Beruflich Dokumente
Kultur Dokumente
UsingGnotobiotic Models
Biologicallysignificantmetabolitesproducedbythegut
microbiota:theiroriginsandfunctions
FedericoE.Rey
UniversityofWisconsinMadison
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).
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
D9-TMA (mM)
15
10
Blank WT cutC
Balskus lab
Questions:
Whathumangutspeciesareabletofermentcholine
toTMA?
Howdoesmicrobialcholinemetabolism/TMA
productionaffecthostbiology?
Usinggnotobiotics tostudyTMAproducing
pathwayinvivo
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+
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%
Linkingmicrobialgenefunctiontohostphysiology
Colonizationwith
Corecommunity+
E.coliwt vs.cutC
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
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
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