects of metformin and lifespan extension that is dependent on the starvation- and hypoxia-induced flavin containing monoxygenase, FMO-2.

INTRODUCTION

The identification of small molecules that extend lifespan and improve health during aging has become a major focus of Geroscience research (1–4). Optimally, such an intervention would be a compound with a consistently robust effect, clear mechanism of action, and well-documented safety profile in people. One possible example of such an intervention is metformin, the most widely prescribed anti-diabetic drug in the United States, which has been reported to increase lifespan in Caenorhabditis elegans, Drosophila melanogaster, and mice, and may reduce mortality in people (5–10). The mechanism of action for metformin remains unclear, however, with multiple mechanisms having been proposed, including activation of AMP kinase, inhibition of mitochondrial electron transport chain complex I, activation of peroxiredoxin, and effects on the microbiome including microbial folate production (5, 8, 11, 12).

The bacteriostatic antibiotic diaminodiphenyl sulfone (DDS, also known as Dapsone) is commonly used in combination with rifampicin and clofazimine as a treatment for leprosy (13), and as a second line treatment for dermatitis herpetiformis, tuberculosis, pneumonia and other infectious diseases (14). It is listed on the World Health Organization’s List of Essential Medicines. The mechanism of action of DDS appears to be primarily antimicrobial, although several studies have also reported anti-inflammatory effects in animal models (14). Anecdotal reports have indicated that leprosy patients in South Korea receiving DDS treatment have enhanced health and longevity relative to the general population, and often continue to take DDS even after their leprosy has been cured, leading to the hypothesis that DDS may promote healthy aging in people (15, 16). In addition, DDS protects cells against oxidative stress in culture and in a mouse model of lung injury, and significantly increases organismal lifespan in C. elegans (17–20).

DDS is a member of the sulfonamide class of antibiotics (also referred to as sulfa drugs) that act as competitive inhibitors of the enzyme dihydropteroate synthetase (DHPS) (14). DHPS is expressed in most bacteria, but not in eukaryotes, and is required for bacterial biosynthesis of folates through conversion of dihydropteroate diphosphate and p-aminobenzoic acid (PABA) into dihydropteroic acid which can then be converted into tetrahydrofolate (Figure 1A). Folates are required for a number of critical biosynthetic processes, including biosynthesis of purine and pyrimidine nucleic acids, biosynthesis of some amino acids, and key methyl donors such as S-adenosylmethionine (21).

Figure 1. Sulfa antibiotics extend C. elegans lifespan.

(A) Sulfa antibiotics inhibit bacterial folate synthesis by inhibiting the enzyme dihydropteroate synthetase. (B) DDS extends the lifespan of adult N2 C. elegans at concentrations between 1–10 μM. (C) Sulfadiazine (10 μM), sulfacetamide, (50 μM) and sulfasalazine (10 μM) all significantly extended lifespan relative to control treated animals. p < 0.05 in all cases.

A prior report indicated that addition of the sulfonamide antibiotic sulfamethoxazole to the nematode growth medium (NGM) was also sufficient to extend lifespan in C. elegans and reduce both bacterial and nematode folate levels (22). We therefore set out to test the hypothesis that DDS increases lifespan in C. elegans through a similar microbiome-mediated mechanism. Here we report that DDS and three additional sulfonamide antibiotics increase C. elegans lifespan and reduce folate availability from the microbiome. We also provide evidence that inhibition of microbial folate synthesis results in reduced folate uptake and activation of a starvation signal requires activation of the flavin-containing monooxygenase FMO-2 to extend lifespan.

RESULTS

Sulfa antibiotics extend C. elegans lifespan independent of effects on bacterial growth

DDS inhibits production of folates by the bacterial microbiome (Figure 1A) and has been previously shown to extend lifespan in C. elegans fed an E. coli food source pre-cultured in medium containing the drug (20). We observed a similar lifespan-enhancing effect when DDS was added directly to the surface of the nematode growth medium (NGM) containing E. coli OP50 (Figure 1B). DDS treatment significantly extended lifespan when added at a final concentration of 1 μM, 5 μM, or 10 μM, with the largest effect observed at a concentration of 5 μM.

Since DDS acts as an anti-bacterial agent by inhibiting bacterial folate synthesis, we wished to determine whether this activity was related to its effects on worm lifespan. Consistent with prior studies of sulfamethoxazole (23), the lifespan extending effects of DDS are unlikely to be caused by reduced bacterial proliferation, as our experiments were performed on UV-treated, growth arrested E. coli as the food source, per our previously reported methodology for nematode lifespan experiments (24). Under these conditions, we found that three additional sulfa antibiotics (sulfadiazine, sulfasalazine, and sulfacetamide) with a similar mechanism of action also significantly increased lifespan of C. elegans on growth arrested food (Figure 1C).

DDS results in dietary folate limitation that is necessary for lifespan extension

In order to confirm that DDS is extending lifespan by acting on bacterial folate synthesis, we utilized E. coli OP50 that are resistant to DDS as the food source. Bacterial resistance was accomplished through expression of a drug resistant allele of dihydropteroate synthase from the R26 plasmid (22, 25), which allows normal folate production in the presence of DDS, while OP50 transformed with the plasmid backbone alone (vector control) remain sensitive to DDS. Worms fed growth arrested OP50 carrying the R26 plasmid in combination with 5 μM DDS were not long-lived, whereas worms treated identically but fed OP50 carrying the vector control were long-lived (Figure 2A).

Figure 2. DDS extends lifespan by decreasing bacterial folate production.

(A) DDS fails to extend the lifespan of worms fed E. coli OP50 that are resistant to DDS due to presence of the R26 plasmid. (B) Lifespan extension from DDS is suppressed by additional of exogenous folate to the E. coli OP50 food source.

To directly assess whether a reduction in dietary folate is required for lifespan extension from DDS, we supplemented DDS-treated worms with folic acid and determined the effect on lifespan. Addition of folic acid was sufficient to completely abrogate lifespan extension from DDS (Figure 2B). Taken together, these observations indicate that lifespan extension from DDS requires inhibition of bacterial dihydropteroate synthase and reduced bacterial folate.

Prior work has suggested that the anti-diabetes drug metformin increases lifespan in C. elegans by inhibiting bacterial folate synthesis and thereby reducing methionine availability (8). To assess whether DDS and sulfadiazine may be acting by a mechanism similar to metformin, we quantified the levels of folate cycle and methionine cycle metabolites in the E. coli food source and in worms (Table 1). As previously reported for metformin (8), DDS and sulfadiazine had little effect on the folate cycle intermediates tetrahydrofolate (THF) or N⁵,N¹⁰-methyleneTHF in the bacteria or worms. In contrast, significant changes were detected in the methionine cycle intermediates S-adenosyl methionine (SAM), S-adenosyl homocysteine (SAH), homocysteine (HC) and methionine (Figure 3). Specifically, treatment with the sulfa drugs caused increased levels of SAH, and reduced levels of SAM, HC, and methionine. These changes resulted in a dramatic reduction in the SAM/SAH ratio, which is an indicator of methylation potential, along with total methionine levels in the worms. In each case, treatment with metformin resulted in changes similar to those induced by the sulfa antibiotics.

Table 1. Metabolites quantified from C. elegans and E. coli OP50.

Worms and bacteria were treated with either vector control, 5μM DDS, 10μM sulfadiazine, or 50mM metformin. Concentrations shown as mmol metabolite per mg protein. Mean value shown with standard deviation in parentheses. Shading indicates significant differences from vector control (p<0.05).

		Control	DDS	Sulfadiazine	Metformin
C. elegans	Hcy	58.7 (17.4)	19.1 (6.1)	13.5 (2.2)	16.4 (2.5)
	SAH	0.29 (0.01)	0.41 (0.04)	0.47 (0.11)	0.46 (0.07)
	Methionine	295 (52)	165 (10)	146 (25)	150 (20)
	SAM	27.0 (4.6)	10.6 (2.2)	11.7 (3.2)	11.4 (1.1)
	THF	0.96 (0.16)	0.56 (0.09)	0.46 (0.16)	0.53 (0.07)
	5-methyl THF	1.2 (0.2)	0.6 (0.1)	0.5 (0.1)	0.7 (0.1)
	5,10-methyl THF	0.4 (0.09)	0.2(0.02)	0.2 (0.03)	0.2 (0.03)
	Folic Acid	1.1 (0.2)	0.4 (0.03)	0.4 (0.05)	0.4 (0.06)
E. coli	Hcy	5.2 (0.8)	4.0 (0.8)	4.2 (0.7)	4.3 (0.5)
	SAH	0.04 (0.003)	0.05 (0.005)	0.05 (0.007)	0.05 (0.005)
	Methionine	31.1 (2.5)	24.0 (0.3)	23.1 (2.1)	20.2 (3.2)
	SAM	2.0 (0.1)	2.7 (0.1)	2.8 (0.2)	2.6 (0.2)
	THF	0.2 (0.02)	0.07 (0.01)	0.08 (0.01)	0.07 (0.01)
	5-methyl THF	0.06 (0.02)	0.06 (0.01)	0.06 (0.01)	0.06 (0.01)
	5,10-methyl THF	0.2 (0.07)	0.05 (0.01)	0.05 (0.01)	0.04 (0.01)
	Folic Acid	0.1 (0.02)	0.06 (0.01)	0.06 (0.01)	0.05 (0.01)

Figure 3. Sulfa antibiotics mimic the effects of metformin on methionine cycle intermediates.

The ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and methionine abundance (mmol/mg protein) in (A) E. coli OP50 and (B) wild type N2 C. elegans treated with either DDS, sulfadiazine (SD), or metformin (Metf). Because SAM and SAH represent the substrate and product, respectively, for many cellular methylation reactions, the ratio of SAM/SAH is frequently used as a measure of total cellular methylation potential. *Significantly different from Control, p<0.05. Quantitation and additional metabolites shown in Table 1.

DDS mimics lifespan extension by dietary restriction

We reasoned that if DDS and other sulfa antibiotics were increasing lifespan by limiting folate production by the bacterial food source, then this may represent a form of dietary restriction. As such, we predicted that DDS should not further extend the lifespan of bacterially deprived (BD) worms, which are long-lived due to complete removal of the bacterial food source (26, 27). Consistent with this model, DDS failed to further extend the lifespan of animals subjected to BD (Figure 4A). DDS also failed to further extend lifespan in combination with rapamycin (Figure 4B), a small molecule inhibitor of the nutrient-responsive mechanistic target of rapamycin (mTOR), which has been previously shown to extend lifespan in yeast, worms, fruit flies, and mice (28, 29).

Figure 4. DDS mimics lifespan extension from dietary restriction.

(A) DDS fails to further extend the lifespan of N2 C. elegans subjected to dietary restriction by bacterial deprivation (BD). (B). DDS extends the lifespan of control treated N2 C. elegans but fails to further extend lifespan upon treatment with rapamycin.

The enzyme flavin containing monooxygenase 2 (FMO-2) has recently emerged as an important component downstream of multiple longevity pathways in worms, including dietary restriction (30). FMO-2 is transcriptionally induced by dietary restriction or fasting (31, 32), hypoxia (33–35), mitochondrial stress (36), and inhibition of the pentose phosphate pathway (37), and is required for full lifespan extension in each of these cases. To assess the importance of FMO-2 in lifespan extension by DDS, we first confirmed induction of fmo-2 following treatment with DDS or sulfadiazine by RT-qPCR. Expression of fmo-2 was significantly increased upon treatment with either sulfa drug (Figure 5A), and DDS failed to extend lifespan in animals lacking endogenous fmo-2 (Figure 5B).

Figure 5. DDS induces fmo-2 expression and requires fmo-2 to extend lifespan.

(A) DDS or sulfadiazine (SD) treatment significantly (p<0.05) increases expression of fmo-2 mRNA. (B) DDS treatment fails to extend lifespan in fmo-2 mutant C. elegans.

DISCUSSION

In this study we have confirmed that multiple sulfa antibiotics, including DDS, extend lifespan in C. elegans by a mechanism that involves limitation of folate production by the bacterial microbiome and reduced uptake by the worms. Like metformin, lifespan extension from DDS appears to be distinct from reduced insulin-like signaling and overlapping with dietary restriction (8, 20)(Figure 4). This model seems reasonable, since dietary folate availability would also be reduced during dietary restriction. Interestingly, like dietary restriction, DDS treatment induces FMO-2 and requires FMO-2 for full lifespan extension.

The identification of FMO-2 as a downstream target of DDS is of particular interest, given that this enzyme is also involved in lifespan extension from activation of HIF-1, dietary restriction, inhibition of the pentose phosphate pathway, and mitochondrial stress in worms (30). Intriguingly, independent analyses of gene expression changes associated with longevity interventions in mice have also identified FMOs as among the most significantly induced genes in multiple longevity models, including dietary restriction, methionine restriction, rapamycin treatment, Snell dwarf mice, Ames dwarf mice, growth-hormone receptor knockout mice, and Little mice (38, 39). Flavin containing monooxygenases generally oxidize sulfur or nitrogen containing compounds and have also been shown to perform oxidative demethylation (30). As such, several components of the folate cycle or the closely linked methionine cycle are potential substrates for FMO-2, although none have yet been demonstrated to be FMO substrates in any organism. Given the dramatic changes in relevant metabolite levels upon DDS treatment, however, it is intriguing to speculate that FMO-2 may be induced in response to altered levels of one or more folate or methionine cycle intermediates. Future studies are likely to shed light on this potential mechanism.

The data presented here also support the model that DDS and other sulfa antibiotics extend lifespan in C. elegans by the same microbiota-mediated mechanism as metformin (8, 22). Specifically, both DDS and metformin reduce the amount of microbial folate available to the worms. Whether DDS and metformin have these effects on microbial folate production in other organisms remains uncertain. It is interesting to note, however, that both DDS and metformin have been proposed to delay age-related disease and increase longevity in human populations (15, 40). Recent work has also indicated that metformin treatment has a substantial impact on the gut microbiome of human patients (41), including one study indicating that inhibition of bacterial degradation of bile acids by metformin may be linked to the drug’s efficacy as a treatment for type II diabetes. (42). Intriguingly, a large body of clinical data indicates that metformin treatment is associated with deficiency in vitamin B12, and supplementation with both vitamin B12 and folate is commonly prescribed to metformin patients with vitamin B12 deficiency (43). To the best of our knowledge, the effects of DDS on folate uptake and abundance in patients has not been clearly established.

In this study, the positive effects of DDS on lifespan appeared to be largest when added to the media at a final concentration of 5 μM, although the positive effect was also seen at concentrations both lower and higher than this. Two of the other sulfa antibiotics tested, sulfadiazine and sulfasalazine, also increased lifespan robustly at 10 μM, but lifespan extension was maximized for sulfacetamide at 50 μM. While it is not surprising that a dose response would be observed for DDS or that optimal doses might be different for different drugs, even if the mechanism of action is similar, these observations do suggest that different sulfa antibiotics may have different potencies. While the simplest model is that all of these drugs are mediating lifespan extension via the same mechanism, namely reduced folate availability from the bacterial diet, we cannot formally rule out the possibility that there may also be additional targets for these interventions, and one prior study supported the model that DDS can inhibit pyruvate kinase in C. elegans (20).

Taken together with prior work, the data presented here support the model that metformin and sulfa drugs act by a similar, microbial-mediated mechanism to increase lifespan in C. elegans. Future studies to establish whether similar mechanisms of action underlie the effects of these drugs in patients are warranted. Given the intense interest in metformin as a putative vehicle to directly target aging in humans, it may also be useful to assess whether DDS or other sulfa antibiotics can achieve similar benefits. Unlike metformin, the biochemical activity of sulfa antibiotics is well-established and relatively specific, perhaps providing more consistent efficacy with fewer off-target effects.

Methods

Strains, growth conditions, and drug treatments.

The following C. elegans strains were used in this study and obtained from the Caenorhabditis Genetics Center: N2 and VC1668 fmo2(ok2147). The R26 plasmid (44) and R26-transformed E. coli were used as previously described (8). C. elegans strain maintenance and manipulation were performed using standard methods, as previously described (45–47). Unless otherwise stated, animals were maintained on solid nematode growth medium (NGM) [50mM NaCl, 0.25% Bacto Peptone (BD Biosciences), 2% agar, 1mM MgSO₄, 1mM CaCl₂, 12.9μM Cholesterol, 9.75mM K₃PO₄ pH 6.0] supplemented with 50μg/ml ampicillin with UV-arrested E. coli OP50 as the food source. For treatments with sulfa antibiotics, metformin, and rapamycin, drugs were added topically in 100 μL volume at the appropriate concentration to yield a final concentration as indicated upon diffusion throughout the NGM agar.

Survival analysis and statistics.

Lifespan studies were performed according to our previously published protocol (24) and were carried out at 20°C, unless otherwise stated. All experiments were performed on at least three biological replicate plates. Ruptured animals were not censored from lifespan experiments. Animals that foraged off the surface of the plate during the course of the experiment were not considered. Oasis 2 (https://sbi.postech.ac.kr/oasis2/surv/) was used to calculate mean lifespan, p-values (Wilcoxon Rank-Sum Test), and to plot survival curves (23).

Reagent Preparation.

DDS (4-Aminophenyl Sulfone; Sigma-Aldrich A74807, USA), sulfadiazine (Sigma-Aldrich S8626, USA), sulfasalazine (Sigma-Aldrich S0883, USA) and sulfacetamide (Sigma-Aldrich S8627, USA) were dissolved in ddH₂O at a stock concentration 1mM then autoclaved. Folate (folic acid; Sigma-Aldrich F7876, USA) was dissolved in ddH₂O at a stock concentration 20 μM and 200 μM, then filter-sterilized. Sulfa antibiotic solutions and folate solutions were always prepared fresh one day prior to the experiment and kept in foil-wrapped tubes until use. Experimental plates were also kept in foil to minimize exposure to light.

Sample preparation and metabolomic analysis of folates.

E. coli OP50 and L4 larvae were grown on vehicle (ddH₂O), 5μM DDS, 10μM sulfadiazine or 50mM metformin plates for 4-days and prepared by washing from plates using M9 buffer. The bacteria were then centrifuged at 4°C, 4,000 rpm for 20 min. The supernatant was discarded and the bacterial pellet kept at −80°C until analysis.

Samples (bacteria and worms) were extracted by homogenization in buffer containing internal standard (methotrexate), deconjugation of polyglutamylated folates and removal of proteins for determination of folate concentration as a reduced form (48). For each sample, an aliquot of homogenized extract was used to measure protein concentration using the Bio-Rad RC DC protein assay kit. The extracted and filtered samples were dried using a refrigerated Speed Vac and stored at −80°C prior to analysis. Analytes were separated on a Hypersil GOLD aQ column (100 × 2.1 mm, i.d., 1.9 μm particle size) (Thermo Scientific, USA), maintained at 30°C, ACQUITY® ultra performance liquid chromatography (UPLC) system (Waters, USA). The mobile phase consisted of a gradient elution of 0.1% formic acid (FA) water (A) and 0.1% FA in acetonitrile (B). Initial conditions were as follows: 0% B for 2 min, 0% B to 15% B for 8 min, 15% B to 90% B for 1 min, 90% B for 3 min and held for 5 min in 0% B to re-equilibrate the column at a flow rate of 250 μL/min. Mass spectrometric analyses were performed using ACQUITY® triple quadruple mass spectrometer (Waters, USA) operated in positive ionization mode with optimized following conditions: capillary 3.4 kV, source temperature 140°C, desolvation temperature 500°C, cone gas flow rate 90 L/h and desolvation gas flow rate 1000 L/h. The multiple reaction monitoring (MRM) of m/z 460.2 → 313.1 at cone 34 V, collision energy (CE) 20 eV for 5-methyl THF, 456.2 → 412.1 (cone 56V, CE 30eV) for 5,10-methenyl THF, 442.2 → 295.1 (cone 22V, CE 13eV) for folic acid, 446.2 → 299.1 (cone 32V, CE 22eV) for THF and 455.1 →308.1 (cone 36V, CE 21eV) for methotrexate (IS) were used for simultaneously analysis. The matrix-matched calibration curve was prepared by adding standard to an unknown sample. The concentration of analytes was determined by its area ratio to that of the IS using a daily prepared standard curve.

Sample preparation and metabolomic analysis of folates.

Bacteria and worms were grown and harvested as described in the preceding section. Extraction was performed as previously described (49).The homogenates in 90% methanol with 0.03% (v/v) trifluoroacetic acid containing internal standard (d3-methionine) were reduced with dithiothreitol, and proteins were precipitated by centrifugation. The clear supernatants were dried in a refrigerated Speed Vac and stored at −80°C prior to analysis. The reconstituted sample with mobile phase A (10 μL) was injected to an ACQUITY® UPLC system (Waters, USA) and the analytes were resolved by a Hypersil GOLD aQ column (100 × 2.1 mm, i.d., 1.9 μm particle size) (Thermo Scientific, USA), maintained at 30°C. The mobile phase consisted of 0.1% FA in water (solvent A) and 0.1% FA in acetonitrile (solvent B). A gradient elution profile started with 0% B for 2 min, 0% B to 15% B for 8 min, 15% B to 90% B for 1 min, 90% B for 3 min, 90% B to 0% B for 1 min, and then 0% B held for 5 min to re-equilibrate column at a flow rate of 250 μL/min. The UPLC was coupled to an ACQUITY® triple quadrupole mass spectrometer (Waters, USA) operating in positive-ion mode using the follow settings: capillary 1.2 kV, source temperature 140°C, desolvation temperature 500°C, cone gas flow rate 90 L/h and desolvation gas flow rate 900 L/h. Methionine-related metabolites were measured by multiple reaction monitoring (MRM) with the transition m/z 150.0 to 103.9 (methionine, cone 20V, CE 10eV), m/z 136.0 to 90.0 (homocysteine, cone 22V, CE 11eV), m/z 399.1 to 250.0 (S-adenosylmethionine, cone 28V, CE 19eV), m/z 385.1 to 135.9 (S-adenosylhomocysteine, cone 30V, CE 23eV) and 153.0 to 106.9 (IS, cone 23V, CE 10eV).