ConnectingMicrobestoMetabolism

UsingGnotobiotic Models
Biologicallysignificantmetabolitesproducedbythegut
microbiota:theiroriginsandfunctions

FedericoE.Rey
UniversityofWisconsinMadison

Host-associated microbial communities are

major contributors to chemical diversity

Exposed to thousands of microbially-derived metabolites.

Origin:
Primary metabolism (e.g., fermentation, amino acid
biosynthesis).
Secondary metabolism (e.g., antibiotics, peptides).
Biotransformations of dietary compounds, drugs,
host metabolites (e.g., bile acids).

Many known microbial metabolites affect community

dynamics or have dedicated host receptors that allow for
microbe-host cross-talk.

Gutbacteriaandfood

Complexplant
polysaccharides

ShortChainFattyAcids:
(e.g.,acetate,propionate,
butyrate)

modulateintestinalmotility
modulatePTMhistones
protectiveagainstdietinduced
obesityanddiabetes
modulatebloodpressure!!

Gutbacteriaandfood
Flavonoids
( e.g.,anthocyanins)

Complexplant
polysaccharides

ShortChainFattyAcids:
(e.g.,acetate,propionate,
butyrate)

modulateintestinalmotility
modulatePTMhistones
protectiveagainstdietinduced
obesityanddiabetes
modulatebloodpressure!!

3,4Dihydroxybenzoicacid
andother phenolicacids
Preventscardiovascular
diseaseanddiabetes

Gutbacteriaandfood
Flavonoids
( e.g.,anthocyanins)

Complexplant
polysaccharides

ShortChainFattyAcids:
(e.g.,acetate,propionate,
butyrate)

3,4Dihydroxybenzoicacid
andother phenolicacids
PreventsPrevents
cardiovasculardiseaseand
diabetes

Choline,carnitine

modulateintestinalmotility
modulatePTMhistones
protectiveagainstdietinduced
obesityanddiabetes
modulatebloodpressure!!

Trimethylamine
Linkedwith
cardiovasculardisease

Largeinterpersonaldifferencesinmicrobiota
composition
Flavonoids
( e.g.,anthocyanins)

Howourmicrobialdifferencesmodulatethenutrients
wegetfromthefoodweconsume?Howthisaffectsour
3,4Dihydroxybenzoicacid
health?
Complexplant
polysaccharides

andother phenolicacids

Preventsatherosclerosis
anddiabetes

Personalizenutritionbasedonthemetabolicpotential
ofapersonsmicrobiota

ShortChainFattyAcids:
(e.g.,acetate,propionate,
butyrate)

Choline,carnitine

modulateintestinalmotility
protectiveagainstdietinduced
obesityanddiabetes
modulatebloodpressure!!

Trimethylamine
Promotesatherosclerosis

Definedmicrobialcommunityconsistsofsequencedisolatesofthe
humandistalgut

Eliminatestheuncertaintysurroundingwhosthere
Iseasilymanipulatedtotesthypotheses(hostandmicrobialgenetics)

Gutmicrobialmetabolismofcholine

KymRomano
(UWMadison)

DanielAmadorNoguez
(UWMadison)

EmilyBalskus
(Harvard)

Cholineismodifiedbybacterialandhost
metabolism

Proatherogenic metabolite:Trimethylamine
oxide(TMAO)
PlasmaLevelofTMAOisagoodpredictor
ofcardiovasculardiseaseinhumans

TMAOexacerbates
atherosclerosisinmice
inhibitsreversecholesterol
transport
Contributestoplatelet
hyperresponsiveness and
enhancesthrombosispotential

Hazenlab
Lusis lab

TMAOanddisease

Wangetal,Nature2011
TangetalNEJM2013
LeveretalPLOSOne2014
Trseid M,JInt Med2015
Schugar er al,C2015
Ufnal M,Nutrition2015

Questions:
Whathumanassociatedgutbacteriausecholine?
WhichconvertcholinetoTMA?
Howdomicrobialcholinemetabolismand
TMA/TMAOproductionaffecthostbiology?

Identifyinghumangutisolatesthatgenerate
TMAfromcholine

Screen~100sequencedhumangutisolatesrepresenting91
differentspecies(37genera)fortheircapacitytoconvert

cholineintoTMA
6.0E+07

PeakArea

5.0E+07

Collect
supernatants

4.0E+07
3.0E+07
2.0E+07
1.0E+07
0.0E+00

Incubatehumangutisolates
withlabeledcholine[choline
chloride(trimethyld9)]inrich
medium(anaerobicconditions)

Measure
choline/TMA
(UHPLCMS)

OnlyTMAproducingbacteriaconsumesignificantlevelsofcholine

BacterialCholineutilization/TMAproducingpathway

Balskus lab

DeletionofCholineTMAlyase (cutC)abolishesTMA
productionbyE.coliMS2001
Escherichia coli MS 200-1
20

E. coli strain MS 200-1

Type strain in the HMP
Isolated from the ileum
Patient with normal histology

D9-TMA (mM)

15

10

Blank WT cutC

Balskus lab

Questions:
Whathumangutspeciesareabletofermentcholine
toTMA?

Howdoesmicrobialcholinemetabolism/TMA
productionaffecthostbiology?

Usinggnotobiotics tostudyTMAproducing
pathwayinvivo

Photo by Bryce Richter

Usinggnotobiotics tostudyTMAproducing
pathwayinvivo
Plasmalevels
60

80

****
M Choline

M TMAO

60
40

40

20

20

Colonizationwith
Corecommunity+
E.coliwt

Colonizationwith
Corecommunity+
E.coliwt vs.cutC

CNUC
-

CC

CUC+

CC

CNUC
-

CC

CC
CC
Corecommunity:Bacteroides caccae, Bacteroides ovatus,
Bacteroides thetaiotaomicron, Eubacterium rectale,and
Collinsella aerofaciens

CCCC+

CUC+

CC

MicrobialTMAproductionreduceslevelsofonecarbon
metabolismintermediatesinplasmaandliver
Protein

AMP

IMP

Methionine Sulfoxide
Adenosine

Inosine

Dimethylglycine
(DMG)
FOLATE CYCLE
Betaine

**

Homocysteine
Serine

Xanthine

105
e

Kynurenine

li n

Uric Acid

ho

Cysteine

106

et
h

im

Glutathione

Choline

Hypoxanthine

ne
yl
gl
yc
M
et
M
in
hi
et
e
on
hi
on
in
e
i
Su ne
l fo
xi
de

Methionine

107

ai

S-adenosyl Methionine
(SAM)

108

et

METHYLATION

****

DNA

***

109

Lipid

MS Peak Area

RNA

CCCC+

Romano et al., In Preparation

TMAproductionsignificantlyaltersglobalDNA
methylation
4
P = 0.07

**
*

CCCC+

2
1
0
Li
ve
r
Li
ve
r
C
ol
on
C
ol
on
B
ra
in
B
ra
in
H
ea
rt
H
ea
rt

5-mC%

Romano et al., In Preparation

Linkingmicrobialgenefunctiontohostphysiology

Colonizationwith
Corecommunity+
E.coliwt vs.cutC

Inguinal Fat Pad (mg)/

Body Weight (g)

Mouse Weight (g)

25
20
15
10
5
-

CC

CC

40

C.

**

30

20

10

CC

CC

8 week(HFdiet)

D.

10000
8000
6000
4000
2000
0

40

*
Serum TG (mg/dL)

B.

30

age: 810 wks

Liver TG (g/mg protein)

A.

GF
C57BL/6 mice

CC-

CC+

30

20
10

CC-

CC+

Cholinesupplementationiscriticalduringpregnancy

At40monthsfoundthatthechildrenwhohadreceived
cholinesupplements inutero andafterbirthhadfewer
attentionproblemsandlesssocialwithdrawal(AmJof
PsychiatryMay2016).

Parentalmicrobialcholineconsumption
affectsmethylationinbrainofneonates
Neonates(n=11)
DNAmethylation

A.

C.

175
150

2.5

60

*
2.0

75
20
15

40

***
20

10

1.5
1.0

0.5

5
0

5-mC%

100

M Choline

M TMAO

125

CNUC
CUC

CNUC

CUC

CNUC

CUC

0.0

Brain

Youngadults
Behavior
Percent of Marbles Burried

Mother(plasma)

Liver

****

100
75
50
25
0

CC-

CC+

Summary
Microbialcholineutilizationpathwayiswidely/discontinuously
distributedamonggutbacteria(impossibletopredictby16S).
MicrobialcholinedependentTMAproducingpathwayresultsin
lowerbioavailabilityofcholine(anddownstreammetaboliteskeyfor
CH3donation),lowerDNAmethylationinmultipletissues,
increasedadiposityandTGand(possibly)alteredbehavior.
Microbialcholineutilizationpathwaymaylimitcholineavailability
duringpregnancyandaffectbraindevelopment.
Currentlydietaryrecommendationsdonotconsiderinterpersonal
differencesinabundanceofcholineconsumingTMAproducing
species

Challengesinthefield
1000sofmicrobialmetabolites:whichonesare
biologicallyrelevant?Howdoweprioritizetheir
study?
Howdowelinkspecificmicrobialmetabolic
activitieswithhostbiology?
Lackofgenetictoolstomodifycommensals.

Welfareissues
Accesstognotobiotic resources(especiallyof
geneticmousemodels)wouldhelpreducethe
numberanimalsproducedbutnotusedby
individualinvestigators.

Thankyou!

BobKerby

JuliaKreznar

KymRomanoNachoVivas

DanishKhan

LindsayTraeger Kazu Kasahara LaurenCallahanKimDillMcFarland AmySoeun

DanielAmadorNoguez (UWMadison)
AlanAttie (UWMadison)
KarlBroman(UWMadison)
EmilyBalskus (Harvard)
JakeLusis (UCLA)
PamelaHerd(UWMadison)

JohnDenu (UWMadison)
JoshCoon(UWMadison)
BarbaraBendlin (UWMadison)
LauraKiessling (UWMadison)

Funding:UWMadison,ICTR,NIDDK,USDA