netbiosig2014-keynote by marian walhout

Interspecies Systems Biology:

Nutritional Regulatory Networks

A.J. Marian Walhout

Interspecies Systems Biology

Intraspecies Systems BiologyDevelopment

BehaviorPhysiology

Disease

Intraspecies systems biology - Networks

Protein-protein interaction networks

Gene regulatory networks Metabolic networks

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Protein-protein interaction networks

Gene regulatory networks Metabolic networks

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Intraspecies systems biology - Networks

Nutrition

Interspecies biology

Symbiosis

Pathogenesis

Interspecies systems biology

Symbiosis

Nutrition

Pathogenesis

Two effects of nutrition

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How is metabolism regulated in response to diet?

?heterogeneous

heterogeneous

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?

How is metabolism regulated in response to diet?

Bacteria

(diet/prey)

C. elegans

(predator)

Superworm: nematode C. elegans

• Hermaphrodite • ~3 days development• ~2 week life span• Lineage known - 959 cells• Transparent – GFP transgenics• RNAi, transgenics, mutants• Well annotated genome

• Feed individual bacterial species• Model for diet• Model for microbiota

Does bacterial diet affectC. elegans physiology?

Life history traits

DevelopmentFertility

Lifespan (aging)

E. coli OP50Standard Laboratory Diet

E. coli HT115Used for RNAi feeding

Comamonas DA1877Soil isolate

Measure developmental time, reproduction and lifespan

MacNeil et al., Cell 2013

The life cycle of the worm

Dietary changes in life-history traits

Reproduction

Comamonas: faster development, fewer offspring, shorter lifespan

Development Lifespan

OP50 = E. coliDA1877 = Comamonas

MacNeil et al., Cell 2013

Dietary changes in gene expression

Nutrigenomics

Microarray expression profiling of worms fed different bacterial diets

A set of core diet-response genes

MacNeil et al., Cell 2013

Are these changes transcriptional?

A proxy gene

acdh-1Repressed >300-fold on Comamonas diet

MacNeil et al., Cell 2013

Transgenic C. elegans reporter strains

GFPPacdh-1

E. coli OP50

Comamonas DA1877

The dietary response is transcriptionalPacdh-1::GFP – 1.5 kb promoter

A dietary sensor in living animals

E. coli OP50

Comamonas DA1877

MacNeil et al., Cell 2013

Starvation represses Pacdh-1

E. coli OP50

Starvation

Does a Comamonas diet mimic starvation?

MacNeil et al., Cell 2013

E. coli OP50GFP ON

Comamonas DA1877GFP OFF

Prediction: if Comamonas is nutrient poor, mixing it with E. coli should alleviate this

Comamonas DA1877+

E. coli OP50

GFP ?MacNeil et al., Cell 2013

The dietary and starvation responses are distinct

DA1877

OP50**Exposure time 1/20

DA18771/200

DA18771/500

DA18771/1000 DA1877

1/10 000 DA1877

1/100 000 DA1877

OP501/20 exposure

1/100 DA1877Also accelerated development and similar gene expression

changes under 1/1000 dilution

Small amounts of one diet can have dramatic effects on gene expression

and physiology, even when mixed withanother diet

Unhealthy?+ =

Healthy?+ =

Comamonas DA1877 produces dilutable compound to which worms respond

Two interpretations with very different implications

Physiology – life history traits

??

How does C. elegans respond toE. coli and Comamonas diets?

C. elegans genetics

Which bacterial molecules affect gene expression and life history traits?

Bacterial genetics

Interspecies systems biology

C. elegans target of rapamycin (TOR) and insulin signaling pathways

are not involved in the response to Comamonas

MacNeil et al., Cell 2013

What are the gene regulatory networks involved in the response to Comamonas?

The response to Comamonas is transcriptional

Comamonas makes a compound or signal

Transcription factors?

Signaling pathways?

What are the gene regulatory networks involved in the response to Comamonas?

Forward and reverse genetic screens

Activators

146

Worms cannot respond to Comamonas diet

Repressors

35Multiple NHRs

(worms have 271!)

Most repressors are metabolicgenes, not transcription factors

Watson, MacNeil et al., Cell 2013

BCAA breakdown

TCA cycle

Glycine cleavage

Methionine/SAM

cycle

Mutations in human homologs of repressors give rise to inborn metabolic diseases treated by dietary intervention

? ?How does C. elegans respond to

E. coli and Comamonas diets?C. elegans genetics

Which bacterial molecules affect gene expression and life history traits?

Bacterial genetics

Interspecies systems biology

Screen I: E. coli deletion collection

Watson et al., Cell 2014

70 genes

7 genes

Screen II: Comamonas transposon mutagenesis

5 hits Lesley MacNeil

Watson et al., Cell 2014

From mutants to metabolites

Sequence Comamonas genome – identify genes

Map E. coli and Comamonas mutant genes onto metabolic network using KEGG

Annotate biological processes/pathways

Annotate candidate metabolites – mutant gene leads to buildup of precursor and lack of product

A bacterial metabolic network implicated in the C. elegans dietary response

Watson et al., Cell 2014

Metabolite screen:

Supplement metabolites to C. elegans dietary sensoron either bacterial diet in increasing concentrations

Watson et al., Cell 2014

Metabolites increasing GFP on Comamonas

Watson et al., Cell 2014

Metabolites decreasing GFP on E. coli

Watson et al., Cell 2014

Ado-Cbl and Me-Cbl are two forms of vitamin B12

Mutation in either C. elegans gene

interferes with the response to Comamonas

Watson, MacNeil et al., Cell 2013

Vitamin B12 is in the C. elegans dietary response network

Is vitamin B12 theComamonas dilutable

molecule?

Mass spectrometry of bacteria

Amy Caudy (U Toronto)

Vitamin B12 levels in Comamonas are orders of magnitude higher than in E. coli

Watson et al., Cell 2014

Vitamin B12 pathway status correlatesperfectly with dietary sensor activity

Vitamin B12 pathway status correlatesperfectly with dietary sensor activity

A metabolic network connecting the two vitamin B12 pathways

Me-Cbl

Ado-Cbl

Propionyl-CoA

Watson et al., Cell 2014

How does the gene regulatory network respond to Comamonas or E. coli?

B12 fails to repress GFP expression in propionyl-CoA breakdown mutants

Vitamin B12 is not sufficient todrive sensor repression

Model: balance betweenVitamin B12 and propionyl-CoA

Chemical epistasis: add both B12 and propionic acid

Excess propionic acid is epistatic toVitamin B12

Watson et al., Cell 2014

Excess propionic acid is epistatic toVitamin B12

Watson et al., Cell 2014

So far all experiments with sensor

Does vitamin B12 mimic broadChanges in C. elegans gene

Expression elicited by Comamonas diet?

qRT-PCR of 28 Comamonas-response genes

Vitamin B12 mimics Comamonas-induced changes in C. elegans gene expression

Two types of B12-regulated genes

Type 1: responsive to propionic acid

Type 2: not responsive to propionic acid

Watson et al., Cell 2014

Does vitamin B12 supplementation by Comamonas Explain its effects on C. elegans life history traits?

Vitamin B12 accelerates C. elegans development

B12 reduces fertilityB12 has no effect on lifespan

Watson et al., Cell 2014

How does B12 accelerate development?

Watson et al., Cell 2014

Vitamin B12 developmental accelerationoccurs via methionine/SAM cycle

Mutants in propionic acid breakdown are accelerated by B12

Mutants in methionine/SAM cycle are not accelerated by B12

Watson et al., Cell 2014

Which metabolite drivesdevelopmental acceleration?

Watson et al., Cell 2014

SAM production is important for developmental acceleration

Watson et al., Cell 2014

What is the physiological role of the Other B12 pathway?

Metabolites increasing GFP on Comamonas

Comamonas E. coli

Watson et al., Cell 2014

Vitamin B12 mitigates propionicacid toxitiy

Watson et al., Cell 2014

Functions of vitamin B12

development

PA toxicity

Vitamin B12

Propionic acid

Conclusions

C. elegans and bacteria are a powerful interspecies system:

Nutritional networks

Effects of microbiota (SCFAs/vitamin B12)

Acknowledgements

Lesley MacNeilEmma Watson

Safak YilmazAshlyn RitterJote BulchaJuan Fuxman BassAurian Garcia GonzalesGabrielle GieseAmy HoldorfMike HoyRudi KaushikAkihiro MoriShaleen ShresthaAlex Tamburino

Efsun Arda (now at Stanford)

Julie Zhu (UMMS)

Adam RosebrockAmy Caudy (U Toronto)

NIDDKNIGMS