Cabreiro F et al. (2013) EMBO Mol Med "Worms need microbes too: microbiota, health and aging in Caenorhabditis elegans."

Cabreiro F, Gems D

[EMBO Mol Med, 2013]

Many animal species live in close association with commensal and symbiotic microbes (microbiota). Recent studies have revealed that the status of gastrointestinal tract microbiota can influence nutrition-related syndromes such as obesity and type-2 diabetes, and perhaps aging. These morbidities have a profound impact in terms of individual suffering, and are an increasing economic burden to modern societies. Several theories have been proposed for the influence of microbiota on host metabolism, but these largely remain to be proven. In this article we discuss how microbiota may be manipulated (via pharmacology, diet, or gene manipulation) in order to alter metabolism, immunity, health and aging in the host. The nematode Caenorhabditis elegans in combination with one microbial species is an excellent, defined model system to investigate the mechanisms of host-microbiota interactions, particularly given the combined power of worm and microbial genetics. We also discuss the multifaceted nature of the worm-microbe relationship, which likely encompasses predation, commensalism, pathogenicity and necromeny.

Cabreiro, F. et al. (2011) International Worm Meeting "Gut microbiota as a therapeutic target of biguanides to extend C. elegans lifespan."

Aston, P., Cabreiro, F., Slack, C., Partridge, L., Au, C., Gems, D.

[International Worm Meeting, 2011]

The biguanide drugs metformin and phenformin have been used to treat type 2 diabetes since the 1950s. Metformin can also extend lifespan in worms1 and in short-lived mice2. Biguanides activate AMP-dependent kinase (AMPK), but their exact mode of action remains unclear. One possibility is that they might affect mammalian physiology via their effects on intestinal microbiota. Notably, metformin causes gastrointestinal upset. We are studying the role of E. coli, which plays a dual role as the worm's food source and microbiota3, in effects of biguanides on lifespan. Phenformin is active in humans at lower concentrations than metformin, and we find the same to be true of its effects on worm lifespan. For both drugs, the concentrations optimal for life extension on E. coli OP50 also retard bacterial growth. By contrast, E. coli HT115 are resistant to such growth inhibition, and on this strain metformin does not increase worm lifespan. Moreover, on UV-irradiated OP50 metformin does not extend lifespan either. This suggests that E. coli mediate the effects of metformin on worm lifespan. In the absence of E. coli, metformin only shortens lifespan, suggesting drug toxicity. Metformin increases lifespan in daf-16 but not aak-2 or skn-1 mutants, perhaps because AMPK and SKN-1 protect against metformin toxicity. Consistent with this, larval development in aak-2 but not daf-16 mutants was retarded by metformin (50mM, the optimal dose for life extension). Thus, metformin seems to have a dual effect on C. elegans, extending lifespan via their effect on E. coli, and shortening lifespan via their direct effect on the worm. How do biguanides affect E. coli to increase worm lifespan? One possibility is that they reduce E. coli pathogenicity. However, metformin does not increase lifespan in the cilium structure mutants che-3, daf-10 and osm-5. This could suggest that metformin increases lifespan by changing perceived food quality; moreover, metabolic defects in E. coli can increase worm lifespan. Thus, effects of biguanides on the metabolic status of the worm's E. coli microbiota may increase its lifespan. In fruitflies, metformin treatment activates AMPK and disturbs gut microbiota but does not increase lifespan. One possibility is that flies' microbiota do not influence their lifespan. Alternatively, concentrations of metformin high enough to sufficiently alter microbiota may not outweigh direct toxicity to the fly, as is the case in C. elegans cultured on E. coli HT115. 1. PLoS One 5 (2010). 2. Cell Cycle 7, 2769 (2008). 3. J. Gerontol. 63, 2422 (2008).

Cabreiro F et al. (2013) Cell "Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism."

Cocheme HM, Gems D, Greene ND, Vergara-Irigaray N, Schuster E, Noori T, Cabreiro F, Leung KY, Au C, Weinkove D

[Cell, 2013]

The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy-and also to its side effects, which include folate deficiency and gastrointestinal upset.

Cabreiro, F. et al. (2011) International Worm Meeting "Why do elevated levels of the SOD-1 cytosolic superoxide dismutase increase C. elegans lifespan?"

Cabreiro, F., Araiz, C., Ackerman, D., Doonan, R., Gems, D.

[International Worm Meeting, 2011]

According to the oxidative damage theory, molecular damage caused by the superoxide radical (O2.-) is a significant contributor to aging. This suggests a role in longevity assurance for superoxide dismutase (SOD), which eliminates O2.-. In C. elegans, over-expression of sod-1, the major cytosolic Cu/ZnSOD, does indeed increase lifespan (Doonan, R. et al. Genes Dev. 22: 3236, 2008). Yet is this extended lifespan observed really due to enhanced O2.- scavenging? Elevation of SOD can actually increase levels of reactive oxygen species (ROS), particularly H2O2, the product of its activity. Moreover, we previously noted that elevated SOD actually reduces resistance to oxidative stress. We therefore investigated further the consequences of sod-1 over-expression in C. elegans. As predicted, worms over-expressing sod-1 showed increased levels of H2O2. Moreover, their longevity required the DAF-16/FoxO transcription factor. We therefore wondered whether elevated H2O2 levels might extend lifespan via a hormetic activation of DAF-16. However, sod-1-induced longevity was not suppressed by co-over-expression of catalase, arguing against this. Elevated SOD-1 levels did not reduce markers of oxidation of protein, lipid or glycation; in fact protein oxidation levels were increased. Over-expression of sod-2, the major mitochondrial MnSOD, also increased lifespan in a daf-16-dependent manner and here again oxidative damage was not reduced. So, why does SOD-1 over-expression increase lifespan? SOD-1 is one of the most abundant cytosolic proteins, and our over-expresser lines show up to a seven-fold increase in SOD-1 protein levels. Potentially, levels of SOD-1 are so high as to challenge protein folding homeostasis, thereby eliciting a stress response. Consistent with this, longevity here is largely dependent upon the heat shock transcription factor HSF-1. Moreover, expression of hsp-4::gfp, an ER stress reporter, is induced by sod-1 over-expression. We are currently testing whether longevity here is the result of an unfolded protein response. Overall, these results imply that the longevity resulting from over-expression of sod-1 are not attributable to enhanced ROS scavenging, and therefore may not be taken as support for the oxidative damage theory.

Cabreiro F et al. (2011) Free Radic Biol Med "Increased life span from overexpression of superoxide dismutase in Caenorhabditis elegans is not caused by decreased oxidative damage."

Araiz C, Gems D, Back P, Doonan R, Cabreiro F, Ackerman D, Papp D, Braeckman BP

[Free Radic Biol Med, 2011]

The superoxide free radical (O(2)(-)) has been viewed as a likely major contributor to aging. If this is correct, then superoxide dismutase (SOD), which removes O(2)(-), should contribute to longevity assurance. In Caenorhabditis elegans, overexpression (OE) of the major cytosolic Cu/Zn-SOD, sod-1, increases life span. But is this increase caused by enhanced antioxidant defense? sod-1 OE did not reduce measures of lipid oxidation or glycation and actually increased levels of protein oxidation. The effect of sod-1 OE on life span was dependent on the DAF-16/FoxO transcription factor (TF) and, partially, on the heat shock TF HSF-1. Similarly, overexpression of sod-2 (major mitochondrial Mn-SOD) resulted in life-span extension that was daf-16 dependent. sod-1 OE increased steady-state hydrogen peroxide (H(2)O(2)) levels in vivo. However, co-overexpression of catalase did not suppress the life-span extension, arguing against H(2)O(2) as a cause of longevity. sod-1 OE increased hsp-4 expression, suggesting increased endoplasmic reticulum (ER) stress. Moreover, longevity was partially suppressed by inactivation of ire-1 and xbp-1, mediators of the ER stress response. This suggests that high levels of SOD-1 protein may challenge protein-folding homeostasis, triggering a daf-16- and hsf-1-dependent stress response that extends life span. These findings imply that SOD overexpression increases C. elegans life span, not by removal of O(2)(-), but instead by activating longevity-promoting transcription factors.

Cabreiro F et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Elevated Expression of SOD-1 Cu/Zn Superoxide Dismutase Increases Lifespan but also Protein Oxidation in C. elegans"

Cabreiro F, Doonan R, Gems D, Ackerman D, Araiz C

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

According to the oxidative damage theory, molecular damage caused by the superoxide radical (O2.-) is a significant contributor to aging. This suggests a role in longevity assurance for superoxide dismutase (SOD), an enzyme that catalytically eliminates O2.-. In C. elegans, over-expression of sod-1, which encodes the major cytosolic Cu/Zn SOD, does indeed increase lifespan1. However, elevation of SOD levels can actually increase levels of reactive oxygen species (ROS), particularly the product of its activity H2O2 (ref. 2).We also noted previously that elevated SOD increased rather than reduced sensitivity to oxidative stress1.This lead us to wonder whether the longevity of worms with elevated SOD levels is really due to enhanced O2.- scavenging. We therefore examined further the consequences of over-expression of sod-1 in C. elegans. As suspected we found that elevated expression of sod-1 increased levels of H2O2 production. We also found that longevity resulting from sod-1 over-expression requires the DAF-16/FoxO transcription factor. We hypothesized that elevated H2O2 levels might extend lifespan via a hormetic activation of DAF-16. However, sod-1 induced longevity was not suppressed by co-over-expression of catalase, arguing against this. Elevated SOD-1 levels did not reduce markers of oxidation of protein (carbonyl), lipid (4-hydroxynonenal) or glycation (carboxymethyl-lysine); in fact, surprisingly, protein oxidation levels were significantly increased. Over-expression of sod-2 (the major mitochondrial Mn SOD) also increased lifespan in a daf-16-dependent manner and, here again, oxidative damage was not reduced. Overall, these results imply that the longevity resulting from over-expression of sod-1is not attributable to enhanced ROS scavenging. We postulate that elevated levels of SOD-1 cause some form of stress (as yet unidentified) that induces a longevity mechanism requiring daf-16. What is striking is that this mechanism is associated with increased rather than reduced oxidative damage. This work implies that the longevity of our C. elegans lines over-expressingsod-1should not be taken as support for the oxidative damage theory of aging. 1Doonan, R. et al. Genes Dev. 22, 3236 (2008). 2Buettner, G. R. et al. Free Radic Biol Med 41, 1338 (2006).

Pryor, R. et al. (2017) International Worm Meeting "Metabolic superbugs regulate the effect of drugs and diet on host health and lifespan."

Temmerman, L., Bryson, K., Maier, L., Pryor, R., De Haes, W., Norvaisas, P., Typas, A., Bala-Krishnan, K., Cabreiro, F.

[International Worm Meeting, 2017]

Animals are typically colonised by a diverse community of commensal and symbiotic microorganisms. Recent studies have linked perturbations to the gut microbiota with several nutrition-associated pathologies including obesity, type-2 diabetes and potentially ageing1. Therefore, in order to fully understand the physiology of an organism, it is important to consider both the host and its microbiota as a unique metabolic entity- collectively referred to as the holobiont. Metformin has been found to extend lifespan in C. elegans by altering the metabolic profile of the gut bacteria2. Similarly, a role for the microbiota in mediating the therapeutic effect of the drug has been uncovered in both mice and humans3,4. Nevertheless, precisely how the microbiota mediates the effects of metformin on the host remains unclear. To this purpose, we have created 35 biguanide-resistant bacterial strains which regulate the effects of metformin on C. elegans lifespan. To systematically elucidate which bacterial mechanisms confer resistance against metformin we utilised a chemical genomics approach and screened a genome-wide overexpression and deletion library in E. coli. This strategy allowed us to identify novel bacterial genes regulating the effects of metformin on the host. In order to establish how bacterial-dependent effects of metformin regulate host heath, we have employed a combination of diverse "omic" techniques at the holobiont level using C. elegans and germ-free mice. Utilising metformin-sensitive and resistant bacterial strains we have disentangled the bacteria-dependent effects of metformin from those that result from direct action of the drug in C. elegans. This has led to the identification of a unique pro-longevity signature mediated by the effects of metformin on bacterial metabolism. Finally, we have discovered a "legacy effect" whereby microbial exposure to metformin triggers long-lasting metabolic rearrangements that have beneficial or detrimental consequences in the host in a nutrient-dependent manner. Ultimately these findings underscore the importance of microbes as molecular integrators of the effects of drugs and diet on host physiology. 1- Pryor R., & Cabreiro F. Biochem J (2015); 2- Cabreiro F. et al. Cell (2013); 3- Shin N.R. et al. Gut (2014); 4- Forslund K. et al. Nature (2015)

Storelli G et al. (2013) Cell Metab "Metformin, microbes, and aging."

Leulier M, Storelli G, Tefit M

[Cell Metab, 2013]

The mechanisms underlying the biological activity of metformin, a widely prescribed drug to treat type 2 diabetes, remain elusive. In a recent issue of Cell, Cabreiro et al. report that in C. elegans, metformin indirectly impacts lifespan by altering the methionine metabolism of its microbial partner E. coli (Cabreiro et al., 2013).

Quintaneiro, L. et al. (2017) International Worm Meeting "Host-microbe co-metabolism dictates cancer drug efficacy in C. elegans."

Bryson, K., Cabreiro, F., Greene, N. D. E., Norvaisas, P., Typas, A., Quintaneiro, L., Lui, P. P., Herrera-Dominguez, L., Scott, T. A.

[International Worm Meeting, 2017]

Fluorouracil (5-FU) is a fluoropyrimidine and uracil analogue commonly used in the treatment of colorectal cancer. 5-FU exerts cytotoxicity by inhibiting nucleotide synthesis and by misincoorporating into RNA and DNA, leading to cancer cell damage and death1. Despite the widespread use of this drug, new approaches are needed to boost efficacy and decrease toxicity. Furthermore, 5-FU treatment outcome is variable between patients and cannot be completely explained by genetic factors, suggesting that environmental factors could contribute to its efficacy. The gut microbiota is defined as the collective community of microbes that reside a host gastrointestinal tract, and have recently been linked to other elements of host fitness, such as immunity2 and metabolism3. However, not much is known about the interplay between drug, gut microbes and the host. To unravel the mechanism by which the microbiota is affecting the metabolism of 5-FU, we used the nematode C. elegans as a host model and microbe E. coli as a gut microbe model. Via high-throughput screens and metabolomic approaches we found that microbes can affect drug conversion and efficacy in the host. We discovered this effect to be influenced by bacterial metabolism and synthesis of ribonucleotides and vitamins B9 and B6. Additionally, we find deoxynucleotide pools imbalances in bacteria increased autophagy and cell death on host cells upon 5-FU treatment. Interestingly, this effect was found to be dependent and regulated by the host gene nucleoside diphosphate kinase ndk-1. In summary, our data suggests bacteria can contribute to drug efficacy on host metabolism through two distinct but complementary processes4. These findings emphasize the importance of the gut microbiome in the co-metabolism of host-targeted drugs. Moreover, this study reveals the potential therapeutic power of manipulating gut microbial communities to help treat disease and improve treatment outcomes. References: 1. Longley, D.B., et al. Nat. Rev. Cancer (2003) 2. Nicholson, J.K., et al. Science (2012) 3. Cabreiro, F., et al. Cell (2013) 4. Scott, A.T., Quintaneiro, L. M., et al. Cell (in press)

Scott TA et al. (2017) Oncotarget "Microbiome genetics underpins chemotherapy."

Cabreiro F, Scott TA, Quintaneiro LM

[Oncotarget, 2017]