Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host

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Washington University School of Medicine
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2006
Genomic and metabolic studies of the impact of
probiotics on a model gut symbiont and host
Justin L. Sonnenburg
Washington University School of Medicine in St. Louis
Christina T. Chen
Washington University School of Medicine in St. Louis
Jeffery I. Gordon
Washington University School of Medicine in St. Louis
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Sonnenburg, Justin L.; Chen, Christina T.; and Gordon, Jeffery I., ,"Genomic and metabolic studies of the impact of probiotics on a
model gut symbiont and host." PLoS Biology.4,12. 2213-2226. (2006).
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PLoS
BIOLOGY
Genomic and Metabolic Studies of the Impact
of Probiotics on a Model Gut Symbiont
and Host
Justin L. Sonnenburg, Christina T. L. Chen, Jeffrey I. Gordon*
CenterforGenomeSciences,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri,UnitedStatesofAmerica
Probioticsaredeliberatelyingestedpreparationsoflivebacterialspeciesthatconferhealthbenefitsonthehost.Many
of these species are associated with the fermentation of dairy products. Despite their increasing use, the molecular
details of the impact of various probiotic preparations on resident members of the gut microbiota and the host are
generallylacking.Toaddress thisissue,wecolonized germ-freemice withBacteroides thetaiotaomicron,aprominent
component of the adult human gut microbiota, and Bifidobacterium longum, a minor member but a commonly used
probiotic.Simultaneouswholegenometranscriptionalprofilingofbothbacterialspeciesintheirguthabitatandofthe
intestinal epithelium, combined with mass-spectrometric analysis of habitat-associated carbohydrates, revealed that
the presence of B. longum elicits an expansion in the diversity of polysaccharides targeted for degradation by B.
thetaiotaomicron(e.g.,mannose-andxylose-containingglycans),andinduceshostgenesinvolvedininnateimmunity.
Although the overall transcriptome expressed by B. thetaiotaomicron when it encounters B. longum in the cecum is
dependentuponthegeneticbackgroundofthemouse(asassessedbyamixedanalysisofvariance[ANOVA]modelof
co-colonization experiments performed in NMRI and C57BL/6J animals), B. thetaiotaomicron’s expanded capacity to
utilizepolysaccharides occurs independently of host genotype, and is also observed with a fermenteddairy product-
associatedstrain,Lactobacilluscasei.Thisgnotobioticmousemodelprovidesacontrolledcasestudyofhowaresident
symbiontandaprobioticspeciesadapttheirsubstrateutilizationinresponsetooneanother,andillustratesboththe
generality and specificity of the relationship between a host, a component of its microbiota, and intentionally
consumed microbial species.
Citation:SonnenburgJL,ChenCTL,GordonJI(2006)Genomicandmetabolicstudiesoftheimpactofprobioticsonamodelgutsymbiontandhost.PLoSBiol4(12):e413.
DOI:10.1371/journal.pbio.0040413
throughtheresidentmicrobiotaversusdirectsignalingtothe
Introduction
host,andtheimpactofhostgenotypeonprobioticresponses
Over the millennia, our species has evolved to cope with, remain open questions. The answers have important impli-
and perhaps benefit from, consuming microbes present in cations for designing and interpreting clinical trials that
decomposing food. Some have postulated that ingestion of explore the specific and general effects of different pro-
food-bornemicrobialspecieswascommoninthePleistocene biotics,andwhethertheirconsumptionshouldbedictatedby
diet of hominins practicing subterranean food storage [1,2]. the microbial ecology or other properties of the host,
Eventually these microbes were co-opted by humans to includingdiet andgenotype.
transformfoodsviafermentation[3].Duringthelastcentury, The task of determining the mechanism of action of
technological improvements, such as refrigeration and probioticsinhumansisdaunting.Ourintestinescontain10–
pasteurization, have resulted in dramatic decreases in 100 trillion microbes. A recent comprehensive 16S rRNA
consumption of food-borne microbes in many countries. sequence-basedenumerationhasprovideddetailedsnapshots
Thissanitizationhascoincidedwithincreasedprevalenceofa of the composition of this community in a few healthy
number of disorders, both within and outside of the gastro- individuals [8]. Our 21st century microbiota contains mem-
intestinal tract, including inflammatory bowel disease and
atopic states (e.g., asthma and food allergies). Although links
AcademicEditor:JonathanEisen,UniversityofCaliforniaDavisGenomeCenter,
between certain ‘‘western’’ diseases and decreased exposure UnitedStatesofAmerica
tomicrobesarebeingconsideredatthepresenttime(e.g.,the ReceivedJune28,2006;AcceptedSeptember25,2006;PublishedNovember28,
hygiene hypothesis in the case of atopic disorders) [4], the 2006
positive health effects of consuming fermented foods were DOI:10.1371/journal.pbio.0040413
notedmorethan100yagowhenElieMetchnikoffcorrelated
Copyright: (cid:2)2006 Sonnenburg etal.Thisisanopen-accessarticledistributed
theiringestionwithincreasedlifespaninBulgarianpeasants under the terms of the Creative Commons Attribution License, which permits
[5]. More recently, the effects of consuming live microbial unrestricted use, distribution, and reproduction in any medium, provided the
originalauthorandsourcearecredited.
supplements with presumptive health benefits on human
physiologyhavebecomethesubjectofvariousclinicalstudies Abbreviations:ANOVA,analysisofvariance;CAZy,CarbohydrateActiveenZymes
database; CFU, colony forming units; COG, cluster of orthologous groups; FDR,
(reviewedin[6,7]).However,theimpactofthese‘‘probiotic’’ falsediscoveryrate;GC-MS,gaschromatography–massspectrometry;LCM,laser
speciesonthecompositionandfunctioningofthehumangut capturemicrodissection;qRT-PCR,quantitativereversetranscriptasePCR
microbiota, the degree to which their effects are mediated *Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected]
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Probiotic-Symbiont-HostInteractions
bersofatleastnineofthe70describeddivisionsofBacteria bacteria was determined in the cecum of each mouse at the
[9], although it is dominated by just two: the Firmicutes and timeoftheirsacrifice.Thisguthabitatwaschosenasthesite
the Bacteroidetes. One methanogenic archaeon, Methanobre- for our analyses because it is a readily defined anatomic
vibacter smithii, which belongs to the Euryarchaea, is very structure, interposed between the distal small intestine and
prominent,comprisingupto10%ofmicrobesinthecolonic colon, that normally houses a very dense and diverse
microbiota [10]. Variations in the microbial composition of microbiota (.1012 organisms/ml luminal contents; seven
the gut exist between individuals, primarily at the level of divisions of bacteria in a recent comprehensive 16S rRNA
speciesandsubspecies[8].Moreover,withinagivenintestinal gene sequence-based survey) [16]. The cecal microbiota is
habitat (address), some microbial members appear to be sustained, at least in part, by its ability to access large
entrenched‘‘residents’’(autochthonouscomponents),where- quantitiesofplant-derivedpolysaccharidesthataredelivered
as others act more like hitchhikers (allochthonous members) from the small intestine in an undigested state because the
that pass through together with food, water, and other mouse(andhuman) hostlackstherequisiteglycoside hydro-
components ofthe environment[11]. lases to break them down (e.g., xylans, arabinogalactans, and
These considerations emphasize the importance of creat- polygalacturonates/pectins) [17,18].
ing simplified and defined model systems for studying the B. thetaiotaomicron and B. longum colonized the cecum in a
functional properties and operating principles of our gut highlyreproduciblefashion.Moreover,microbialdensitywas
microbial communities, and the consequences of adding not significantly different for either species when they were
probiotics. In this report, we colonize germ-free mice with introduced alone (1010–1011 CFU per milliliter of cecal
Bacteroides thetaiotaomicron with or without Bifidobacterium contents;p.0.05accordingtoStudentt-test).Furthermore,
longum. B. thetaiotaomicron was chosen because it is a theco-colonization(bi-association)experimentrevealedthat
prominentsaccharolyticmemberofourdistalgutmicrobiota thetwobacterialspeciescanco-existinthececumwithouta
(6%ofallbacteriaintheenumerationstudydescribedabove) statistically significant impact on one another’s population
[8], that possesses the largest arsenal of glycoside hydrolases size(unpublished data).
and polysaccharide lyases among sequenced human gut To gain a genome-wide view of the effects of co-
symbionts [12]. Thus, its highly evolved ‘‘glycobiome’’ helps colonization on each organism’s transcriptome, RNA was
us to process otherwise indigestible glycans in our diet. B. isolated from the cecal contents of each animal in each
longum is a normal, albeit less prominent member of our treatment group. The resulting RNA preparations were
microbiota [8,13] that belongs to the Actinobacteria and is highly enriched for microbial RNA (.90% as judged by
commonly used as a probiotic [14]. Since both bacterial quantitative reverse transcriptase PCR [qRT-PCR] assays of
genomes have been sequenced, we were able to use a B. 16S and 18S rRNAs) [17]. Transcripts expressed in B.
thetaiotaomicron/B. longum ‘‘community’’ GeneChip to simulta- thetaiotaomicron and B. longum were profiled using a custom
neously examine how each organism affects the other’s ‘‘community’’GeneChip(Affymetrix,SantaClara,California,
transcriptome in a specified intestinal habitat of a host United States) that contains oligonucleotides representing
consuming a consistent and defined diet. Using a combina- 4,719 of B. thetaiotaomicron’s 4,779 predicted chromosomal
tion of in silico reconstructions of microbial metabolism protein-coding genes (98.7%) [17] and 1,722 of B. longum’s
based on these transcriptional profiles, mass spectroscopic 1,730 genes (99.5%). Each known or predicted gene in each
analysis of glycan utilization, and whole genome transcrip- genome was represented by an average of 13 perfect match
tional profiling of laser capture microdissected intestinal and control mismatch 25-mer ‘‘probe pairs’’ (together
epitheliumfromgerm-freeandcolonizedmice,wewereable designated as a gene’s ‘‘probe set’’) (Table S1). Probe pairs
to ascertain how a resident symbiont and probiotic species were designed to minimize cross-reactivity between genes
affect one another’s carbohydrate substrate range and host containedwithinandbetweenthetwobacterialgenomes:this
biology. The generality of our findings was explored by was confirmed experimentally by hybridizing the GeneChips
testing the effect of two different host genetic backgrounds tocDNAtargetspreparedfromeachoffivececalRNAsfrom
onthebehaviorofthismodelbi-componentmicrobiota,and one or the other group of mice colonized with a single
bycomparingtheresponsesofB.thetaiotaomicrontotwoother bacterialspecies(‘‘mono-associated’’animals).Whenthefive
probiotic strains added to a variety of fermented milks: B. thetaiotaomicron mono-association–derived cDNA targets
Bifidobacterium animalis and Lactobacillus casei. werehybridizedtotheB.thetaiotaomicron/B.longumGeneChip,
a‘‘present’’call(definedbyGeneChipsoftware)wasobtained
fromanaverageof67.7%oftheB.thetaiotaomicronprobesets,
Results/Discussion
butfromonly4.3%ofB.longumprobesets.Similarly,cDNAs
Bi-associationofGerm-FreeMicewithB.thetaiotaomicron generatedfromthececalRNAsofB.longummono-associated
and B. longum mice produced ‘‘present’’ calls for 63.2% of B. longum probe
Three groups of 7-wk-old, germ-free male mice belonging setsversus 5.6%ofB. thetaiotaomicron probesets.B. thetaiotao-
to the NMRI inbred strain and maintained on a standard micronprobesetscalled‘‘present’’inatleastthreeofthefive
polysaccharide-rich chow diet, were colonized with a single GeneChipshybridizedwithB.longumtargets(153probesets)
gavage of 108 colony forming units (CFU) of either B. were designated as cross-reacting, and eliminated from
thetaiotaomicron (ATCC 29148; isolated from the fecal micro- further analysis using an electronic probe mask. The
biota of a healthy adult human) [15], B. longum (NCC2705; reciprocal analysis resulted in electronic masking of 84
recoveredfromaninfant)[14],orbothbacterialspecies(n¼5 cross-reactingB. longumprobe sets.
animals/group). Mice were sacrificed 10 d later, a period of ThefiveGeneChipdatasetsproducedfromthegroupofco-
timesufficientfortheintestinalepitheliumanditsoverlying colonized animals were then referenced to five GeneChip
mucuslayertoturnoverseveraltimes.Thenumberofviable datasets from the B. thetaiotaomicron mono-associated group,
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Figure1.ImpactofCo-colonizationonB.thetaiotaomicronandB.longumGlycosideHydrolaseandPolysaccharideLyaseGeneExpression
(A) and (B) Bacterial glycoside hydrolase and polysaccharide lyase genes (rows) exhibiting differential expression in mono-associated versus co-
colonizedmice.Colorsindicatethedeviationofagene’ssignalabove(red)andbelow(green)itsmeanexpressionvalueacrossalltensamples.Thirty-
three B. thetaiotaomicron enzymes and seven B. longum enzymes meet the selection criteria for differential expression described in Materials and
Methods(FDR,0.01).Fold-differencesinaverageexpressionlevelsbetweenthetwocolonizationstatesareindicatedinparenthesisforeachgene.
(C)and(D)Averageaggregatesignalintensityinthetwocolonizationstatesforthesevengroupsofglycosidehydrolases/polysaccharidelyasesthatare
commontoB.thetaiotaomicron(C)andB.longum(D).Thenumberofenzymesineachgroupisindicatedinparenthesis.Valuesobtainedfrommono-
associatedmiceareshownwithblackbars,andfromco-colonizedmicewithwhitebars.SeeTablesS14andS15foralistoftheB.thetaiotaomicronand
B.longumgenes,respectively.SeeMaterialsandMethodsforadescriptionofhow‘‘%maximumaggregatesignal’’wascalculated.Meanvalues6
standarderror(S.E.)offivesamplesareshown:anasterisk(*)indicatesp,0.05,anddoubleasterisks(**)indicatep,0.001accordingtoStudentt-test.
DOI:10.1371/journal.pbio.0040413.g001
yieldingacohortofB.thetaiotaomicrongenesshowingchanged involved liberating oligo- and monosaccharides from poly-
expressionasafunctionofcolonizationstate.Atotalof681B. saccharides (classified in Table S3 according to the nomen-
thetaiotaomicron genes were designated as differentially ex- clature employed by the Carbohydrate Active enZymes
pressed upon co-colonization using selection criteria that database [CAZy; http://afmb.cnrs-mrs.fr/CAZY/]). The pres-
maintainedafalsediscoveryrate(FDR)oflessthan0.01:359 ence of B. longum produced statistically significant (FDR ,
(53%) of these genes were expressed at higher levels in the 0.01)changesinexpressionof31B.thetaiotaomicron glycoside
presence of B. longum, whereas 322 were expressed at lower
hydrolases and two polysaccharide lyases when compared to
levels.Functionalcategorizationofthedifferentiallyexpressed
B. thetaiotaomicron residing in the ceca of mono-associated
genesusingclustersoforthologousgroups(COGs)revealeda
mice: the vast majority of these genes (29/33) were up-
‘‘metabolism’’-biased response to co-colonization, including
regulated(Figure1A).Thesubstratespecificitiesrepresented
statisticallysignificantover-representationofup-regulatedB.
inthe29up-regulatedglycosidehydrolases(20CAZyfamilies;
thetaiotaomicrongenesinvolvedin‘‘carbohydratetransportand
Figure 1A and Table S3) suggests that B. thetaiotaomicron
metabolism’’ and ‘‘energy production and conversion’’ (p ,
undergoesanexpansioninthediversityofpolysaccharidesit
0.01basedonabinomialdistributiontest;FigureS1AandS1B;
targetsfordegradation wheninthe presence of B. longum.
seeTableS2foralistofgenessortedbyCOGs).
B.thetaiotaomicroncontains163paralogsoftwocomponents
Co-colonization Prompts B. thetaiotaomicron to Expand of a well-characterized starch utilization system (Sus): 106
Expression of Genes Involved in the Acquisition and paralogs of the gene encoding SusC, a TonB-dependent, b-
Breakdown of Polysaccharides barrel-type outer membrane porin that participates in
The B. thetaiotaomicron genome encodes 226 known or energy-dependent transport of malto-oligosaccharides into
predicted glycoside hydrolases and 15 polysaccharide lyases theperiplasmicspace,and57paralogsofthegeneencoding
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SusD, a secreted starch-binding protein with an N-terminal
lipidtailthatallowsittoassociatewiththeoutermembrane
[12]. These paralogs likely confer the ability to bind and
import diverse carbohydrate substrates encountered in the
gut[19].Expressionof31B.thetaiotaomicronSusC/Dgeneswas
significantly altered (1.4–6.7-fold) by B. longum: nearly two
thirds (19 of 31) display higher expression with co-coloniza-
tion;12ofthesearepresentinthegenomeasadjacentpairs
ofSusC/Dparalogs(Figure S2).
Sixty-one B. longum genes showed significantly changed
expression upon co-colonization with B. thetaiotaomicron (26
up-regulated, 35 down-regulated; FDR , 0.01). Together,
these genes represent a much smaller proportion of the B.
longum genome than does the cohort of B. thetaiotaomicron
genesaffectedbyB.longum(3.5%versus14.2%,respectively).
Comparison of B. longum genes up- versus down-regulated
upon co-colonization revealed that the only significant
difference in COG representation involved down-regulated
genes associated with ‘‘carbohydrate transport and metabo-
lism’’ (p , 0.01; Figure S3; Table S4). Moreover, all down-
regulatedgenesinthisCOGarelocatedinoneregionofthe
B. longum genome (BL1327–BL1337) that is dedicated to the
degradation, import, and metabolism of mannosylated
glycans.Allfourglycosidehydrolaseswithreducedexpression
arepositioned withinthis genecluster(see below).
Co-colonizationResultsinDifferentialExpressionofGenes
Encoding Mannosidases and Related Enzymes
Mannose is highly coveted as a source of energy for many
saccharolytic bacteria: it is a ubiquitous monosaccharide
foundinbothplantandanimalglycans,andiseasilyshunted
intotheglycolyticpathwayviaanisomerizationofmannose-
6-phosphate to fructose-6-phosphate. The desirability of
mannose as a substrate is evidenced by the fact that the B.
thetaiotaomicron genome dedicates over 15% of its repertoire
of glycoside hydrolases to the liberation of glycosidically
linked mannose (30 mannosidases; ten mannanases). B.
thetaiotaomicron’s 40 mannose-liberating enzymes contrasts
withthe three mannosidases presentinB. longum.
ThreeofB.thetaiotaomicron’smannosidasesaremorehighly
expressedwithbi-association(BT2199,BT3530,andBT4093;
1.9-,2.3-,and1.7-fold,respectively),whereastwoofB.longum’s
mannosidasesshowreducedexpression(BL1327andBT1329;
2.8-and6.7-fold,respectively)(Figure1).ThesetwoB.longum
a-mannosidasesarepositionedinthegeneclusterreferredto
above that contains two linked permease genes (BL1331 and
Figure2.CarbohydrateUtilizationbyB.thetaiotaomicronandB.longum BL1332; down-regulated 7.4- and 10.1-fold, respectively) and
inthePresenceorAbsenceofOneAnother two other glycoside hydrolase activities (BL1333 [substrate
(A)and(B)Averageexpressionlevelsforfivececalsamplesareshownfor specificity unknown]; BL1335 [endo-b-N-acetylglucosamini-
genesencodingenzymesthatshunttheindicatedmonosaccharidesinto dase]; down-regulated 8.8- and 14.1-fold, respectively; Figure
the glycolytic or pentose phosphate pathways. Values obtained from
1Band1D).Expression ofnineofthe tengenesencoding B.
mono-associatedmiceareshownwithblackbars,andfromco-colonized
mice with white bars. Significant differences in expression levels were longumenzymesthatdelivermonosaccharidestotheglycolytic
determinedusingStudentt-test;doubleasterisks(**)indicatep,0.01, and pentose phosphate pathways is unchanged by the
andtripleasterisks(***)indicatep,0.001.
presence of B. thetaiotaomicron (Figure 2). The lone exception
(C)GC-MSdeterminationofmonosaccharidecompositioninthececaof
germ-free (GF), B. thetaiotaomicron mono-associated (Bt), B. longum is another component of the gene cluster, a mannose-6-
mono-associated(Bl),orB.thetaiotaomicron/B.longumco-colonized(Bt/ phosphate isomerase (BL1337) that shows 4.2-fold lower
Bl) mice. Mean values 6 S.E. of biological triplicates are plotted. expressionwithco-colonization(p,0.001;Figure2B).Thus,
Significant changes in the abundance of a monosaccharide were
B. thetaiotaomicron and B. longum exhibit changes in their
identified using a one-way ANOVA followed by a Tukey post test; a
singleasterisk(*)indicatesp,0.05,doubleasterisks(**)indicatep, transcriptomethatsuggestdifferencesineithertheextentor
0.01,andtripleasterisks(***)indicatep,0.001. efficiencyofmannosideutilizationoccurduringco-coloniza-
DOI:10.1371/journal.pbio.0040413.g002
tioncomparedtowheneitherspeciesisthesoleoccupantof
the cecum. The apparent decreased catabolism of mannose
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up-regulation of xylose-degrading enzymes by both B.
by B. longum is consistent with its decreased expression of
thetaiotaomicron and B. longum, xylose was depleted to a
mannosidases. Nonetheless, despite the relative disparity in
significant degree when both bacterial species were present
mannoside-degrading enzymes between the two organisms
inthe cecalhabitat (Figure2C).
(40inB.thetaiotaomicronversusthreeinB.longum),wemustbe
To determine if depletion of xylose in the co-colonized
cautiousbeforeconcludingthatB.longumisarelativelypoor
state could be attributed to differential usage by either
competitorformannosylatedglycans.Forexample,intheco-
bacterial species, we performed in silico metabolic recon-
colonized cecum, other substrates may be available that B.
structionstovisualizeexpressionofgenesencodingenzymes
longumprefersovermannose,resultingindown-regulationof
that shunt monosaccharides liberated by the glycoside
its mannosidase genes, increases in mannose levels, and
hydrolases/polysaccharide lyases into either the glycolytic or
subsequentup-regulationofB.thetaiotaomicron’smannosidase
pentosephosphatepathways.Withco-colonization,B.thetaio-
genes. Alternatively, co-colonization could result in changes
taomicronincreasesexpressionofonlyoneofitstengenesthat
intheenvironment,suchaspHorosmolarity,thatrenderB.
can be assigned to these pathways: a xylulokinase (BT0792)
longum’s mannosidases more stable, or more efficient, result-
that catalyzes a key step in D-xylose catabolism (3.1-fold
ingin theirsubsequent down-regulation.
increase with co-colonization, p , 0.01; Figure 2A). In
B. thetaiotaomicron Up-Regulates Machinery Involved in contrast, neither of the two enzymes assigned to xylose-
specific metabolism in B. longum—a xylose isomerase
Xyloside Degradation and Xylose Catabolism in the
(BL1704) and xylulokinase (BL1709)—showed differences in
Presence of B. longum
theirexpressionbetween thetwo colonizationstates.
In contrast to their disparate responses to mannose-
The observed up-regulation of genes involved in both
containing glycans, expression of enzymes dedicated to
liberation and catabolism of xylose by B. thetaiotaomicron
degradationofxylose-containingglycansisenhancedinboth
indicates that it is responsible for at least some of the
bacterial species when they co-exist in the cecum. B.
depletion that occurs during co-colonization. The up-
thetaiotaomicron increases expression of four xyloside-degrad-
regulation of xyloside-degrading machinery by B. thetaiotao-
ing glycoside hydrolases: an endo-xylanase predicted to be
micronandB.longummaybearesultofbothbacterialspecies
secreted (BT3662; 1.8-fold), an exo-xylanase (BT3663; 2.4-
turning to an available, but less-coveted substrate that is
fold),andtwosecretedxylosidases(BT4095andBT4185;1.8-
neglected in mono-association, but accessed when their
and2.3-fold, respectively) (FDR ,0.01; Figure 1Aand1C).
preferred substrates are depleted in the more competitive
Two of the three glycoside hydrolases that B. longum up-
environmentofco-colonization.Althoughlessparsimonious,
regulates are secreted xylanases that occur in a putative
an alternative possibility is that each bacterial species, when
operon:BL1543,anendo-xylanase(2.3-fold),andBL1544,an
present alone in the cecum, possesses a complement of
exo-xylanase (1.8-fold), both of which contain sortase motifs
glycosidehydrolasesthatisinsufficientforefficientdegrada-
[20], indicating that they are located on the surface of this
tion of available xylose-containing glycans. However, when
Gram-positive bacterium (Figure 1B). Xylose-liberating hy-
their repertoires of glycoside hydrolases are combined,
drolasesaretheonlygroupofB.longumglycosidehydrolases
complementary enzymes activities allow the two bacterial
that show significantly higher expression when B. thetaiotao- speciestoparticipateina‘‘synergistic’’harvestofthisclassof
micronis present(p, 0.001;Figure 1D). glycans, a phenomenon observed in other mixed microbial
communities that degrade cellulose [21,22]. A third, formal
VerificationofBacterialGlycanUtilizationBehaviorswithin
possibility is that in mono-associated mice, each species
the Ceca of Colonized Mice
restrains expression of these genes (despite the presence of
The coincident regulation of glycoside-degrading machi-
substrate), but with co-colonization, in response to some
nery by B. thetaiotaomicron and B. longum suggests that when
signal from the other species, they up-regulate the genes to
both components of this model community are present in
thecombinedbenefitofbothspecies.Thisiscooperation(the
their cecal habitat, utilization of a subset of available
sacrificeofindividualfitnessforthefitnessofthegroup)and
polysaccharide substrates changes. To examine this notion
is unlikely to be taking place because of the inevitability of
further, gas chromatography–mass spectrometric (GC-MS)
‘‘cheating’’ [23].
analysis was employed to establish which monosaccharide
componentsofpolysaccharidesweresignificantlydepletedin B. longum Induces an Expansion of B. thetaiotaomicron’s
each colonization state. When compared to germ-free Substrate Range Independent of Host Genotype
controls, mono-association with B. thetaiotaomicron produced MicebelongingtotheNMRIinbredstrainwereselectedas
apreferentialdepletionofthethreemostprominenthexoses the initial host for our experiments because of the ease of
(glucose, galactose, and mannose), whereas there were no rearingtheminagerm-freestate.Todeterminethegeneral-
significant changes in the most prominent pentoses (arabi- ity of B. thetaiotaomicron’s response to co-colonization with B.
noseandxylose).B.longummono-associationalsoresultedin longum, we also performed a series of experiments, identical
significant depletion of galactose, glucose, and mannose: to those described above (including use of the same
however, unlike B. thetaiotaomicron mono-association, the autoclaved chow), but in animals belonging to the C57BL/6J
pentose arabinose was also depleted (Figure 2C). This latter (B6)inbredstrain.Tenday–longmono-andbi-associationsof
observation is consistent with B. longum’s considerable germ-free 6–8-wk-old male B6 mice produced levels of
arabinoside-degrading capability (11 arabinosidases, ex- colonization similar to that documented in their NMRI
ceeded only by glucosidases as the most highly represented counterparts (p.0.05;unpublished data).
class of glycoside hydrolases in its genome; Table S3). Weidentifiedgeneswhoseexpressionchangessignificantly
Consistent with the documented co-colonization–associated asafunctionofhostgenotype,byapplyingatwo-stagemixed
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analysis of variance (ANOVA) model to our 20 GeneChip hydrolase family 20) and a-fucosidases (glycoside hydrolase
datasets (i.e., five NMRI B. thetaiotaomicron mono-associated, family 95), the enzymes that liberate monosaccharides
fiveNMRIB.thetaiotaomicron/B.longumco-colonized,fiveB6B. commonly found in host mucus glycans, which are coveted
thetaiotaomicron mono-associated, and five B6 B. thetaiotaomi- substrates of B. thetaiotaomicron in vivo [17]. Together, these
cron/B. longum co-colonized). The ANOVA model identified a data indicate that B. longum induces a shift in B. thetaiotaomi-
totalof2,369B.thetaiotaomicrongenesthatweredifferentially cron substrate preferences away from B6 and NMRI mucus
expressed in the two host mouse strains independent of glycans and towards arabinose- and xylose-containing plant
colonizationstate(50.2%ofthe4,719genessurveyed;FDR, polysaccharides in the host diet. This change in glycan
0.01; see Figure S4, plus Table S5 for a full list of genes foraging behavior could also be responsible for altering the
identified by the ANOVA model and the associated p-values functional relationships between B. thetaiotaomicron and its
and q-values of each gene, as well as Tables S6 and S7, plus host(see below).
Figure S5 for a COG-based functional categorization of the
Expansion of the Carbohydrate Substrate Range of B.
1,168 B. thetaiotaomicron genes that exhibit higher expression
thetaiotaomicron Can Be Induced by Multiple Probiotic
in NMRI versus B6 mice, and the 1,201 genes that exhibit
higherexpression inB6versus NMRI). Species
Despite the large effect of host genotype on B. thetaiotao- Probioticbacterialspecies varyintheirabilitytoestablish
micron gene expression, the B. longum–induced expansion in residency within the human intestinal microbiota. Another
thecarbohydratesubstraterangeofB.thetaiotaomicronoccurs Bifidobacteriumspecies, B.animalis, andL.casei,amemberofa
independently of host genotype. The ANOVA model identi- different bacterial division (the Firmicutes), are common
fied1,806geneswhoseexpressionissignificantlychangedasa ingredientsoffermentedmilkproducts[24,25].Bothspecies,
function of colonization state (mono-association or co- althoughlesswelladaptedthanB.thetaiotaomicronorB.longum
colonization; Figure S6A). Comparison ofthe 801 genes that to take up residency in the human gut, can remain viable
areexpressedathigherlevelsduringco-colonization,withthe during their transit through our gastrointestinal tracts [26–
1,005 genes expressed at reduced levels (Tables S8 and S9), 28].
revealed that ‘‘carbohydrate transport and metabolism’’ is TodeterminethespecificityofB.thetaiotaomicron’sresponse
oneofthemostsignificantlyover-representedCOGs(15%of to B. longum, we performed expression profiling of B.
the response, p , 10(cid:2)10; Figure S6B). Nearly twice as many thetaiotaomicron in the presence of either B. animalis DN-173
glycoside hydrolases/polysaccharide lyases exhibit higher 010 (Danone Vitapole, Jouy-en-Josas, France) or L. casei DN-
expression during co-colonization compared to mono-asso- 114001(DanoneVitapole,CNCMnumber,I-1518).Toensure
ciation (61 up-regulated versus 33 down-regulated; Figure continuous interaction between B. thetaiotaomicron and these
S7A and S7B). Similarly, SusC/D paralogs whose expression two frequently ingested strains, B6 mice were mono-
changesasafunctionofcolonizationstateshowastrongbias associated with each of the three species, or were first given
toward up-regulation with co-colonization (43 up-regulated B. thetaiotaomicron followed by B. animalis or L. casei every 3 d
versus 22 down-regulated; Figure S7A and S7B). In several overa10-dspan(fivemice/treatmentgroup).Importantly,at
instances, co-regulated SusC/D paralogs and glycoside hydro- thetimeofsacrifice,thelevelofcecalcolonizationwassimilar
lases/polysaccharide lyases are present together in putative inmono-orbi-associatedanimalsforallbacterialspecies(83
operons, a genomic feature that would allow B. thetaiotaomi- 1010 to331012;p.0.1).
crontoefficientlycoordinateglycanbindingandimportwith Whole genome transcriptional profiling of cecal popula-
glycan degradation (Figure S7Aand S7B). tions of B. thetaiotaomicron revealed that the impact of B.
A comparison of the glycoside hydrolase/polysaccharide animalis and L. casei on the B. thetaiotaomicron transcriptome
lyasefamiliesrepresentedinthegroupsofgenesthatincrease was modest when compared with the response elicited by B.
ordecreasetheirexpressionduringco-colonizationirrespec- longum. Fifty-eight genes showed increased levels of expres-
tive of host genotype further substantiated that B. thetaiotao- sion in the presence of B. animalis and 53 increased in the
micron’s polysaccharide substrate preferences change in the presence of L. casei (FDR , 0.05; Figure 3A; see Tables S10
presence of B. longum. The 61 polysaccharide-degrading and S11 for gene lists). Moreover, the principal COG
enzymesthatshowincreasedexpressionfallinto27different categories representing the response to B. animalis were
glycoside hydrolase/polysaccharide lyase families (44 families ‘‘transcription’’ and ‘‘replication’’ (Figure 3B), whereas the
are represented in the entire B. thetaiotaomicron genome), twoCOGcategoriesthatdominatetheresponsetoL.caseiare
consistent with a broad diversification of glycan utilization. ‘‘carbohydratetransportandmetabolism’’and‘‘inorganicion
The most highly represented glycoside hydrolase family, 43 transport and metabolism.’’ These latter two categories are
(11members),providesevidencethatsubstratescontainingb- composed largely of glycoside hydrolase genes and SusC/D
xylose and a-arabinose become a major focus of B. paralogs, respectively, and represent over 60% of B.
thetaiotaomicron when B. longum is present (Figure S7C). thetaiotaomicron’s functionally categorized response (Figure
Alpha-mannosidases and b-galactosidases (glycoside hydro- 3B). The enrichment of glycoside hydrolases (11 genes; four
lase families 2 and 92, respectively) are also abundantly galactosidases,threehexoaminidases,twoarabinosidases,one
represented (Figure S7C). amylase,andonesialidase)andSusC/Dparalogs(13genes)in
The 33 glycan-degrading enzymes with decreased expres- the dataset of 53 B. thetaiotaomicron genes up-regulated by L.
sionalsodisplaydiverseactivities:theyrepresent18glycoside casei (Figure 3C) is functionally similar to the B. longum–
hydrolase/polysaccharide lyase families, and show a distribu- inducedresponse.ThisstandsincontrasttoB.animalis,which
tion dissimilar to the up-regulated enzymes (Figure S7C). does not significantly impact expression of any glycoside
Highly represented activities distinct from those present in hydrolaseorSusC/DparalogintheB.thetaiotaomicrongenome.
theup-regulatedgroupincludeb-hexosaminidases(glycoside The L. casei up-regulation of three B. thetaiotaomicron
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Probiotic-Symbiont-HostInteractions
Figure3.ChangesintheB.thetaiotaomicronTranscriptomeinResponsetoCo-colonizationwithB.animalisorL.casei
(A)B.thetaiotaomicrongenesthatshowsignificantlyincreasedexpressionlevelsuponco-colonizationwitheitherofthetwofermenteddairyproduct–
associatedstrainsareshown.Selectioncriteria:(1)a1.2-foldorgreaterdifferenceinexpressionusingthe90%confidenceboundofthefold-change;(2)
differenceinsignal(cid:3)25;(3)p-value(cid:4)0.025usingStudentt-test;(4)transcriptcalled‘‘present’’in66%ormoreoftheGeneChipsintheco-colonized
group;and(5)amedianFDRlessthan0.05basedon50permutatedcomparisons.
(B)COG-basedfunctionalclassificationofgenesidentifiedin(A).Thirty-fiveof53geneswithhigherexpressioninthepresenceofL.caseiand30of58
geneswithhigherexpressioninthepresenceofB.animaliscouldbeassignedtoCOGs.Functionalcategorieswithsignificantover-representationinthe
dataset of differentially expressed genes, compared to their representation in the B. thetaiotaomicron genome, were determined using a
hypergeometricdistribution;anasterisk(*)indicatesp,0.01.
(C) All B. thetaiotaomicron SusC/D paralogs and glycoside hydrolases/polysaccharide lyases that show differential expression between the three
colonizationstatesareup-regulatedinthepresenceofL.casei.Fold-differencesareindicatedinparenthesisandreflectaverageexpressionacrossthree
cecalsampleswhenB.thetaiotaomicronisco-colonizedwithL.casei,comparedtotheiraverageexpressioninthreececalsamplesobtainedfromB.
thetaiotaomicronmono-associatedanimals.
DOI:10.1371/journal.pbio.0040413.g003
glycosidehydrolasefamily20hexosaminidasescontrastswith up-regulation of three enzymes that target arabinose-con-
the down-regulation of this family’s hexosaminidases that taining polysaccharides (BT0360, endo-a-arabinosidase;
occurs in the presence of B. longum (Figure S7C). Two of the BT0368,a-arabinofuranosidase;andBT4668,arabinogalactan
induced hexosaminidases (BT0459 and BT0460) are present endo-b-galactosidase) suggests that accessing dietary plant-
in a putative operon containing an up-regulated a-sialidase derived substratesis also partofthe responseto L. casei.
andb-galactosidasededicatedtodegradingsialylatedglycans Insummary,comparisonofB.thetaiotaomicron’sresponseto
fromhostmucus(BT0455–BT0461;Figure3C).Inadditionto the three bacterial species revealed that they are able to
theexpandeddegradationofhostglycans,B.thetaiotaomicron’s induce distinctresponses inB. thetaiotaomicron, bothinterms
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of the magnitude of the response (i.e., the number of B. Togainabroaderperspectiveofthehostresponsestoeach
thetaiotaomicron genes that undergo significant changes in of the three colonization states, we categorized all up-
expression)andthefunctionofaffectedgenes.Thedisparity regulated host genes using Ingenuity Pathways Analysis
inidentityanddiversityofpolysaccharidesubstratestargeted software (http://www.ingenuity.com). A right-tailed Fisher
by B. thetaiotaomicron in response to B. longum versus L. casei exact test was used to determine significantly over-repre-
suggests that the nature of the expansion, the resulting sented(p,0.05)functionalcategoriescontainingaminimum
impact on the intestinal nutrient environment, and ulti- of eight genes (Table S12). Gene interaction networks were
mately the effect on the host, are likely to show significant then generated for each colonization state using these up-
specificity with regardto themicro-organism consumed. regulated genes as ‘‘focus genes’’ among the thousands of
genes called ‘‘present’’ for each condition (B. thetaiotaomicron
Host Epithelial Response to B. thetaiotaomicron plus B.
mono-association, 10,795 genes; B. longum mono-association,
longum Is Characterized by a Synergistic Induction of
10,479 genes; and co-colonization, 12,138 genes). Unsuper-
Interferon-Responsive Genes visedgenerationofmolecularinteractionnetworks,basedon
The transcriptional responses of B. thetaiotaomicron and B. (1) focus genes, (2) genes called ‘‘present’’, and (3) unrepre-
longumtoco-colonization,andtheresultingtransformationin sented but highly connected genes was performed by the
the cecal nutrient environment raised the question of how Ingenuity Pathway Analysis software using a knowledge base
host epithelial responses differ between colonization states. that consists of more than 1,000,000 functional and physical
Therefore, we performed comprehensive transcriptional interactionsformorethan23,900mammaliangenes(includ-
profiling on cecal epithelia harvested from the same groups ing 8,200 for mouse). These networks reveal that the host
of NMRI mice that were used to profile bacterial gene response to B. thetaiotaomicron is centered on tumor necrosis
expression(i.e., B. thetaiotaomicron mono-associated; B. longum factor-a (TNF-a; Figure S8A), an LPS-responsive cytokine
mono-associated, and B. thetaiotaomicron/B. longum co-colon- producedbynaturalkiller(NK)andTcellsandmacrophages.
ized). A group of age-matched, untreated male germ-free This contrasts to the network generated by the B. longum
NMRI mice that were given the same batch of autoclaved responsewhichiscenteredontheTcell–producedcytokine,
chow served asadditional controls. interferon-c(IFN-c;Figure S8B).
Laser capture microdissection (LCM) was performed on The network generated from the co-colonization gene set
cecalcryosectionspreparedfromfourmice/treatmentgroup revealed that both TNF-a and IFN-c are central highly
(;5,000epithelialcellsrecovered/cecum).RNAwasprepared connected nodes (Figure S8C), suggesting that the cytokine
from epithelial cells harvested from the cecum of each milieu in the cecal epithelium of co-colonized mice shares
individual mouse in each treatment group. Equivalent characteristics witheach mono-associationstate.
amounts of RNA from each cecum were then pooled from Consistent with the Ingenuity Pathway Analysis network
each mouse per group, targets were generated by two construction,the groupofgenes showing highestexpression
independent amplifications of each pooled RNA sample, in the co-colonized state relative to germ-free controls
and each amplified RNA preparation was hybridized to a includes three members of the interferon-inducible 47kDa
separate Affymetrix mouse 430v2.0 GeneChip. GTPases (Igtp, 5.6-fold; Ifi47, 6.6-fold; and Iigp2, 3.7-fold) and
Expression profiles from each of the three colonization three members of the interferon-inducible p56 family (Ifit1,
states were referenced to datasets obtained from germ-free 12.3-fold; Ifit2, 16.0-fold; and Ifit3, 3.2-fold). The interferon-
controls to identify significantly up-regulated genes using stimulated gene-15 (Isg15), a ubiquitin family member
criteria described in Materials and Methods. These compar- implicated in mediating viral resistance [29,30], is induced
isons yielded 50 genes in the cecal epithelium of B. 17.1-fold with co-colonization (20.5-fold by qRT-PCR) com-
thetaiotaomicron mono-associated mice, 24 genes in B. longum paredto2.0-fold(2.1-foldbyqRT-PCR)and1.2-fold(1.8-fold
mono-associatedhosts,and52genesinco-colonizedanimals, by qRT-PCR) with B. thetaiotaomicron and B. longum mono-
whose expression was defined as significantly increased associations, respectively (Figure 4B and 4C). Two other
relative to germ-free epithelium. Comparison of genes up- interferon-responsive genes, Zbp-1, a Z-DNA binding adeno-
regulated in each colonization state revealed that the host sinedeaminase,andMuc4,amucincapableofinhibitingviral
responsetoco-colonizationwasdistinctfromtheresponseto capsid binding to target glycans [31] are up-regulated in all
mono-association with either organism (Figure 4A and 4B): colonization states but show highest expression with co-
i.e.,ofthe52genesidentifiedintheepithelialresponsetothe colonization(Figure 4B).
two-component B. thetaiotaomicron/B. longum model commun- In addition to inducing host cytokine-responsive genes, B.
ity, only 15 genes are shared with the response to B. longum appears to possess the capacity to regulate host
thetaiotaomicronalone,andonlysevengeneswiththeresponse antibacterial proteins. Reg3c (regenerating islet-derived-3c)
toB. longum. isaC-typelectinthatissecretedbyanepithelialcelllineage
SincetheseconclusionswerebasedonaGeneChipanalysis that plays an important role in innate immune responses.
ofpooledRNAs,weperformedfollow-upqRT-PCRassaysof Reg3cbindswithhighaffinitytopeptidoglycan,andexhibits
laser capture microdissected cecal epithelial RNAs isolated broad microbicidal activity against Gram-positive bacteria
from the individual animals used to prepare the pooled [32]. We found that although Reg3c is induced in mice
samples (n ¼ 4/colonization state). Reg3c and Isg15 were colonized with B. thetaiotaomicron alone (5.2-fold above germ
selectedforverificationbecausetheyhadexhibiteddifferent free; 3.6-fold by qRT-PCR), it is significantly down-regulated
expression profiles across the three colonization states. The when B. longum is present by itself (repressed 8.5-fold
qRT-PCR results established that GeneChip data obtained compared to germ free; 9.7-fold by qRT-PCR) (Figure 4B
from pooled RNA samples were representative of individual and4C).Pancreatitis-associatedprotein(Pap;Reg3b),another
animals withineachgroup (Figure 4C). memberoftheRegfamilythatalsocontainsalectindomain,
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Figure4.GeneExpressionChangesintheCecalEpitheliumInducedbyColonization
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