(12) Patent Application Publication (10) Pub. No.: US 2005/0239706A1 Backhed Et Al
US 2005O2397O6A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0239706A1 Backhed et al. (43) Pub. Date: Oct. 27, 2005
(54) MODULATION OF FIAF AND THE Related U.S. Application Data GASTRONTESTINAL MICROBOTAASA MEANS TO CONTROL ENERGY STORAGE (63) Continuation-in-part of application No. 10/432,819, INA SUBJECT filed on Oct. 31, 2003. (75) Inventors: Fredrik Backhed, St. Louis, MO (US); (60) Provisional application No. 60/591.313, filed on Jul. John Rawls, St. Louis, MO (US); 27, 2004. Justin Sonnenburg, St. Louis, MO (US); Lora V. Hooper, Coppell, TX Publication Classification (US); Jeffrey I. Gordon, St. Louis, MO (US) (51) Int. Cl...... A61K 38/17 (52) U.S. Cl...... 514/12 Correspondence Address: POLSINELL SHALTON WELTE (57) ABSTRACT SUELTHAUS PC. 700 W. 47TH STREET The invention provides compositions and methods to modu SUTE 1000 late fat Storage and weight loSS in a Subject. In certain KANSAS CITY, MO 64112-1802 (US) aspects of the invention, fat storage (adiposity) and weight (73) Assignee: Washington University in St. Louis loSS is modulated by altering the Subject's gastrointestinal microbiota population. In other aspects of the invention, fat (21) Appl. No.: 11/080,755 Storage and weight loSS is modulated by altering the amount of or the activity of the protein, fasting-induced adipocyte (22) Filed: Mar. 15, 2005 factor, in the Subject.
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Patent Application Publication Oct. 27, 2005 Sheet 1 of 41 US 2005/0239706A1 Fig. 1
Epithelium Mesenchyme
Before capture Before capture Patent Application Publication Oct. 27, 2005 Sheet 2 of 41 US 2005/0239706A1 Fig. 2
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lactase O OB OS OA O2 Golipase
angiogenin-3 O
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Patent Application Publication Oct. 27, 2005 Sheet 4 of 41 US 2005/0239706A1 Figure 4: Angiogenin-4 and -3 nucleotide sequence alignment
angiogeniu- l -gaacrracaceaasaccorsiclecassascacacagorasacriticariccaeill 2. t s angiogeniu-3 Cuscicarecscacisco Stecca'ssacACSAASCUAACACAccCCs Consensus agott adao Og aggadootgtotodaggagdao agotaged to to go to g 6. Bl 9. ill angiogenin- Sec.ScGSCCGCSA CAAccCAS analogerlin-3 TSGAGSAAGonggccaccTLSSAATCGEAAG start Casersus ttggaggaaagatggoopagotttggaatu stgttgaaagagats atgagtega st 2 lil s lsi augiogenil- s (CPS angiogenit-3 exact sect CC sease NCAGS-Ace ecCACNGCCF Cascal Coasess otttgttgttggtott tutg gttctg. ittgat oatge actatggotoag at l 2D 2. 2. angiogenin-s gaa-...--AGGTACGAAAAATTCCTACGICAGCACIAGATGCCAASCCAAAGGGCCGGGac angiogenin-3 AACTACASGAAAAAAccreacticaGCACAARSCCAGCCACCSCCGSGAE Consensus aggtao aaaattgot to aggagtatatgcaa.go Gala godggga 2. as s 2. 2B 2. anoriocrenit-4Ai is Cussless al 3. 32. 33 3. 35. angiogenil anogenin-5 a Salf Yu is a WAY is v Y WAY Wor Consensus aaoacotttat datg tado agaa aadata go oatatgttgga agaa gigaag 35 37 3. 3 4. angiogeniu-4. CCITATGGAGAAAACTTCAGAATAASCAATTCTCCCTCCAGATCACGACTTGEACGCAC angiogenin-3 ce accusetta seates Cease Consensus gettatag aaa.ott agaataagaattute tteoag to assactites augao s2. s t 45 3. angiogenia- ICAAGAGGGIGLXCCCLX3GCGTCGAIGCGSGIACCGAgCCTTTAAASATTCAGACATAT angiogen-3 Acciacoscoes seccuccius CAct Costases a gagggittgct giggoto gatga gtag gotstttaaagattittaga atatt El al SO 52 3. creat- graecceeaaga SCCEGGCGNPCCAcacca's Crack GE E-3 Gasco SGAEGSGCGGCCXCCNCCASCANAGIC stop Consensus ttattooitgttgaagatggotggaetgtoca uttegatagtotttitato agbo cigtag E. iogenin- eccleocococcCaesacagogic coccacAcces Willis CASCGCCCCCGCACAGACCASPSPs acceCCCAcces Corpseuss aigiocreli Eli s sy, A., M. War. O Consensus atgaatgttca d tactitta giged tgaattatuttgaaatatattot 55 S. 7. ogeniu-4 ccTCATIATAATGCACASAAAAAAGATATOEXCAAAAMCCATAAAAAAAAAAAAAAAAA EEis CCS is a GCACSAAAAAAAACAAAAACAAAAAAAA------sees got atttataatguagagaaataaagatatotaaraa dua aaaaaa 72. 73 7. 75 7. 77 angiogenail- ABAAAAAAA SEQDNO29 aloren-3 ------SEOD NO30 Cocsetes Patent Application Publication Oct. 27, 2005 Sheet 5 of 41 US 2005/0239706A1 Figure 5: Alignment of mouse angi genin family members
l ll 2. 3. 4. angiogenin TiSPCPFFWGYWPTA-IRYKFIREDAKKRDDRC angiogenin ASGPLF,F,GVWFADDSETEFTDARPERDDRYC angiogelin-3 EVESFGSLLIVFLISLDVIPPTLAnNYRYIKFTTEYDAKPTGRDYRYC angiogenin RB ESPGPLFFFTLGVWPPTSDDSKYKFTYEAKPKGRDDRYC Conseals In mappillwflglwvipptland ry kfilthydarpkgridryc 5 5. 7. Bl 9. angiogenin-6 ESSKERKLTSPCKDWTFEGTKKRRICKKGSPYEREISNSPF angiogelin-l ERKRRSLTSPCKDYRTFEG, KSRRAICG.GSPYRNassPFC ESKKRKTSPCKETFESTKRRKACGETRYGFRSNESRF Esi KRRTSFCKDWTFETKRKACGESPYRIRISKRF esmkrkltepckdwateih tk nikai.cg aspyg n ris B to lol lill 2. 3. l SEID angiogelin-6 TTCTBSRGSPPPCGRFKDRWADGEESFS 3 . angiogenin WTTKTGGSFRPFCYRASAGFREWWACEGLUEFDESEFSI. . 32 angiogenin-3 vTTCTHKGGSPRPPCOYNAFKDFRYIVLACEDCPVHFDESFISP 33 angiogenir WTTCTERGRSPRPPCRRASKGFRIIIGCEGFWBFDESISP 34 CDs easis wittcth ggsprppc yra k fryiviace gapvhfdesfisp Patent Application Publication Oct. 27, 2005 Sheet 6 of 41 US 2005/0239706A1 Fig. 6 Locations of primers specific for mouse angiogenin family members
angiogenin-d angiogenin-3 angiogenin-1 i angiogening? Consensus
angiogenin-d angiogenit-3 angiogenin-1. angiogenin RP Consensus O). ll l2 3. 4. angiogenin-4 ACGS RS angiogenin-3 angiogenin-1 : W angiogeninrPSA Consensus 15 l6 l 8. 19 angiogenin-4 angiogenin-3 angiogenil angiogeninkp SS SittigiassissiTTEgg Consensus gaaagtatgatgaagaaaagaaagctaaccto co tigcaaagatgtcaa 20 21 22 23. 24l angiogenin-d angiogenin-3 angiogeninri angiogen?inEP 35 OE. E. s Consensus cacctttatcCatg ceccaagaacaa.catceaeggc.catctgtgga age 2S 25i 27 28 29 angiogenin-d Saayaaaass s s angiogenin-3 T. angiogenin-i alagiotein Siii.55:iii.5iSigEcESi3S2, Consensus a gigaegcc.cttatggag aaactu agaataegcaa tactc ctitccag 30 3. 32. 34 angiogenium-4 E332 says: saxa se:sawaxy's angiogenin-3 angiogenin-1 angiogenirrP Consensus 35 36 3. 38 39 angiogenin f - area 3. angiogenin-3 &E. M angiogenin-1 E. angiogenin RP
C Casess SEO O NO.
angiogenil- 61 angiogenin-3 62 angiogenin-1 63 angiogenin RP Egg 64 Consensus ggcc tigtocacttcgatgegtCttittetcagtcc tag Patent Application Publication Oct. 27, 2005 Sheet 7 of 41 US 2005/0239706A1 Figure 7: Tissue distribution of Angiogenin-4 mRNA
qRT-PCR analysis:
6.0 S.O
Agarose gel analysis (with Gapdh control) Patent Application Publication Oct. 27, 2005 Sheet 8 of 41 US 2005/0239706A1 Figure 8: Tissue distribution of angiogenin-1 mRNA
qRT-PCR analysis:
Patent Application Publication Oct. 27, 2005 Sheet 9 of 41 US 2005/0239706A1 Figure 9: Tissue distribution of angiogenin-3 mRNA
Quantitative real-time RT-PCR analysis:
Patent Application Publication Oct. 27, 2005 Sheet 10 of 41 US 2005/0239706A1 Figure 10: RT-PCR analysis showing absence of angiogenin-related protein expression
- as a 2 a SA 2 S 22 R as & R & K SS bp 194 194 118- 18 72- -72 expected amplicon size: 130 bp Note that Gapdh expression levels for each of these tissues are shown in Figure 4
l. distal small 14. brain 2. middle Small S. heart 3. proximal small 16. bladder 4. Squamous 17. skin 5. glandular stomach 18. tranchealthyroid 6. ascending colon 19. pancreas 7. descending colon 20. Salivary gland 8. rect 21. testes 9. Cec 22. prostate 10. kidney 23. ovary 11. liver 24. uterus 12. lung 25. mammary gland 13. spleen w 26. genomic DNA 27. Water Patent Application Publication Oct. 27, 2005 Sheet 11 of 41 US 2005/0239706A1
Figure 11: Microbial regulation of angiogenin-4 expression in the small intestine
Pair 1
-0-germ-free ys -- conventionalized8 is asi gs s
3 5 7 9 11 13 15 intestinal segment
Pair 2 2.5 w -0-germ-free 2.0 -- conventionalized S. s 15 s 3. 1.0 O. 5
O. O 1 3 5. 7 9 11 13 15 inte tinals gment Patent Application Publication Oct. 27, 2005 Sheet 12 of 41 US 2005/0239706A1 Figure 12 Regulation of angiogenin-4 expression during postnatal development
germ-free 11
5 10 15 20. 25 30 days after birth Patent Application Publication Oct. 27, 2005 Sheet 13 of 41 US 2005/0239706A1 Figure 13 Cellular localization of angiogenin-4 expression in small intestige: qRT-PCR analysis feels is lated from the crypt base
wild-type CR2-tox176 (lacking Paneth mouse strain Patent Application Publication Oct. 27, 2005 Sheet 14 of 41 US 2005/0239706A1 Figs. 14A-14D
O (Kep/6)uo?duunsu00MO?O LO<+croÇN•Q.D. Patent Application Publication Oct. 27, 2005 Sheet 15 of 41 US 2005/0239706A1 Figs. 15A-15C eye A 3 15 12 2 2 2 1.0 8 CD cy S c 8 1 0.5 2 4 - (D O O $ S dš -NQ S S OS C C i? CONV-D C B 300 -- GF 100 () r g 8 75 g200 o e CD 50 f 8 100 2 as 25 CD O SS O O 3O 6O 90 120 O 30 6O 90 120 time (min) time (min) Patent Application Publication Oct. 27, 2005 Sheet 16 of 41 US 2005/0239706A1 Figs. 16A-16D rt G in t S15 g D E a g 200 c 10 SS 22 100 9> 5 E3ed & 9 O eso D ChREBP 5 Patent Application Publication Oct. 27, 2005 Sheet 17 of 41 US 2005/0239706A1 Fig. 17A-17F A B 125 $5100 de 2 75 is59 50 E23 25 C o CC cr’ sS. (° 3 4 WA Heart 2. arra g3 S 52 s 5 o s $sCS &sC D 2 F. 80 5 150 gas 60 a g g t sq 100 o 40 6 sol E3 r as c 8.3 kb gi20 O .S. c. X Martinspotte see-H*3. ++ + -i- *S. \s S if 47 gi Fiafgenotype w Souther to Norther to 0-f v- 84 -- - -- Patent Application Publication Oct. 27, 2005 Sheet 18 of 41 US 2005/0239706A1 Fig. 18 increased hepatic u-1 (ChREBPISREBP-1)lipogenesis Processing of dietary Microbial u- polysaccharides colonization of the gut Suppression of u s Fiaf in the gut Triglyceride epithelium LPL activity - adipocytesstorage in Patent Application Publication Oct. 27, 2005 Sheet 19 of 41 US 2005/023970.6 A1 Fig. 19 CONV-R Donor rClostridium Bacteroides CONV-D Eubacterium Recipient 1 OLactobacillus CONV-D Anaerophaga Recipient 2 Shewanella Ruminococcus CONV-D s a Porphyromonas Recipient 3 Tannerella Turicibacter CONV-D Eirit Ešš. Other Recipient 4 is s 20% 40% 60% 80% 100% % representation in Cecal microbiota Patent Application Publication Oct. 27, 2005 Sheet 20 of 41 US 2005/0239706A1 Fig. 20 O 5 10 15 20 25 30 35 Age (days after birth) Patent Application Publication Oct. 27, 2005 Sheet 21 of 41 US 2005/0239706A1 Figs. 21A-21B A PhyloCon Prediction Closest TRANSFAC entry Motif 1. B Other Conserved MotifFamilies Representative Logo Corresponding Matrices Fork head box, recognized by HNF3, HNF4, HFH8 and other fork head homologs E-box, recognized by E12, E47, MyoD, Myogenin and other HLH proteins ISRE, interferon responsive element, recognized by interferon regulatory factors 1 to 7. Patent Application Publication Oct. 27, 2005 Sheet 22 of 41 US 2005/0239706A1 Figs. 22A-22C A 150 B 16 . res C 1 OO C)g 12 dSir CD 75 100 SS E a Se 8 > 50 $250D isO 4 toO 25 Q- O NS S way o O O O f9 Sd Sis S.s S. S.s S. Ppara+1+ genotype -/- S Cs O Ppara genotype Patent Application Publication Oct. 27, 2005 Sheet 23 of 41 US 2005/0239706A1 Figs. 23A-23D A Zebrafish Angptl3 C 120 L GF Fugu Angptl3b 100 Esa CONV-D 9 Fugu Angptl3a g 80 Human ANGPTL3 : i 60 Mouse Angptl3 É E 40 s Human ANGPTL4/FEAF t E 8 20 Mouse Angptl4/Fiaf a 0. Zebrafish Angptl4/Fiaf" Fed Fasted Fugu Angpti4/Fiaf D Human ANGPTL1 . 5 S ce t e C Z E s C Mono- 0.41m Heat associated membrane killed O CONV-D A.h. Pa. Patent Application Publication Oct. 27, 2005 Sheet 24 of 41 US 2005/0239706A1 Figs. 24A-24L Patent Application Publication Oct. 27, 2005 Sheet 25 of 41 US 2005/023970.6 A1 Figs. 25A-25C 4 O Epithelium Mesenchyme and Muscle Patent Application Publication Oct. 27, 2005 Sheet 26 of 41 US 2005/0239706A1 Figs. 26A-26D Saal 0.8 O Fiaf CONV-D Ah. Pa. Slc31a1 CONV-D A.h. Pa. Patent Application Publication Oct. 27, 2005 Sheet 27 of 41 US 2005/023970.6 A1 Figs. 27A-27D Patent Application Publication Oct. 27, 2005 Sheet 28 of 41 US 2005/0239706A1 Figs. 28A-28D A. G M BTO367-BT0369; plant glycans endo-arabinosidase, arabinofuranosidase.xyanase) BT3654-BT3657;plant glycans arabinosidases (2), galactosidase, xylosidase) BT4667-BT4672; plant glycans (arabinogalactan-specific galactosidase, galactosidase, SusC/D paralogs (2) BT0455-BT0461;sialylated glycans (sialidase, 9-O-acetylesterase, hexosaminidases (3), mannosidase) BT1272-BT1277; fucose utilization operon (fucose permease, fucose metabolisrn (3) SusG-SusA; starch utilization system (Sus) amylase/glucosidases (3), SusC, Suso) Sooo." B 2OOOO MM-G oMMM 5. 15000 E. ECecum 10000 E. ca s : e 5000 O t SN y SN N N SN g SS g S S g S co& s & g?S. c S. S SS :S Sp S S S S s f SF 9 c s es s 9 SS SP st SP Y S QS cy S Q 3 is o Glycoside hydrolase C 10000 B.theta lysate (MM-G) D Germ-free cecum a B.theta lysate (heat kitled) B.theta Colonized cecum et e OOO s s a s 100 S g E 10 V s s c S ; : cy & w" &S s SS p-nitrophenylglycoside Monosaccharide Patent Application Publication Oct. 27, 2005 Sheet 29 of 41 US 2005/023970.6 A1 Fig. 29 MMG standard diet sucrose hydrolasel. unique hydrolase lyase specificities lyase specificities hexosamine (9) galactose (3) fucose (2) xylose (2} fucose (2) glucose sialic acid arose sialic acid Tannose (10) galactose (2) glucuronic acid (2) N-acetylglucosamine (2) glucose (2) chitin rhamrose (2) chondroitin sulfate chtin chondroitin sulfate unspecified (6) glucose (7) arabinose (6) galactose (6) xylan (6) pectin (4) arabinose (6) fructose (3) pectin (4) hexosamine (2) fructose (3) xylose (2) galacturonic acid galacturonic acid glucuronic acid glucuronic acid arose rthamnose unspecified (7) so Patent Application Publication Oct. 27, 2005 Sheet 30 of 41 US 2005/023970.6 A1 Fig. 30 time (h) Patent Application Publication Oct. 27, 2005 Sheet 32 of 41 US 2005/023970.6 A1 Fig. 32A A Translation ribosomal structure and biogenesissils ...le Transcription Exam Up in vio relative to MM Replication, recombinationand repair Down in wo relative to MM-G Estheta genome Cetcyde control cell division dhromosome partitioning Defense medhanismse Posttranslational modification protein turnovel chaperones site Energy production and conversions Carbohydrate transport and metabolisms Amino acid transport and metabolisms Nudeotidetransport and metabolism Coenzyme transportand metabolisms Lipid transportand metabolism said Function unknown 09 598 109 1596 20 259 Fercentage of B.theca genes with assignable CoGs Patent Application Publication Oct. 27, 2005 Sheet 33 of 41 US 2005/023970.6 A1 Fig. 32B &lucose's Rose dhow is Standard Polysaccharide-rich chow expt 1) Standard Polysaccharide-rich chow expt2) Ofense mechanisms rate stss Function unknown is ca 5. 5, 209. 258 - 30 Percentage of 8.thetaganes upregulated in vivo (relative to MMG Patent Application Publication Oct. 27, 2005 Sheet 34 of 41 US 2005/0239706A1 Fig. 33A A. is e -i -if 3 - N m a f r i i Si Si S 3 Gere Annotation f Suzhenclog 9. OS Suschemolog 16 Sushorndog F. 49 Susichomolog 53. T Susthomolog 85 45 Suzhormclog OS Susch.ormclog FOS Susthomolog 855 Susthomolog 8. 857 Suzhonolog 28 TOS Susthomolog 15 Susic homolog 55 -2 Suschemolog 25 T5 Sushorndog 8 59 Sushornclog 9 T58. Suthorolog 24 T-3 Suschomolos S. 8 Sushomolog gs Susholmclog 29 Sushon clog 5 ES5 Sushomolog 2. E. Susthomolog 255 Suschemolog 288 Sushornclog 8) Sushorndog 89 82 Susthomolog 90 SS Sushonolog 92 Suschemolog s Sushonolog 3. 3. sus homolog 83 Sushcrnolog T55 Sushonolog f TES Sushcrno F5 Sus g F. Sus 16 398 Sushomolog 55 Og Sushomolog O TASS Susthomolog 4 sus honolog O 4 Sushorndog 34 T2 Sushonolog T22 Sushonolog 224 T 4 Suzhornclog 55 Sustomolog T468 Suthormclog TS sus homolog TF Sushchdog 9. Sushomolog 30 T457 Sushorticlog 44. sus homolog E. 46 Sushromolog 23 57 Sushomolog 252 4.F Sushomolog 8 s" Patent Application Publication Oct. 27, 2005 Sheet 36 of 41 US 2005/023970.6 A1 Fig. 34A 5-Dehydro-4-deoxy- SEE O xy 25-dioxohexanoate(43-4f6-Dihydro iSE.5ame d-Attite 2-dehydro-3-deoxy 4. d-gluconate f - ticksovie sugars 27.5 L-outonate L-Lyronate - --- 53."glucomae-SP L-xylome Sisie".4R5S)-45A-Trihydro Xylono 14-actone D-Frucose 6-phosphate d-oxo-6rheulose 3-phosphate Patent Application Publication Oct. 27, 2005 Sheet 37 of 41 US 2005/023970.6 A1 Fig. 34B C L-Xylose M S. D-xylose Xylit D-xylonoaconese-lis-e- e 11.2 2H)-L-Arabinose 3.168 D-xylonate 4.3.162 2-Dehydro 3-deoxy-D- xylomate D-Rito-SP D-Ribose 4 -SP O004 42 Patent Application Publication Oct. 27, 2005 Sheet 38 of 41 US 2005/0239706A1 Fig. 35 glucosef standard charide-rich diet sucrose diet is is se e s is S. S. e - s: a. . a ...... g. . . . s in5 a n g g : E G differenceAwe fold 2 S S is S ; ; ; ; ; ; ; ; ; ; ; Gene Arnotation vs. MM-G is ---is a a is is a s BT4267 SusChomolog s BT4246 susdhonolog S3 BT4247 Suschomolog s 3 BT3670. Suchomolog SB367 Susdhoralog s BT1619 Suschomolog 78 BT4357 SusChomolog 97 3 BTC46 SusChonolog A. BT1631 Susc homolog 7 BT1025 SusChorolog g BTO866. Suso honolog 839 2 BTOB67 Susc homolog 82 BT3495 Suesohomolog 7 Boo Suso honolog 8. BT1042 Susc honolog 277 BT3346. Susc homolog s BTO317 Suschomolog 3. BTO45. Suso homolog s BTO452 Suschotholog 2S EBT0439 Susc homolog 599 stO440 Suso honolog B2S60 Susthorolog 4. BT2821 Suso homolog 651 B288 Susc homolog 42 BT2820 SusChomolog 600 BT2805 SusChomolog 702 BT3332 Sushonolog 9 BT3519 SusChomolog 4 B3520 Suso homolog 7 BT4404 SusChomolog 8 BT4297 Suso homolog 106 BT4298 SusChomolog 229 33983 SusChomolog 6 BT4038 Suso honolog go BT4039 Suschomolog 86 BTT683 Sushomolog s BT3311 Suso homolog B2202 SusChomolog 48 B3604 Sushonolog 4. 82625 Suso homolog BE2626 SusChornolog 220 3483 Sushomolog 2 BT2920 Sushomolog 8 BT2919 Suso homolog 7 B3474. Suso homolog 5 SusChomolog 9 B3102 Susd homolog 3 B3103 Sushomolog 4. BTO483 SusChomolog 14 BTO484 Susd homolog 6 a BT3505 Suschonolog 53 a BT2460 Suso homolog 3. BE2461 SusChomolog a BO754 SusChomolog 4. o 80206 Sushomolog s B3788 SusChomolog s EIGI susshomolog 334 38.1280 Sushonolog BT4707 SusChomolog s 3. 83701 Suso SBT3702 susc 5 E B290s Suschoenolog 7 E3958. SusChomolog 4. ET0.364 SusChomolog BTo362 Suschomolog 144 BT468 Suschomolog 4. BT464 SusChomolog BT4165 Suso homolog BT3680 SusChomolog 39 B1028 Suso honolog 7 BT1682. Suso horolog 2 BT1029 Suschomolog s BT3046 Sushonolog 4. BT207 SusChomolog A. BT4122. Suso homolog 35 BT412 SusChomolog 5 B413 Suso homolog 30 BT414 Suschomolog 296 B762 Suso homolog 4 BT1763 Suschomolog BT2894 SusChomolog BT2393 SusChomolog 8 3.0 -20 -O D 30 Patent Application Publication Oct. 27, 2005 Sheet 39 of 41 US 2005/0239706A1 Fig. 36 Patent Application Publication Oct. 27, 2005 Sheet 40 of 41 US 2005/0239706A1 Fig. 37 Anotation putative transcriptional regulator Conserved hypothetical protein putative nudeoside-diphosphate sugar epimerasesdehydrase UDP-glucose 6-dehydrogenase nucleotide sugar epimerase UDP-N-acetylglucosamine 2-epirrerase UDP-N-acetyl-D-mannosamineronic acid dehydrogenase conserved hypothetical protein putative coenzyme F420-reducing hydrogenase putative polysaccharide export protein hypothetical protein serine O-acetyltransferase glycosyltransferase glycosyltransferase glycosyltransferaselipopolysaccharide biosynthesis protein, putative glycosyltransferase putative artinotransferase putative mentireme protein involved in polysaccharide export putative tyrosine protein kinase in cps region dTDP-4-dehydrorhamnosessenes uridecaprenylphosphate alpha-N-acetylglucosaminyltransferase UDP-glucose 6-dehydrogenase Putative UDP-glucuronic acid epimerase putative capsule biosynthesis protein putative glycosyltransferase glycosyltransferase capsule biosynthesis protein capa conserved hypothetical protein C-abecuose synthase Conserved hypothetical protein CDP-glucose 4,6-dehydratase 9ucose-phosphate cytidylyltransferase Potative glycosyltransferase it glycosyltransferase potative flippase hypothetical protein Putative capsule polysaccharide export protein Conserved hypothetical prote putative transcriptional CPSS Putative glycosyltransferase conserved hypothetical proteinputative integral membrane protein putative glycosyltransferase conserved hypothetical protein Putative techoic acid biosynthesis protein F pyrophosphorylase poPolysaccharide biosynthesis protein hypothetical protein Polysiatic acid transport protein kpsoprecursor s s aegulato 30-20-100 to to 3. Patent Application Publication Oct. 27, 2005 Sheet 41 of 41 US 2005/0239706A1 Fig. 38 Host intestina epithelium SusCID paralogs sloughed epithelial cells Glycoside hydrolase/ Piant polysaccharide polysaccharide lyase Mucus Glycans B. theta US 2005/02397O6 A1 Oct. 27, 2005 MODULATION OF FLAF AND THE that are approved also have side-effects. Currently, two GASTRONTESTINAL MICROBOTAAS A MEANS FDA-approved anti-obesity drugs are orlistat, a lipase TO CONTROL ENERGY STORAGE IN A SUBJECT inhibitor, and Sibutramine, a Serotonin reuptake inhibitor. Orlistat acts by blocking the absorption of fat into the body. CROSS-REFERENCE TO RELATED An unpleasant Side effect with orlistat, however, is the APPLICATIONS passage of undigested oily fat from the body. Sibutramine is an appetite SuppreSSant that acts by altering brain levels of 0001. This application claims priority from Provisional Serotonin. In the process, it also causes elevation of blood Application Ser. No. 60/591,313 filed on Jul. 27, 2004, and preSSure and an increase in heart rate. Other appetite Sup is a continuation-in-part application of application Ser. No. preSSants, Such as amphetamine derivatives, are highly 10/432,819 filed on Nov. 27, 2001, which claims priority addictive and have the potential for abuse. Moreover, dif from Provisional Application Ser. No. 60/252,901 filed on ferent Subjects respond differently and unpredictably to Nov. 27, 2000, all of which are hereby incorporated by weight-loSS medications. reference in their entirety. 0007. In Summary, current surgical and pharmacotherapy FIELD OF THE INVENTION treatments are problematic. Novel non-cognitive Strategies are needed to prevent and treat obesity and obesity-related 0002 The current invention generally relates to the disorders. effects of the gastrointestinal microbiota on the regulation of energy Storage in a Subject. In particular, the invention SUMMARY OF THE INVENTION provides compositions and methods to modulate fat Storage 0008. The applicants have discovered novel treatment in a Subject by increasing either the amount of or the activity Strategies that may be employed to treat obesity and to of the fasting-induced adipose factor protein in the Subject promote weight loSS. Briefly, the present discovery was made by Studying the impact of the gastrointestinal micro BACKGROUND OF THE INVENTION biota on energy Storage in a Subject. The human gut contains 0003. According to the Center for Disease Control an immense number of microorganisms, collectively known (CDC), over sixty percent of the United States population is as the microbiota. There are approximately 500 to 1000 overweight, and almost twenty percent are obese. This Species of microorganisms whose collective genomes (the translates into 38.8 million adults in the United States with “microbiome') are estimated to contain more than 100 times a Body Mass Index (BMI) of 30 or above. Obesity is also a more genes than the human genome. The microbiota is a world-wide health problem with an estimated 500 million metabolic organ that performs functions humans cannot. overweight adult humans body mass index (BMI) of 25.0- These finctions, for example, include the ability to process 29.9 kg/mi) and 250 million obese adults (Bouchard, C otherwise indigestible components of the human diet, Such (2000) N Engl J Med. 343, 1888-9). This epidemic of as plant polysaccharides. obesity is leading to worldwide increases in the prevalence 0009. By studying the impact of the microbiota on a of obesity-related disorders, Such as diabetes, hypertension, Subject's energy balance, the applicants have discovered that as well as cardiac pathology, and non-alcoholic fatty liver the microbiota acts through an integrated host-Signaling disease (NAFLD, Wanless, and Lentz (1990) Hepatology pathway to regulate energy Storage in the Subject. In par 12, 1106-1110. Silverman, et al., (1990). Am. J Gastroen ticular, the applicants have discovered that the microbiota terol. 85, 1349-1355; Neuschwander-Tetri and, Caldwell Suppresses a Subject's transcription of Fiaf in the gas (2003) Hepatology 37, 1202-1219). trointestinal tract. Moreover, the applicants have shown that 0004. According to the National Institute of Diabetes, microbial-mediated Suppression of Fiaf causes a Subject to Digestive and Kidney Diseases (NIDDK) approximately store body fat. While Fiaf has previously been shown to 280,000 deaths annually are directly related to obesity. The inhibit lipoprotein lipase (LPL) in vitro, a direct in vivo NIDDK further estimated that the direct cost of healthcare in causal connection between Fiaf's role in the regulation of the U.S. associated with obesity is $51 billion. In addition, energy Storage in a Subject has not been previously demon Americans spend S33 billion per year on weight loss prod Strated. In particular, the role played by the gastrointestinal ucts. In spite of this economic cost and consumer commit microbiota in this process has not been previously demon ment, the prevalence of obesity continues to rise at alarming Strated. rates. From 1991 to 2000, obesity in the U.S. grew by 61%. 0010. Among the several aspects of the current invention, 0005 Although the physiologic mechanisms that support therefore, is the provision of compositions and methods that development of obesity are complex, the medical consensus may be utilized to regulate energy Storage in a Subject. In is that the root cause relates to an exceSS intake of calories certain aspects of the invention, fat Storage and weight loSS compared to caloric expenditure. While the treatment Seems are modulated by altering the Structure or finction of the quite intuitive, dieting is not an adequate long-term Solution Subject's gastrointestinal microbiota, or by administering for most people; about 90 to 95 percent of persons who lose chemical entities that regulate (host) intestinal Fiaf expres weight Subsequently regain it. Although Surgical interven SO. tion has had Some measured Success, the various types of 0011. Other aspects and embodiments of the invention Surgeries have relatively high rates of morbidity and mor are described in more detail herein. tality. 0006 Pharmacotherapeutic principles are limited. In BRIEF DESCRIPTION OF THE FIGURES addition, because of undesirable side effects, the FDA has 0012 FIG. 1 shows the results of real-time quantitative had to recall Several obesity drugs from the market. Those RT-PCR Studies of colonization-associated changes in gene US 2005/02397O6 A1 Oct. 27, 2005 expression in laser capture microdissected (LMC) ileal cell tionated cecal microbiota harvested from CONV-R donors populations of a colonized mouse. Also shown is the proceSS (conventionalized; CONV-D) were analyzed for: of LCM of the ileum in a colonized mouse. Sections were 0026) 14(A) total body fat content by DEXA (n=21 stained with nuclear fast red. Bars=25 um. 25/group); 0013 FIG. 2 shows the results of real-time quantitative qRT-PCR analyses of mRNA levels in isolated from laser 0027) 14(B) epididymal fat weight (n=10-20/ captured cell populations. Values are expressed relative to group); levels in germ-free mesenchyme using AACT analysis 0028) 14(C) chow consumption (average daily value described below. Each gene product per Sample was assayed over the 3 day period prior to termination of the in triplicate in 3-4 independent experiments. Representative experiment, n=10/group); and results (mean +/-1 S.D.) from pairs of germ-free and colo 0029) 14(D) oxygen consumption (VO, defined by nized mice are plotted. open circuit calorimetry just prior to Sacrifice, n=10/ 0.014 FIG. 3 shows the results of an experiment to group). Mean values+SEM are plotted. illustrate the Specificity of host responses to colonization 0030 FIG. 15 depicts a series of graphs detailing the with different members of the microbiota. Germ-free mice impact of a 14-day conventionalization of wild-type GF B6 were inoculated with one of the indicated organisms, or with mice. Sera were obtained after a 4-hour fast and analyzed a complete ileal/cecal microbiota from conventionally raised for: mice (CONV-R microbiota) (J. M. Friedman, Nat Med 10, 563-9 (2004)). Ileal RNAS, prepared from animals colonized 0031) 15(A) leptin, insulin, and glucose (n=8 ani at 107 CFU/mlileal contents 10 days after inoculation, were mals/group). Numbers represent mean valuestSEM. pooled, and levels of each mRNA shown were analyzed by 0032) 15(B, C) Glucose- and insulin-tolerance tests real time quantitative RT-PCR (qRT-PCR). Mean values were performed after a 4 hour fast (n=8 mice/group). (mean +/-1 S.D.) for triplicate determinations are plotted. Mean values i SEM are plotted 0.015 FIG. 4 shows the nucleotide sequences of mouse 0033 FIG. 16 depicts a series of graphs and images angiogenin-4 and angiogenin-3 in alignment (SEQ ID NOS detailing the impact of conventionalization on hepatic lipo 29 and 30 respectively). genesis and nuclear import of the bFILH transcription factor, 0016 FIG. 5 illustrates the sequence alignment of the ChREBP. amino acid Sequences of mouse angiogenin family members 0034) 16(A) Oil-red O stains of paraformaldehyde (SEQ ID NOS 31-34). fixed liver sections prepared from 8 week-old, wild 0017 FIG. 6 shows the locations of primers specific for type, male GF and CONV-D B6 mice. mouse angiogenin family members. 0035) 16(B) Liver triglyceride levels. 0.018 FIG. 7 is a graph illustrating tissue distribution of 0036) 16(C) qRT-PCR assays of livers from GF and angiogenin-4 mRNA, together with the results of an agarose CONV-D mice n=15/group; mean values-ESEM are gel analysis. expressed relative to levels in GF animals (GF set at 0.019 FIG. 8 is a graph illustrating tissue distribution of 100%)). angiogenin-1 mRNA. 0037) 16(D) Immunohistochemical study of paraformaldehyde-fixed sections of liver from GF or 0020 FIG. 9 is a graph illustrating tissue distribution of CONV-D mice. Sections were stained with rabbit angiogenin-3 mRNA following quantitative real-time RT polyclonal antibodies to mouse CHREBP (green). PCR analysis. Nuclei are labeled dark blue with 4,6-diamidino-2- 0021 FIG. 10 shows the results of RT-PCR analysis phenylindole. Bars, 25 lum. showing the absence of angiogenin-related protein gene 0038 FIG. 17 depicts a series of graphs and images expression. detailing the impact of conventionalization on adipocyte 0022 FIG. 11 is a set of graphs showing the results of hypertrophy and Fiaf expression in the intestine. experiments on the microbial regulation of angiogenin-4 0039) 17(A) Epididymal fat pads (left half of the expression in the Small intestine. panel) from 8 week-old wild-type male GF, CONV 0023 FIG. 12 is a graph showing the regulation of D, and CONV-R B6 mice. The corresponding hema angiogenin-4 expression during postnatal development. toxylin- and eosin-Stained Sections are shown in the right half of the panel. 0024 FIG. 13 is a graph showing cellular localization of 0040 17(B) qRT-PCR assays of epididymal fat pad angiogenin-4 expression in Small intestine: qRT-PCR analy RNAS harvested from wild-type mice reveal that sis of cells isolated from the crypt base. conventionalization does not produce Significant 0.025 FIG. 14 depicts a series of graphs detailing phe changes in expression of mediators or biomarkers of notype characteristics of wild-type gnotobiotic mice. Three lipogenesis and adipogenesis in white fat tissue. groups of 8 week-old adult male C57B1/6J mice (abbrevi ated B6)- those raised in a germ-free State (GF), those 0041) 17(C) LPL activity is increased upon coloni allowed to acquire a microbiota from birth to adulthood Zation in both epididymal fat pads and heart. (conventionally-raised; CONV-R) and those raised GF until 0.042) 17(D) qRT-PCR assays of Fiafexpression in adulthood and then colonized for 2 weeks with an unfrac wild-type animals. US 2005/02397O6 A1 Oct. 27, 2005 0043) 17(E) Generation of Fiafknockout mice. out of 1000 are considered statistically robust. The Structures are shown for the wild-type Fiaf locus, the Zebrafish Fiaf ortholog is indicated by an asterisk. targeting vector, and the mutated locus with exons 1-3 replaced by a BgeopA cassette. The desired 0057 23(B) is a graph showing the impact of colo disruption was verified by Southern blot analysis. nization of 3 dpf germ-free zebrafish with a micro Northern blots of adipocyte RNA establish the biota harvested from conventionally-raised Zebrafish absence of detectable Fiaf mRNA in Fiaf-f-animals. (CONV), or with A. hydrophila (A. h.), P. aerugi noSa (Pa.), or E. coli (E. c.). The downregulation of 0044) 17(F) The absence of Fiaf markedly attenu Fiaf in the digestive tracts of colonized 6 dpf com ates the increase in total body fat content following pared to GF controls shows microbial Specificity. a 14 day conventionalization. 0.058 23(C) is a graph depicting the effects of fast 004.5 FIG. 18 is a diagram illustrating the impact of the ing on Fiaf expression. GF (black bars) and gastrointestinal microbiota on a Subject's energy Storage. CONV-D (white bars) zebrafish were either fed 0.046 FIG. 19 is graph depicting the distribution of the beginning on 3 dpf (fed) or not fed (fasted). Fiaf 10 most abundant microbial genera in the cecal microbiota mRNA levels in their digestive tracts assessed on 6 of conventionalized B6 mice. dpf. 0047 FIG. 20 is a graph depicting developmental regu 0059) 23(D) is a graph depicting the effect of mono lation of Fiaf expression in the Small intestine of germin-free asSociation with E. coli causes mono-associated (GF) and conventionally-raised (CONV-R) mice. downregulation of Fiaf compared to GF the same 0.048 FIG. 21 depicts transcription factor binding sites result occurs when GF fish are separated from live E. conserved in orthologous mouse, rat, human, Zebrafish and coli by a 0.4 um membrane, or are inoculated with fugu Fiaf genes. heat-killed E. coli. 0049 21 (A) depicts two motifs that are predicted by 0060. In panels B and D, the Y-axis indicates FiafmRNA PhyloCon, together with the closest matches in the fold-change relative to a GF baseline (note inverted Scale). TRANSFAC database; In panel C, the Y-axis indicates percent Fiaf mRNA levels relative to fed GF larvae. Quantitative RT-PCR assays of 0050 21(B) depicts selected TRANSFAC motifs, digestive tract RNA in panels B-D were performed in including fork head boxes, E-boxes and inferon triplicate with biological duplicate pools (5-10 animals/ responsive elements. pool) for each treatment, and normalized to 18S rRNA 0051 FIG. 22 depicts a series of graphs detailing the levels. Error bars indicate Standard error of the mean. impact of a 14 day conventionalization on Ppara +/-- and 0061 FIG. 24 depicts a series of photographic images Ppara -/- littermates. detailing the results of morphologic studies of CONV-R, 0.052 22(A) is a graph depicting the expression CONV-D, and GF Zebrafish. levels of the transcription factor Ppar-C. that were examined by qRT-PCR in various tissues from GF 0062 24(A-C) are photographic images of whole-mount and conventionalized CONV-D CB57/B6J animals; preparations of 6 dpf Zebrafish. Rostral is to the left, dorsal is to the top. Panel (A) shows the position of the Swim 0053 22(B) is a graph depicting DEXA measure bladder (SB) and the boundary of intestinal segment 2 (red ments of total body fat content in Ppara +/+ and bracket). Segments 1 and 3 lie rostral and caudal to Segment Ppara -/- mice (n=8/group); and 2, respectively. 0054) 22(C) is a graph depicting qRT-PCR assays of 0063 24(D-F) are photographic images of whole mounts Fiaf expression in Ppara +/-- and Ppara -/- mice of the caudal regions of 9 dpf CONV-R, GF, and CONV-D (n=8/group). Values in panels A and C are expressed (conventionalized at 3 dpf) animals, showing onset of epi as percentages of GF (meani-SEM). dermal degeneration phenotype in GF fish. This phenotype 0.055 FIG. 23 depicts a series of graphs showing that is manifested by loSS of transparency and integrity of the Zebrafish ortholog of mouse and human Fiaf/Angptl4 is epidermis in fin folds (the edges of these fin folds are Suppressed by a Soluble microbial factor. highlighted with open arrowheads in E). CONV-R and 0056 23(A) is a graph showing the phylogenetic CONV-D fin folds remain transparent (edges indicated by comparison of Angptl4/Fiaf and Angpt13 protein filled black arrowheads in D and F). Sequences in Zebrafish (Danio rerio), Fugu (Fugu 0064. 24(G, H, J and K) are photographic images depict rubripes), Mouse (Mus musculus), and Human ing hematoxylin- and eosin-Stained transverse Sections (Homo sapiens). The closely related Angptl4/Fiaf showing intestinal Segment 1 (G and J) and segment 2 (H and Angpt13 protein families are shown with Human and K) in 6-dpf CONV and GF zebrafish. There are no ANGPTL1 used as a root (all other Angiopoietin-like detectable epithelial abnormalities in intestinal Segment 1, and Angiopoietin proteins cluster with ANGPTL1; whether judged by light microscopy (G and J) or by trans data not shown). Sequences were aligned with Clust mission EM (data not shown). In contrast, enterocytes in alW using the BLOSUM matrix, then a parsimony Segment 2 contain prominent Supranuclear vacuoles filled tree was constructed. Numbers at each branch point with eosinophilic material in CONV-D (and CONV-R) fish indicate the Subset of 1000 bootstrap replicates of (e.g., black arrowheads in H). These vacuoles appear clear heuristic Searches in which this topology was Sup in GF animals (e.g., open arrowheads in K). Pigmented ported. Branch points with bootstrap support of >700 melanocytes (m) lie adjacent to the intestine in Hand K. US 2005/02397O6 A1 Oct. 27, 2005 0065 24(I and L) are photographic images depicting EM only 13 of the 4719 genes queried exhibit a 210-fold Study of 6-dpf intestines, showing electron-dense material in difference in their expression. Eight of these genes comprise the Supranuclear vacuoles (v) of segment 2 CONV-D entero a starch utilization System (Sus) operon: its three SuS cytes, and electron-lucent material in GF enterocytes. The alpha-amylases are the only ones among 241 B. thetaio filled black arrowhead in I points to a bacterium in the taOmicron glycoside hydrolases and polysaccharide lyases intestinal lumen. (Bars: 500 um in A-F; 100 um in G and J; whose expression change 210-fold as a result of exposure 20 um in H and K; 5 um in I and L.). to maltotriose, underScoring the Specificity of the organism’s 0.066 FIG. 25 depicts a series of photographic and graph induced responses to the glycosidic linkages that it must images detailing microbiota-Stimulated intestinal epithelial process (e.g., compare alpha- and beta-glucosidases in panel proliferation in Zebrafish. B plus data in panel C). 0075 28(B, C) Selective induction of glycoside hydro 0067. 25(A and B) are photographic images showing lases in Vivo. Panel B, induction of expression of groups of sections prepared from the intestines of 6-dpf CONV-D and glycoside hydrolases in the cecum compared to MM-G and GF Zebrafish after a 24-h exposure to bromodeoxyuridine in MM-M (see Table S4 for a list of genes; the number of genes their environmental water. Sections were incubated with in each group is indicated in parenthesis, Summed GeneChip antibodies to bromodeoxyuridine (magenta) and the nuclear signals for B. thetaiota Omicron transcripts called “Present” Stain bisbenzimide (blue). The mesenchyme and muscle for individual Samples within an experimental group were Surrounding the intestinal epithelium are outlined in white. averaged to calculate the aggregate mean signal-tS.E.M.). 0068 25(C) is a graphic quantitation of S-phase cells in (C) Biochemical evidence of B. thetaiotaomicron’s “pre the intestinal epithelium and mesenchyme. The percentage paredness' for degrading glycans. Lysates were generated of cells in S phase in GF intestinal epithelium is significantly from bacteria during late-log phase growth in MM-G. The lower than in CONV-R or CONV-D animals (P<0.0001, organism produces a portfolio of hydrolases capable of indicated by brackets with three asterisks). Data are processing a wide variety of glycosides, even when exposed expressed as the mean of two independent to a single fermentable monosaccharide. Mean valuestS.D. experiments+SEM (n=19-31 sections scored per animal; >6 of triplicate assays are plotted. animals per experiment). Bars, 25 um in A and B. 0076) 28(D) GC-MS of neutral and amino sugars in cecal 0069 FIG. 26 is a series of graphs showing real-time contents from germ-free versus B. thetaiota Omicron-colo quantitative RT-PCR studies of the microbial species speci nized mice. ficity of selected evolutionarily conserved Zebrafish 0077 FIG. 29 depicts a schematic showing diet-associ responses to the digestive tract microbiota. Expression lev ated changes in the in Vivo expression of B. thetaiota Omi els of Serum amyloid A1 (Saal; A), complement component cron glycoside hydrolases and polysaccharide lyases. Unsu 3 (C3; B), fasting-induced adipose factor (Fiaf, C), and pervised hierarchical clustering yields the following groups solute carrier family 31 member 1 (Slc3lal; D) in digestive of genes upregulated an average of >2.5-fold in Vivo com tracts from 6-dpf conventionalized (CONV-D), A. hydro pared to their average level of expression at all growth phila-monoassociated (A.h.), and P. aeruginosa-monoasso phases in MM-G: Group 1, highest expression on a simple ciated (Pa.) larvae are shown relative to 6-dpf GF larval Sugar diet, includes activities required for degradation of digestive tracts. ASSays were performed in triplicate (n>4 host glycans, Group 2, equivalent expression on both diets, assays per gene). Data were normalized to 18S ribosomal Group 3, highest on a polysaccharide-rich Standard chow RNA and results are expressed as mean log valuestSEM. diet, includes enzymes that degrade plant glycans. 0070 FIG. 27 depicts a series of photographic images 0078 FIG. 30 depicts a graph showing growth of B. showing the distribution of B. thetaiota Omicron within its thetaiotaomicron in a chemostat under various nutrient con intestinal niche. ditions. Curves show the average OD600 of duplicate B. 0071. 27(A) is a low power view of the distal small thetaiota Omicron cultures during growth in minimal intestine of B. thetaiotaOmicron mono-associated gnotobi medium plus 0.5% glucose (MM-G), minimum medium otic mouse showing a villus (arrow) viewed from above. plus 0.5% maltotriose (MM-M), or a control rich medium (TYG; 1% tryptone, 0.5% yeast extract, 0.2% glucose). 0072) 27(B-D) depicts progressively higher power views Bacteria were harvested at the time points noted by open showing B. thetaiotaOmicron associated with luminal con symbols. tents (food particles, shed mucus) (arrows), and embedded in the mucus layer overlying the epithelium (boxed region in 007.9 FIG. 31 is a schematic showing the hierarchical clustering of B. thetaiota Omicron transcriptional profiles in C, and panel D). Bars: A, 50 um; B, C, 5um; D, 0.5um. vitro and in vivo. 0.073 FIG. 28 depicts a series of graphs showing carbo 0080 31(A) The quality of replicates was assessed using hydrate foraging by B. thetaiota Omicron. unsupervised clustering (centroid linkage method) of 0074 28(A) B. thetaiotaomicron gene expression during samples using 4014 of 4823 probe sets that were (i) called growth from log to Stationary phase in minimal medium “Present” by dChip and (ii) had signal values 2100 in at containing 0.5% glucose or 0.5% maltotriose (a simplified least 1 of 16 samples. MM-G samples represent the time Starch composed of three a 1-4 linked glucose residues) points shown in FIG. 30 (A and B refer to samples taken Versus the ceca of mono-associated gnotobiotic mice fed a from independent vessels in the chemostat). Each of the in polysaccharide-rich diet. Predicted operons are shown Vivo Samples was prepared from the cecal contents of a together with their component gene products. All genes gnotobiotic mouse after a 10 day colonization (numbers listed were Significantly upregulated in Vivo relative to refer to individual animals, all of which were maintained on MM-G. Note that during growth in MM-G versus MM-M a high polysaccharide Standard chow diet). US 2005/02397O6 A1 Oct. 27, 2005 0081 31(B) Unsupervised clustering (centroid linkage putative enzymes required for metabolism of arabinose and method) using expression values of 98 B. thetaiotaomicron Xylose (Solid arrows) to intermediates that enter the pentose genes from the “replication, recombination and repair COG phosphate pathway (open arrow). that Satisfy the same criteria used in panel A above. The 42 0089 FIG. 35 depicts a schematic detailing diet-associ B. thetaiota Omicron Samples consist of 12 cecal populations ated changes in the in Vivo expression of B. thetaiota Omi nine from mice fed a polysaccharide-rich Standard chow cron SuSC/D paralogs. UnSupervised hierarchical clustering (purple), three from mice fed a simple Sugar diet (ochre), yields two distinct groups of genes upregulated an average plus five time points during growth in MM-G, MM-M, or of 22.5-fold in vivo compared to their average level of TYG (each time point assayed in duplicate cultures, desig expression at all growth phases in MM-G: Group 1, highest nated A and B). The results reveal that all of the cecal expression on a simple Sugar diet, Group 2, highest expres bacterial populations cluster most closely to log phase cells Sion on a polysaccharide-rich Standard chow diet. An aver irrespective of diet. age fold difference in expression is given for each gene in 0082 FIG. 32 depicts schematics showing COG catego each of the two groups (defined by white boxes) relative to rization of B. thetaiota Omicron genes with increased expres MM-G. Sion in the cecum. 0090 FIG. 36 depicts a schematic showing diet-regu 0.083 32(A) Genes exhibiting significantly different lated operons. Candidate SuSC/D paralogs were checked for expression during growth in the cecum of mice fed a proximity in the B. thetaiotaomicron genome to a chow or Standard polysaccharide-rich chow diet compared to growth host glycan-directed glycoside hydrolase. If a SuS gene Alay ex vivo in MM-G. Three groups of genes with assignable within the same “directon' (defined as all intervening genes COGs are considered: 442 of the 1237 (36%) genes showing transcribed on the same Strand) of a glycoside hydrolase higher expression in Vivo (designed as Up and shown in gene B, then B. thetaiotaomicron operon predictions were blue); 278 of 519 (54%) genes showing lower expression in checked to see whether A and B were likely part of a vivo (Down; yellow) and 1845 of the 4779 genes in the common operon. Operon associations between glycoside genome (green). The X-axis plots the percentage of each hydrolases (left column) and SusC/D paralogs (right col group that falls within a given COG. Note that the largest umn) are shown for genes upregulated in mice fed a simple group of genes upregulated in Vivo belongs to the “carbo Sugar-rich diet (green box) or a polysaccharide-rich diet hydrate transport and metabolism' COG, while the largest (brown box). group of genes downregulated in Vivo are members of the 0091 FIG. 37 depicts a schematic illustrating relative “amino acid transport and metabolism' COG. expression levels of CPS loci genes showing differential 0084 32(B) COG comparisons of genes upregulated in expression in B. thetaiota Omicron grown in vitro and in the ceca of mice fed a Standard polysaccharide-rich chow or vivo. Differential expression relative to MM-G is defined high Sugar diet compared to MM-G. The largest group of using the following criteria: (i) fold difference 21.2 using genes upregulated in all three in Vivo experiments belong to lower 90% confidence bound; (ii) signal difference 2100; the “carbohydrate transport and metabolism' COG. and (iii) upregulated genes (transcripts) called “Present” in 0085 FIG. 33 depicts a schematic showing components 26.6% GeneChip datasets generated from cecal Samples or of B. thetaiota Omicron's polysaccharide acquisition and in 220% of samples harvested during in vitro growth in a degradation machinery upregulated in the ceca of gnotobi given medium (i.e., at least one of the time points). otic mice fed a Standard polysaccharide-rich chow diet. B. 0092 FIG. 38 depicts a schematic view of adaptive thetaiotaomicron contains 106 SuSC paralogs postulated to foraging of glycans by B. thetaiotaOmicron. Bacterial con be conserved components of a Series of multifunctional Sortia assemble on nutrient Scaffolds composed of partially outer membrane porins, and 57 SuSD paralogs thought to digested plant glycans, shed mucus, or exfoliated epithelial function as specificity elements. Thirty-seven SusC and 16 cells. These scaffolds interact with one another, and with the Susld homologs exhibited >10-fold higher levels of expres intact mucus layer, Serve to oppose bacterial washout from sion in the cecum compared to MM-G (range 11-to 2523 the gut bioreactor, and enhance nutrient harvest and fold; panel A). Each induced SusID gene is physically linked eXchange with other members of the microbiota. Insets: to a SuSC paralog in the B. thetaiotaOmicron genome: 13 bacterial attachment to nutrient Scaffolds is promoted by adjacent pairs of upregulated SuSC-SuSD paralogs are mem glycan-specific outer membrane binding proteins (SuSC/D bers of predicted operons. Thirty-Seven glycoside hydro paralogs), induced depending upon the glycan landscape lases and polysaccharide lyases were upregulated 210-fold encountered in the gut micro-habitat. If dietary polysaccha in vivo (Panel B). Fold differences in average level of rides are unavailable, B. thetaiotaomicron forages on mucus expression in Vivo compared to all phases of growth in glycans. MM-G are indicated. 0.086 FIG. 34 depicts schematics detailing an example of DETAILED DESCRIPTION OF THE B. thetaiotaomicron expression data placed on KEGG meta PREFERRED EMBODIMENTS bolic pathways. 0093. I. Methods for Determining Modulation in Gene 0087 34(A) “Pentose and Glucuronate Interconversions” Expression Resulting from Colonization of the Mammalian KEGG map showing average fold difference in expression Intestine with Components of the Gut Microbiota of B. thetaiotaOmicron genes in the mouse cecum compared 0094) Mammals generally, and humans in particular, are to growth in MM-G. home to an incredibly complex and abundant ensemble of 0088 34(B) Higher power view of boxed region in panel microbes. ASSembly of components of this microbiota A, highlighting in Vivo upregulation of genes encoding begins at birth. The adult human intestine is home to an US 2005/02397O6 A1 Oct. 27, 2005 almost inconceivable number of micro-organisms. The size (see for example L. Eckmann, et al., J Biol. Chem., 275, of the population-up to 100 trillion-far exceeds that of all 14084 (2000);. D. A. Relman, Science, 284,1308 (1999); D. other microbial communities associated with our body's A. Relman, Curr. Opin. Immunol., 2, 215 (2000)) little is Surfaces, and is 10-fold greater than the total number of our known about how gut bacteria shape normal human devel Somatic and germ cells. Thus, it seems appropriate to view opment and physiology. This is due partly to a paucity of ourselves as a composite of many species and our genetic defined, experimentally tractable in Vivo model Systems for landscape as an amalgam of genes embedded in our H. examining how nonpathogenic microorganisms regulate Sapiens genome and in the genomes of our affiliated micro host biology. bial partners (the microbiome'). 0100. A mouse model using adult germ-free animals, 0.095 The human gut microbiota can be pictured as a colonized with Bacteroides thetaiota Omieron, has previ microbial organ placed within a host organ: it is composed ously been used to show that this prominent member of the of different cell lineages with a capacity to communicate normal distal human and mouse intestinal microbiota regu with one another and the host; it consumes, Stores and lates production of distal Small intestinal (ileal) epithelial re-distributes energy; it mediates physiologically important fucosylated glycans after it is introduced into germ-free chemical transformations, and it can maintain and repair mice, and to delineate how the microbe controls production itself through Self-replication. The gut microbiome, which of these glycans for its own nutritional benefit (L. Bry, et al., may contain 2100 times the number of genes as the human Science 273, 1380. (1996); L. V. Hooper, et al., Proc. Natl. genome, endows humans with functional attributes we have not had to evolve on our own. Acad. Sci. USA, 96, 9833 (1999)). 0.096 Our relationship with components of this micro 0101 Virtually nothing else is known about how indig biota is often described as 'commensal (one partner ben enous bacteria modulate intestinal gene expression and how efits, the other is apparently unaffected), as opposed to this impacts the host's digestive process. It has been dis mutualistic (both partners experience increased fitness). covered that components of the microbiota make significant However, use of the term commensal generally reflects our contributions to nutrient digestion, and to other aspects of lack of knowledge, or at least an agnostic (noncommittal) gut physiology and maturation. The present invention attitude about the contributions of most citizens of this encompasses (i) methods for testing the impact of compo nents of an animal's gut microbiota on intestinal gene microbial Society to the fitness of other community mem expression, including the effects of Specific components of bers, or ourselves. this microbiota on nutrient harvest and uptake, and the 0097. The guts of ruminants and termites are well-studied pathways used to regulate host Storage of energy extracted examples of bioreactors programmed with anaerobic bac from the diet; (ii) the discovery that Fiaf, a microbiota teria charged with the task of breaking down ingested modulated host gene product, is a regulator of host energy polysaccharides, the most abundant biological polymer on Storage and it, or its derivatives, or activators of Fiaf gene our planet, and fermenting the resulting monosaccharide expression, can be used to promote leanneSS in various Soup to short chain fatty acids. In these mutualistic relation mammalian species, including humans, and (iii) manipula ships, the hosts gain carbon and energy, while their microbes tion of the composition of the microbiota can be used to are provided with a rich buffet of glycans and a protected modulate host energy balance. anoxic environment (A. Brune, et al., Curr Opin Microbiol3, 263 (2000)). The human distal intestine is also an anaerobic 0102) In order to study the changes in intestinal gene bioreactor that harbors the majority of our gut microorgan expression orchestrated by members of the microbiota bac isms: they degrade a varied menu of otherwise indigestible teria, germ-free mice were colonized with various bacterial polysaccharides, including plant-derived pectin, cellulose, Species including Bacteroides thetaiota Omicron. Global hemicellulose, and resistant Starches. intestinal transcriptional responses to colonization were 0098. The adult human GI tract contains all three delineated using high-density oligonucleotide arrays and the domains of life - Archaea, Eukarya, and Bacteria. Bacteria cellular origins of Specific responses established by laser living in the human gut achieve the highest cell densities capture microdissection and real-time quantitative RT-PCR. recorded for any ecosystem (W. B. Whitman, et al., Proc. A similar approach has been used in germ-free Zebrafish to Natl. Acad. Sci. USA. 95,6578 (1998)). Nonetheless, diver discover host responses to the microbiota that have been sity at the division-level (Superkingdom, or deep evolution conserved between Vertebrate Species during the course of ary lineage) is among the lowest (P. Hugenholtz, et al., J Bact evolution, including the response of Fiaf 180, 4765 (1998)): only 8 of the 55 known bacterial divi 0103) The results illustrated hereinafter, reveal that com sions have been identified to date (Fig IA), and of these, five ponents of the human gut microbiota modulate expression of are rare. The divisions that dominate-the Cytophaga-Fla a large number of genes. The genes involved participate in vobacterium-Bacteroidetes (CFB, e.g., the genus Bacteroi diverse and fundamental physiological functions of the gut, des), and the Firmicutes (e.g., the genera Clostridium and including nutrient absorption, mucosal barrier fortification, Eubacterium) each comprise -30% of bacteria in feces and and Xenobiotic metabolism. The microbial Species-Selectiv mucus overlying the intestinal epithelium. Proteobacteria ity of Some of the colonization-associated changes in gene are common but usually not dominant (P. Seksik, et al., Gut expression emphasizes how human physiology can be 52, 237 (2003)). In comparison, soil, the terrestrial bio impacted by changes in the composition of indigenous Sphere's GI tract where degradation of organic matter microbiota. Furthermore, changes associated with the Suck occurs, can contain 20 or more bacterial divisions (J. Dun ling-weaning transition were elicited in adult mice by B. bar, et al., Appl Environ Microbiol. 68, 3035 (2002)). thetaiota Omicron, Suggesting that indigenous intestinal bac 0099 Although the effects of pathogenic or other poten teria play an instructive role in postnatal gut development. tially harmful invasive microorganisms have been Studied Coupling defined in Vivo models with comprehensive US 2005/02397O6 A1 Oct. 27, 2005 genome-based analyses thus provides a powerful approach pression by B. thetaiotamicron occurred in the epithelium. for identifying the critical contributions of resident microbes Microbial regulation of intestinal and villus epithelial to host biology. expression of Fiaf has not been described previously. In 0104 Bacteroides thetaiotaOmicron is a genetically-ma addition, qRT-PCR analysis of intestinal Fiaf expression nipulatable anaerobe and was chosen for initial Study to during postnatal period disclosed that the gene is induced in define the impact of resident bacteria on intestinal (and host) GF mice during the Suckling-weaning transition. Induction biology because it is a prominent member of both the adult does not occur in CONV-R animals, resulting in signifi mouse and human gut microbiota and because it is able to cantly lower levels of Fiaf mRNA in adult CONV-R versus breakdown otherwise indigestible polysaccharides which GF (see FIG. 20). During the suckling-weaning transition, are prominent components of the human diet, and of the the diet Switches from lipid/lactose-rich mother's milk to diets of many animal Species, including domestic animals. low fat/polysaccharide-rich chow, with coincident expan Bacteroides thetaiota Omicron's prodigious capacity for sion of the microbiota and a shift from facultative to obligate digesting otherwise indigestible dietary polysaccharides is anaerobes (e.g., Bacteroides). These developmental studies reflected in the fully sequenced 6.3 Mb genome of the type Suggested that Fiaf could provide a signal that links the strain (ATCC 29148; originally isolated from the feces of a microbiota with a change in host energy partitioning. The healthy adult human) (J. Xu, et al., Science 299, 2074 Significant repression of Fiaf found following colonization (2003)). Its glycobiome' contains the largest ensemble of of adult GF mice with B. thetoaiota Omicron illustrated genes involved in acquiring and metabolizing carbohydrates yet reported for a Sequenced bacterium, including 163 further hereinafter are indicative of a previously unappreci paralogs of two outer membrane proteins (SuSC, SusID) that ated mechanism by which a resident gut bacterium, contrib bind and import starch (J. A. Shipman, etal, J Bacteriol 182, utes to energy homeostasis. 5365 (2000)), 226 predicted glycoside hydrolases, and 15 0106 Additionally, the applicants have found that B. polysaccharide lyases (http://afinb.cnrs-mrs.fr/CAZY/). By thetaiotamicron colonization elicited a concerted response contrast, our 2.85 Gb human genome only contains 98 involving enhanced expression of four genes involved in the known or putative glycoside hydrolases, and is deficient in breakdown and processing of dietary lipids. mRNAS encod the enzyme activities required for degradation of Xylan, ing pancreatic lipase related protein-2 (PLRP-2) and coli pectin, and arabinose-containing polysaccharides that are pase increased an average of 4- and 9-fold, respectively common components of dietary fiber. (Tables 1 and 2). PLRP-2 hydrolyzes tri- and diacylglycer 0105 Colonization of adult GF mice with B. thetaiota ols, phospholipids and galactolipids. Colipase augments the micron produced a prominent decrease in expression of activity of PLRP-2 as well as triglyceride lipase (M. E. fasting-induced adipose factor (Fiaf), previously known to Lowe, et al., J. Biol. Chem. 273, 31215 (1998)). In addition, be expressed in liver and fat (S. Kersten, et al., J. Biol. there was (i) a 4-6-fold increase in L-FABP mRNA, which Chem. 275,28488 (2000)) but not known to be regulated by encodes an abundant cytosolic protein involved in fatty acid microbes in any tissue, or to be Selectively regulated by trafficking within enterocytes, and (ii) an induction of apo microbes in the host intestine. Moreover, qRT-PCR analysis lipoprotein AIV, a prominent component of triglyceride-rich of RNA isolated from laser capture microdissected villus lipoproteins (chylomicrons, VLDL) secreted from the baso epithelium and Villus mesenchyme revealed that Fiaf Sup lateral Surfaces of enterocytes (Table 1 below). TABLE 1. Colonization-associated changes in distal small intestinal gene expression GenBank/TIGR average Gene function Reference fold A Nutrient Uptake and Metabolism carbohydrates Na+/glucose cotransporter glucose uptake AF163846 +2.4 (SGLT1) actase phlorizin-hydrolase lactose hydrolysis AAS21747 -2.2 ipids pancreatic lipase-related lipid metabolism M30687 +4.1 protein 2 colipase lipid metabolism AA611440 +9.4 iver fatty acid binding protein lipid metabolism Y1466O +4.0, +5.6 apolipoprotein A-IV lipid metabolism M13966 +2.2 asting-induced adipose factor regulation of lipid metabolism AF278699 -90 phospholipase B lipid metabolism TC38683 -2.2 CYP27 cholesterol 27-hydroxylation TC25974 -2.2 metals high-affinity copper copper uptake AA1901.19 +2.6 ransporter metallothionein I Cu/Zn sequestration WOO835 -4.6, -6.1 metallothionein II Cu/Zn sequestration KO2236 -5.7, -6.3 erritin heavy chain iron sequestration M24509 -4.5 US 2005/02397O6 A1 Oct. 27, 2005 TABLE 1-continued Colonization-associated changes in distal small intestinal gene expression GenBank/TIGR average Gene function Reference fold A cellular energy production isocitrate dehydrogenase citric acid cycle U68564 +2.4 subunit cytochrome c oxidase subunit 1 mitochondrial electron transport TC106691 +2.4 succinyl CoA transferase ketone body utilization TC18674 +2.0 transketolase Pentose phosphate pathway O5809 +2.4 phosphogluconate Pentose phosphate pathway CS1475 +2.8 dehydrogenase malate oxidoreductase malate-asparate shuttle JO2652 +6.0 asparate aminotransferase malate-asparate shuttle JO2623 +2.5 hormonal/maturational responses adenosine deaminase adenosine inactivation +2.3 omithine decarboxylase regulation of polyamine levels +2.4 antizyme 15-hydroxyprostaglandin prostaglandin inactivation U44389 -3.2 dehydrogenase GARG-16 response to glucocorticoid U43O84 -4.0, -4.5 production FKBPS1 component of steroid receptor U16959 -3.8 complex androgen-regulated vas steroidogenesis JOS663 -3.3, -3.4 deferens protein short chain dehydrogenase steroid?retinoid metabolism AFO56.194 -2.2, -2.8 heat-stable antigen hematopoietic differentiation X53825 +3.0 marker Mucosal barrier function decay-accelerating factor complement inactivation D63679 +5.2 polymeric Ig receptor transepithelial IgA transport UO6431 +2.3 small proline-rich protein 2a crosslinking protein AJOO5559 +10.6, +102 serum amyloid A protein acute phase response U60437 +2.8, +5.4 CRP-ductinct (MUCLIN) mucin-like protein U37438 +2.4 Zeta proteasome chain antigen presentation AFO19661 +2.8 anti-DNA IgG light chain U55583 +2.5 Detoxification/drug resistance glutathione S-transferase GSH conjugation to LO6047 -2.4 electrophiles P-glycoprotein (mdrla) export of GSH-conjugated M33581 -4.6 compounds CYP2D2 4-hydroxylase TC36686 -2.6 Enteric nervous system? muscular layers L-glutamate transporter glutamate uptake U73521 +4.4 L-glutamate decarboxylase GABA production M55253 +2.2 vesicle-associated protein-33 neurotransmitter release AF157497 +2.2 cysteine-rich protein 2 cGMP kinase I target AAO2877O +3.2 smooth muscle (enteric) contractility M26689 +2.3 gamma actin SM-2O growth-factor responsive gene TC33445 +4.8 Calcium channel5 subunit calcium channel regulation A272O46 -2.2 angiogenesis angiogenin-4 unknown SEO ID NO. 29 +10.9 angiogenin-related protein unknown U22519 +6.4 angiogenin family' +2.4, +6.0, cytoskeleton/extra-cellular matrix gelsolin actin binding protein JO4953 +7.9 destrin actin depolymerizing factor W17549 +3.0 alpha cardiac actin contractility M155O1 +3.4 endoB cytokeratin intermediate filament protein m11686 +3.0 fibronectin extracellular matrix protein M1819.4 +2.9, +3.2 proteinase inhibitor 6 serine protease inhibitor U25844 +2.6 US 2005/02397O6 A1 Oct. 27, 2005 TABLE 1-continued Colonization-associated changes in distal small intestinal gene expression GenBank/TIGR average Gene function Reference fold A mpgcGO serine protease inhibitor Y11505 +2.5 alpha 1 type 1 collagen extracellular matrix protein XO6753 +2.2 +4.7 signal transduction Pten protein?lipid phosphatase U92437 +3.2 gp106 (TB2/DP1) unknown U281.68 +6.9 rac2 ras-related GTP-binding protein X53247 +7.0 Semcap2 SemaF-associated protein AFO61262 -2.9 serum and glucocorticoid- serine/threonine protein kinase AF139638 -2.6 regulated kinase STE20-like protein kinase serine/threonine protein kinase AA154321 +2.6 B-cell myeloid kinase unknown JO3O23 +2.1 general cellular functions glutathione reductase maintenance of reduced X76341 +2.9 glutathione calmodulin calcium homeostasis M27844 +2.2 e1F3 subunit translation initiation U70736 +2.7 hsc70 stress response U73744 +2.9 oligosaccharyl transferase protein N-glycosylation U84211 +3.4 subunit fibrillarin ribosomal RNA processing Z22593 +2.4 H+-transporting ATPase intracellular organelle AA108559 +2.9 acidification MSec23 component of the COPII AA116735 +2.8 complex vacuolar protein sorting 35 membrane protein recycling U47024 +2.4 0107 Additionally, the applicants have found that colo nization produces changes in expression of four genes TABLE 2-continued involved in dietary metal absorption. A high affinity epithe Real-time quantitative RT-PCR studies of colonization lial copper transporter (CRT1) mRNA was increased, while associated changes in gene expression metallothionein-I, metallothionein-II, and ferritin heavy chain mRNAs were decreased (Table 1). These changes Fold - difference Suggest that colonization engenders increased capacity to (relative to germ absorb heavy metals (e.g., via CRT1) and a concomitant Gene free) decreased capacity to Sequester them within cells (MT-I/II, adenosine deaminase (ADA) 2.6 0.6 ferritin). This implies greater host demand for these com angiogenin-4 9.1 - 18 pounds, either due to increased utilization by the host's own metabolic pathways or to competition with the microbe. These changes in gene expression (plus those of several 0108) Additionally, the applicants have found that B. thetaiota Omicron colonization produces effects that enhance other mRNAs discussed below), were independently vali intestinal barrier function. An intact mucosal barrier is dated by qRT-PCR (C. A. Heid, et al., Genome Res. 6,986 critical for accommodating the vast population of resident (1996) (see Table 2 below). intestinal microbes. Its disruption can provoke an immune TABLE 2 response that is deleterious to the host and to the stability of microbiota, leading to pathologic States Such as inflamma Real-time quantitative RT-PCR studies of colonization tory bowel disease (reviewed in, for example, P. G. Falk, et associated changes in gene expression al, Microbiol. Mol. Biol. Rev. 62, 1157 (1998); P. J. San Fold - difference sonetti, Nat Rev Immunol., 4,953 (2004)). (relative to germ 0109 B. thetoaiotaOmicron produces no detectable Gene free) inflammatory response, as judged by histologic Surveys (L. Na+/glucose cotransporter (SGLT1) 2.6 O.9 colipase 6.6 1.9 Bry, et al., Science 273, 1380 (1996)) and no discernible liver fatty acid binding protein (L-FABP) 4.4 it 1.4 induction (or repression) of the many genes, represented on metallothionein I (MTI) -5.4 - 0.7 the DNA microarrays, that are involved in these types of polymeric immunoglobulin receptor (pgR) 2.6 O.7 inflammatory responses. An influx of IgA-producing B-cells decay accelerating factor (DAF) 5.7 1.5 small proline-rich protein 2a (sprr2a) 205 - 64 does occur in the ileal mucosa 10 days after introduction of multi-drug resistance protein (mdrla) -3.8 - 1.0 B. thetaiota Omicron; Similar commensal-induced IgA glutathione S-transferase (GST) -2.1 + 0.1 responses have been shown to be T-cell independent and to lactase-phlorizin hydrolase -4.1 + 0.6 enforce barrier integrity (A. J. Macpherson, et al., Science 288, 2222 (2000)). US 2005/02397O6 A1 Oct. 27, 2005 0110 Genes involved in barrier function account for 10% invention. Laser capture microdissection (LCM) experi (7/71) of the changes in gene expression observed with B. ments described below have delineated the cellular origins thetaiotaomicron colonization. DNA microarray and qRT of this response. PCR analyses revealed that the influx of IgA producing 0114. The gut is the site of first contact of innumerable B-cells is accompanied by increased expression of the ingested toxins and Xenobiotics. The relative contributions polymeric immunoglobulin receptor (plgR) that transports of luminal bacteria and the epithelium to detoxification and IgA across the epithelium (Tables 1, 2). There is also metabolism of these compounds has been difficult to delin augmented expression of the CRP-ductin gene, encoding eate in conventionally-raised mammals. It has been found both a component of the protective mucus layer overlying that colonization of germ-free mice with B. thetaiota Omi the epithelium (MUCLIN; R. C. DeLisle, et al., Am. J cron results in reduced expression of Several genes involved Physiol. 275, G219 (1998)) and a putative receptor for in these processes (Table 1). There is a decrease in the host trefoil peptides that participate in fortification/healing of the mRNA encoding glutathione S-transferase, which detoxifies intestinal mucosa (L. Thim, et al., Regul. Pept. 90, 61 a variety of electrophiles, and a corresponding decrease in (2000)). Additionally, there is increased expression of decay multi-drug resistance protein-1 (Mdr-1), which exports glu accelerating factor (DAF), an apical epithelial Surface pro tathione-conjugated compounds from the epithelium (R. W. tein that inhibits complement-mediated cytolysis (M. E. Johnstone, et al., Trends Biochem. Sci. 25, 1 (2000)). Medof, et al., J. Exp. Med 165, 848 (1987)). Coincident Expression of CYP2D2 (debrisoquine hydroxylase) enhancement of plgR, MUCLIN, and DAF expression involved in oxidative drug metabolism in humans (M. should not only help prevent bacteria from crossing the Ingelman-Sundberg, et al., Trends Pharmacol. Sci. 20, 342 epithelial barrier, but should also prevent mucosal damage (1999)), also declines with colonization. A genetic polymor that may ensue from microbial activation of complement phism that produces a deficiency in this cytochrome P-450 components present in intestinal Secretions. is common in humans and associated with altered oxidative 0111. The most pronounced response to B. thetaiota Omi drug metabolism (M. Ingelman-Sundberg, et al., Trends cron was an increase in Small proline-rich protein-2 (sprr2a) Pharmacol. Sci. 20, 342 (1999)). The reduced expression of mRNA (Table 1). qRT-PCR analysis established that there these three host genes Suggests that components of the wass a 205-64-fold elevation in this mRNA with coloniza microbiota, Such as B. thetaiotaomicron, contribute to the tion (Table 2), and that this response had microbial speci detoxification of compounds that could be deleterious to the ficity (FIG. 3). Sprr2a is a member of a family of proteins host. This indicates that a component of the normal intestinal associated with terminal differentiation of Squamous epithe microbiota can modulate host genes involved in drug lial cells. SprrS contribute to the barrier functions of Squa metabolism, and underScore how variations in Such metabo mous epithelia, both as a component of the comified cell lism between individuals may arise from differences in the envelope, and as cross-bridging proteins linked to desmo composition of their resident intestinal microbial commu Somal desmoplakin, a prominent desmosomal constituent (P. nities. Consequently, evaluation of the effect of indigenous M. Steinert, et al., Mol. Biol. Cell 10. 4247 (1999)). Colo gut bacterial Species on expression of these genes using the nization did not produce a notable change (i.e. two-fold or method of the invention may be helpful-both as means for more), in the expression of genes encoding other proteins testing the role of the microbiota in metabolism of drugs, linked to desmosomes (desmoplakin, plakoglobin, plakophi and for identifying novel microbial biotransformation activi lin, plectin), or tight junctions (ZO-1, occludin). ties that could be used to develop more or less active forms of drugs. 0112 Sprr2a expression in the intestine and its microbial regulation are novel findings. The critical contribution of 0115 The motility of the intestine is regulated by its Sprr2a to the Squamous epithelial barrier and the dramatic enteric nervous system (ENS). The relative contributions of response of Sprr2a expression to B. thetaiota Omicron intrinsic and extrinsic factors to ENS activity are poorly together Suggest that this protein plays an important role in understood, despite the fact that irritable bowel Syndrome, intestinal barrier function. It is therefore a particularly which involves dysregulated motor activity, is a major health Suitable target for further investigation in accordance with problem. The impact of components of the microbiota, Such the invention, in particular by evaluating the biochemical as B. thetaiota Omicron, on gut physiology extends to genes pathway in which Sprr2a participates in intestinal barrier expressed in the enteric nervous system (ENS) and in the functions, the mechanism by which B. thetaiota Omicron muscular layerS. mRNAS encoding the L-glutamate trans regulates Sprr2a expression and the utility of using B. porter and L-glutamate decarboxylase, which converts glutamate to GABA, are both increased, Suggesting a colo thetaiota Omicron as a probiotic to enhance intestinal barrier nization-associated effect on the glutamatergic neurons of function. the ENS (M. T. Liu, et al., J. Neurosci. 17, 4764 (1997)). 0113. Using the method of the invention, it has been Enhanced expression of vesicle-associated protein-33, a found that colonization results in increased expression of Synaptobrevin-binding protein involved in neurotransmitter angiogenin-4 which resembles angiogenin-3, a Secreted pro release (P. A. Skehel, et al., Proc. Natl. Acad. Sci. U.S.A. 97, tein with demonstrated angiogenic activity (X. Fu, et al., 1101 (2000)) is also observed. There is a concomitant Mol. Cell Biol. 17, 1503 (1997), X. Fu, et al., Growth increase in two muscle-specific mRNAS. enteric y-actin and Factors 17, 125 (1999)). The 11-fold increase in expression cysteine-rich protein 2. Previous electrophysiological Stud of the angiogenesis factor recognizable by amplification ies of germ-free and conventionally-raised animals have using primers of SEQID NO 12 and SEQ ID NO 25, which Suggested that the microbiota plays a role in gut motility (E. is angiogenin-4 (Table 1, 2) upon B. thetaiota Omicron colo Husebye, et al., Dig. Dis. Sci. 39,946 (1994)). The method nization represents a novel mode of regulation for this or of the invention can provide molecular details about how other new putative angiogenesis factors, and So may be the resident gut microbes, Such as B. thetaiota Omicron, may act Subject of further investigation in accordance with the to modulate intestinal motility. US 2005/02397O6 A1 Oct. 27, 2005 0116 Expression profiling revealed surprisingly that idea that bacteria Serve as upstream effectors of a cascade colonization of adult germ-free mice with B. thetaiota Omi that affects gut maturation. Some changes in gut maturation cron elicits other responses that mimic changes that nor asSociated with the Suckling-weaning transition are thought mally occur in the maturing intestine of conventionally to be regulated by increases in glucocorticoids (S. J. Hen reared animals. Expression of lactase, which hydrolyzes the ning, et al., in Physiology of the Gastrointestinal Tract, L. R. principal milk Sugar (lactose), normally declines during the Johnson, Ed. (Raven Press, New York. 1994), pp. 584-586)). weaning period (S. D. Krasinski, et al., Am. J Physiol. 267, B. thetaiota Omicron colonization as described hereinafter G584 (1994)). Colonization of adult germ-free mice with B. was accompanied by reduced expression of two genes thetaiota Omicron produces a decrease in ileal lactase mRNA whose transcription is known to be Suppressed by glucocor (Table 1, 2). Adenosine deaminase (ADA) and polyamines ticoids: I5-hydroxyprostaglandin dehydrogenase (M. D. (spermine, Spermidine) play important roles in postnatal Mitchell, et al., ProStaglandins Leukot. Essent. Fatty Acids intestinal maturation (G. D. Luk, et al., Science 210, 195 62, 1 (2000)) and glucocorticoid-attenuated response gene (1980); J. M. Chinsky, et al., Differentiation 42, 172 (1990)). 16 (J. B. Smith, et al., J. Biol. Chem 270, 16756 (1995)). It has been found that B. thetaiotaOmicron colonization Furthermore, there was reduced expression of another gene produces an increase in mRNAS encoding ADA and orni whose product interacts with nuclear hormone receptor thine decarboxylase (ODC) antizyme. The antizyme, whose family members, the immunophilin FKBP5I (S. C. Nair, et expression is affected by polyamine levels, is a critical al., Mol. Cell. Biol. 17. 594 (1997)). regulator of ODC turnover (J. Nilsson, et al., Eur: J Biochem. 250, 223 (1997)); an increase in antizyme mRNA levels 0117. As mentioned above, the applicants have found that therefore Suggests that colonization influences ileal a particular member of the angiogenin family, whose gene is polyamine Synthesis. These data demonstrate that genes amplifiable using primers of SEQ ID NO 12 and 25 (Table controlling Synthesis of two classes of regulators of gut 3 below) and is expressed in mouse intestine, is novel. Thus, maturation, adenosine and polyamines, are themselves this protein and the gene encoding it forms a further aspect modulated by a component of the microbiota, leading to the of the invention. TABLE 3 SEQ SEQ ID D gene name forward primer NO reverse primer NO Na+/glucose 5'-CAGAGACCCCATTACTGGAG 1 5'-TCGTTGCACAATGACCTGATC 4 cotransporter ACA (SGLT1) colipase 5-TGACACCATCCTGGGCATT 25'-ACACCGGTAGTAAATCCCATAA 5 AGG liver fatty acid 5'-CTCCGGCAAGTACCAATTGC 35'-TGTCCTTCCCTTTCTGGATGAG 6 binding protein (L-FABP) metallothionein.I 5'-ATGTGCCCAGGGCTGTGT 4 5'-AACAGGGTGGAACTGTATAGGA 7 (MT-I) AGAC polymeric immunoglobulin 5'-CTTCCCTCCTGTCCCAGAGGT 5 5'-GGCGTAACTAGGCCAGGCTT 8 receptor (pIgR) decay accelerating 5'-CAACCCAGGGTACAGGCTAGTC 65'-GGTGGCTCTGGACAATGTAT 9 factor (DAF) TTC small proline-rich 5'-CCTTGTCCCCCCAAGCG 7 5'-AGGGCATGTTGACTGCCAT 20 protein 2a (sprir2a) multi-drug resistance 5'-GCCGCTTCTTCCAAAGTCTACA 8 5'-CGTGTCTCTACTCCCGGTTTCC 21 protein (mdrla) glutathione S-transferase 5'-CATCCAGCTCCTAGAAGCCATT 9 5'-GGGTTGCAGGAACTTCTTAATT 22 (GST) GTA lactase-phlorizin 5'-TTGAATGGGCCACAGGCT 105'-AGCGGACTATGGAGGCGTAG 23 hydrolase adenosine deaminase 5'-GCGCAGTAAAGAATGGCATTC 11 5'-CTGTCTTGAGGATGTCCACAGC 24 (ADA) angiogenin- 4 5'-TCGATTCCAGGTCACCACTTG 12 5'-CACAGGCAATAACAATATATCT 25 GAAATCT US 2005/02397O6 A1 Oct. 27, 2005 TABLE 3-continued SEQ SEQ ID ID gene name forward primer NO reverse primer NO glyceraldehyde 5'-TGGCAAAGTGGAGATTGTTGCC 135'-AAGATGGTGATGGGCTTCGCG 26 3-phosphate dehydrogenase 0118. A further aspect of the invention provides a protein of SEQ ID NO 29 as shown in FIG. 4 hereinafter, or an TABLE Z-continued allelic variant thereof or a protein which has at least 85% amino acid sequence identity with SEQ ID NO 29. In Species PubMed Ref. particular, the invention provides a protein of SEQ ID NO. SuS Scrofa AY307772 29. In yet a further aspect, the invention provides a nucleic BoS taurus AY192008 acid that encodes a protein as described above. These Pan troglodytes AY411895 proteins are useful as a target for the Screening process of the invention. 0.124. In certain aspects, a polypeptide that is a homolog, 0119) II. Modulation of Fiaf and the Gastrointestinal ortholog, mimic or degenerative variant of a Fiaf polypep Microbiota as a Means to Control Energy Storage in a tide is also Suitable for use in the present invention. In Subject particular, the subject polypeptide will typically inhibit LPL 0120) The applicants have discovered, as detailed in when administered to the Subject. Section I, that B. thetaioatOmicron alone, or a more complex 0.125. A number of methods may be employed to deter microbiota, modulates expression of a Subject's Fiaf. It has mine whether a particular homolog, mimic or degenerative further been discovered, as detailed in the examples below, variant possesses Substantially similar biological activity that the microbiota regulates a Subjects energy storage in relative to a Fiaf polypeptide. Specific activity or finction part by Selectively Suppressing a Subject's gastrointestinal may be determined by convenient in Vitro, cell-based, or in transcription of Fiaf Referring to FIG. 18, the gut micro Vivo assays, Such as measurement of LPL activity in white biota effects a Subject's energy Storage through Fiaf by adipose tissue or in the heart. In order to determine whether coordinating increased digestion of dietary polysaccharides, a particular Fiaf polypeptide inhibits LPL, the procedures increased hepatic lipogenesis and increased LPL activity in detailed in lo the examples may be followed. adipocytes, thereby promoting Storage of calories harvested from the diet to fat. Taking advantage of these discoveries, 0.126 In addition to having a substantially similar bio the present invention provides compositions and methods logical function, a homolog ortholog, mimic or degenerative that may be employed for decreasing body fat and for variant Suitable for use in the invention will also typically promoting weight loSS in a Subject. share Substantial Sequence Similarity to a Fiaf polypeptide. In addition, Suitable homologs, ortholog, mimic or degen 0121 (A) Modulation of Fiaf erative variants preferably share at least 30% sequence 0.122 One aspect of the present invention provides a homology with a Fiaf polypeptide, more preferably, 50%, method to regulate fat Storage and weight loSS in a Subject and even more preferably, are greater than about 75% by modulating the amount of or the activity of Fiaf. To homologous in Sequence to a Fiaf polypeptide. Alternatively, decrease body fat and promote weight loss, the amount of or peptide mimics of Fiaf could be used that retain critical the activity of Fiaf is increased in the subject. molecular recognition elements, although peptide bonds, Side chain Structures, chiral centers and other features of the 0123. In one embodiment, Fiaf may be increased by parental active protein Sequence may be replaced by chemi administering a Suitable Fiaf polypeptide to the Subject. cal entities that are not native to Fiaf protein yet, neverthe Typically, a Suitable Fiaf polypeptide is one that can Sub less, confer activity. stantially inhibit LPL when administered to the subject. A number of Fiaf polypeptides known in the art are suitable for 0127. In determining whether a polypeptide is substan use in the present invention. Generally Speaking, the Fiaf tially homologous to a Fiaf polypeptide, Sequence Similarity polypeptide is from a mammal. By way of non limiting may be determined by conventional algorithms, which typi cally allow introduction of a Small number of gaps in order example, Suitable Fiaf polypeptides and nucleotides are to achieve the best fit. In particular, "percent homology of delineated in Table Z. two polypeptides or two nucleic acid Sequences is deter mined using the algorithm of Karlin and Altschul (Proc. TABLE Z Natl. Acad. Sci. USA 87, 2264 (1993)). Such an algorithm Species PubMed Ref. is incorporated into the NBLAST and XBLAST programs of Homo Sapiens NM 139314 Altschul, et al. (J. Mol. Biol. 215, 403 (1990)). BLAST NM O16109 nucleotide searches may be performed with the NBLAST Mus musculus NM 020581 program to obtain nucleotide Sequences homologous to a Rattus norvegicus NM 1991.15 nucleic acid molecule of the invention. Equally, BLAST protein searches may be performed with the XBLAST US 2005/02397O6 A1 Oct. 27, 2005 program to obtain amino acid Sequences that are homolo activates expression through interactions with components gous to a polypeptide of the invention. To obtain gapped of host regulatory networks that control Fiaftranscription. alignments for comparison purposes, Gapped BLAST is For example, Such an agent could be identified by Screening utilized as described in Altschul, et al. (Nucleic Acids Res. natural product and/or chemical libraries using the gnotobi 25, 3389 (1997)). When utilizing BLAST and Gapped otic Zebrafish model described below as a bioassay. BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are employed. See 0.134. In another embodiment, a chemical entity could be http://www.ncbi.nlm.nih.gov for more details. used that interacts with Fiaftargets Such as LPL to reproduce the effects of Fiaf (e.g., in this case inhibition of LPL 0128 Fiaf polypeptides suitable for use in the invention activity). are typically isolated or pure and are generally administered as a composition in conjunction with a Suitable pharmaceu 0135) In another alternative of this embodiment, Fiaf tical carrier, as detailed below. A pure pplypeptide consti expression and/or activity may be increased by administer tutes at least about 90%, preferably, 95% and even more ing a Fiaf agonist to the Subject. In one preferred embodi preferably, at least about 99% by weight of the total ment, the Fiaf agonist is a peroxisome proliferator-activated polypeptide in a given Sample. receptor (PPARs) agonist. Suitable PPARs include PPARC, PPARB/ö, and PPARY. Fenofibrate is another suitable 0129. The Fiaf polypeptide may be synthesized, pro example of a Fiaf agonist. Additional Suitable Fiaf agonists duced by recombinant technology, or purified from cells and methods of administration are further described in using any of the molecular and biochemical methods known Manards, et al., J. Biol Chem, 279, 34411 (2004), and U.S. in the art that are available for biochemical Synthesis, Patent Publication No. 2003/0220373, which are both molecular expression and purification of the Fiaf polypep hereby incorporated by reference in their entirety. tides see e.g., Molecular Cloning, A Laboratory Manual 0.136. In yet another a further alternative of this embodi (Sambrook, et al. Cold Spring Harbor Laboratory), Current ment, Fiafis increased in a Subject by altering the microbiota Protocols in Molecular Biology (Eds. Ausubel, et al., Greene population in the Subject's gastrointestinal tract Such that the Publ. Assoc., Wiley-Interscience, New York). microbial-mediated Suppression of Fiaf in the Subject is 0130 Expression vectors that may be effective for the decreased. Suitable methods for altering the microbial popu expression of Fiaf polypeptides include, but are not limited lation are described in detail in section II (B). to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.), 0.137 (B) Alteration of the Gastrointestinal Microbiota PCMV-SCRIPT, PCMV-TAG, PEGSHIPERV (Stratagene, Population La Jolla Calif.), and PTET-OFF, PTETON, PTRE2, 0.138 Another aspect of the present invention provides a PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). Fiaf method to regulate fat Storage and weight loSS in a Subject polypeptides may be expressed using (i) a constitutively by altering the microbial population in the Subject's gas active promoter, (e.g., from cytomegalovirus (CMV), Rous trointestinal tract. To decrease body fat and promote weight sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), loSS, the microbiota is altered Such that at least one micro or P f-actin genes), (ii) an inducible promoter (e.g., the bial-mediated Signaling pathway in the Subject that regulates tetracycline-regulated promoter (Gossen, et al., Proc. Natl. energy Storage is either Substantially inhibited or Stimulated, Acad. Sci. USA, 89,5547 (1992); M. Gossen, et al., Science, whereby Stimulating or inhibiting the Signaling pathway 268, 1766 (1995); F. M., Rossi, et al., Curr. Opin. Biotech causes a decrease in body fat or promotes weight loSS in the nol. 9, 451 (1998), commercially available in the T-REX Subject. In one embodiment, the microbiota population may plasmid (Invitrogen)); the ecdysone-inducible promoter be altered Such that microbial-mediated transcriptional Sup (available in the plasmids. PVGRXR and PIND; Invitrogen); pression of a LPL inhibitor, such as Fiaf, is decreased in the the FK506/rapanmycin inducible promoter; or the RU486/ Subject and results in a decrease of triglyceride Storage in the mifepristone inducible promoter (F.M. Rossi, et all, Supra)), adipocytes of the Subject. In a certain embodiment, Fiaf is or (iii) a tissue-specific promoter or the native promoter of Selectively increased only in the gastrointestinal tract of the the endogenous gene encoding Fiaf from a normal indi Subject. In yet another embodiment, the microbiota popula vidual. tion may be altered Such that a signaling pathway that 0131 Commercially available liposome transformation regulates hepatic lipogenesis is Substantially inhibited, kits (e.g., the PERFECT LIPID TRANSFECTION KIT, thereby resulting in a decrease of triglyceride Storage in the available from Invitrogen) allow one with ordinary skill in adipocytes of the Subject. In one embodiment, hepatic the art to deliver Fiaf polynucleotides to target cells in lipogenesis is Substantially inhibited as a result of a decrease culture, and require minimal effort to optimize experimental in microbial processing of dietary polysaccharides. parameters. Alternatively, transformation is performed using 0.139. Accordingly, in one embodiment, the subject's the calcium phosphate method (F. L. Graham, et al., Virol gastrointestinal microbial population is altered So as to ogy, 52, 456 (1973), or by electroporation (E. Neumann, et decrease body fat and promote weight loSS in the Subject. In al., EMBOJ, 1,841 (1982)). one alternative of this embodiment, the presence of 0132) A Fiaf peptide can be synthesized using traditional microbes that Suppress Fiaf transcription may be decreased. Solid-phase methods. In one alternative of this embodiment, the presence of Saccharolytic microbes, Such as Bacteroides, is decreased. 0133. In another alternative of this embodiment, an agent (Saccharolytic microbes typically degrade complex, other can be delivered that Specifically activates Fiaf expression: wise indigestible dietary polysaccharides that the Subject this agent could represent a natural or Synthetic compound cannot.) In another alternative embodiment, the presence of that directly activates Fiaf gene transcription, or indirectly microbes that ferment Sugars to short chain fatty acids is US 2005/02397O6 A1 Oct. 27, 2005 decreased. In Still another embodiment, the presence of 0145 An additional embodiment of the invention relates microbes that increase the uptake of microbial and diet to the administration of a composition that generally com derived monosaccharides (e.g., glucose, fructose and galac prises an active ingredient formulated with a pharmaceuti tose) by the host is decreased. cally acceptable excipient. Excipients may include, for 0140. To decrease the presence of any of the microbes example, Sugars, Starches, celluloses, gums, and proteins. detailed above, methods generally known in the art may be Various formulations are commonly known and are thor utilized. In one embodiment, a Suitable probiotic is admin oughly discussed in the latest edition of Reminton's Phar istered to the Subject. Generally Speaking, Suitable probiotics maceutical Sciences (Maack Publishing, Easton Pa.). Such include those that alter the representation or biological compositions may consist of a Fiaf polypeptide or Fiaf properties of microbiota populations that are involved in a peptidomimetic. Subjects uptake of energy. By way of non-limiting example, 0146 The compositions utilized in this invention may be Suitable probiotics include Lactobacillus, Acidophillus and administered by any number of routes including, but not Bifidobacteria, each of which is commercially available limited to, oral, intravenous, intramuscular, intra-arterial, from Several Sources. In another embodiment, microbes that intramedullary, intrathecal, intraventricular, pulmonary, induce Fiaf expression in the Subject's gastrointestinal tract transdermal, Subcutaneous, intraperitoneal, intranasal, may be administered to the Subject. In yet another embodi enteral, topical, Sublingual, or rectal means. ment, Selective reduction in the representation of compo 0147 The actual effective amounts of compound nents of the microbiota, Such as Saccharolytic bacteria, is described herein can and will vary according to the Specific achieved by administering an antibiotic to the Subject. In yet composition being utilized, the mode of administration and another embodiment, Selective reduction in the representa the age, weight and condition of the Subject. Dosages for a tion of components of the microbiota, Such as Saccharolytic particular individual Subject can be determined by one of bacteria, is achieved with antibiotics. ordinary skill in the art using conventional considerations. 0.141. In yet another embodiment, a subject may be Those skilled in the art will appreciate that dosages may also administered a diet that alters the microbiota population So be determined with guidance from Goodman & Gilman's as to decrease body fat and promote weight loSS in the The Pharmacological Basis of Therapeutics, Ninth Edition Subject. (1996), Appendix II, pp. 1707-1711 and from Goodman & Gilman's The Pharmacological Basis of Therapeutics, Tenth 0142 (C) Combination Therapy Edition (2001), Appendix II, pp. 475-493. 0143 Another aspect of the invention encompasses a combination therapy to regulate fat Storage and weight loSS 0148 (D) Methods for Treating Weight-Related Disor in a Subject. In one embodiment, the invention encompasses ders a composition for decreasing body fat or for promoting 0149. A further aspect of the invention encompasses the weight loSS. Typically, the composition comprises a Fiaf use of the methods to regulate fat Storage and weight loSS polypeptide and an agent that alters the microbiota popula gain in a Subject as a means to treat weight-related disorders. tion in a Subject's gastrointestinal tract Such that microbial In one embodiment, weight-related disorders are treated by mediated transcriptional Suppression of a LPL inhibitor in modulating the amount of or the activity of Fiaf, as detailed the Subject is decreased. Suitable Fiaf polypeptides and in II(A). In another embodiment, weight-related disorders agents that alter the microbiota population are detailed are treated by altering a Subject's gastrointestinal microbial above. population, as detailed II(B). In still another embodiment, weight-related disorders are treated by administering the 0144. In other embodiments, any of the proteins or polypeptides, agonists, of the invention as detailed in Section combination therapy, as detailed II (C). II may be administered in combination with other appropri 0150. In one particularly preferred embodiment, the ate therapeutic agents. Selection of the appropriate agents weight-related disorder is obesity or an obesity-related dis for use in combination therapy may be made by one of order. A Subject in need of treatment for obesity is diagnosed ordinary skill in the art, according to conventional pharma and is then administered any of the treatments detailed ceutical principles. Generally Speaking, agents will include herein, Such as in Sections II (A), (B), or (C). Typically, a those that decrease body fat or promote weight loSS by a subject in need of treatment for obesity will have at least one mechanism other the mechanisms detailed herein. In one of three criteria: (i) BMI over 30; (ii) 100 pounds over embodiment, acarbose may be administered with any com weight; or (iii) 100% above an “ideal” body weight. In pound described herein. Acarbose is an inhibitor of C-glu addition, obesity-related disorders that may be treated by the cosidases and is required to break down carbohydrates into methods of the invention include metabolic Syndrome, type Simple Sugars within the gastrointestinal tract of the Subject. II diabetes, hypertension, cardiovascular disease, and non In another embodiment, an appetite Suppressant Such as an alcoholic fatty liver disease. amphetamine or a Selective Serotonin reuptake inhibitor Such 0151 (E) Biomarkers and Screenine for Compounds that as Sibutramine may be administered with any compound Modulate Fiaf Expression or Activity described herein. In Still another embodiment, a lipase inhibitor such as orlistat or an inhibitor of lipid absorption 0152. A further aspect of the invention provides biomar Such as Xenical may be administered with any compound kers that may be utilized in predicting whether a Subject is described herein. The combination of therapeutic agents at risk for becoming obese or Suffering from an obesity may act Synergistically to decrease body fat or promote related condition. In one embodiment, the biomarker is weight loss. Using this approach, one may be able to achieve serum Fiaf levels. In a further embodiment, the biomarker is therapeutic efficacy with lower dosages of each agent, thus gastrointestinal levels of microbiota that SuppreSS Fiaf tran reducing the potential for adverse Side effects. Scription. US 2005/02397O6 A1 Oct. 27, 2005 0153. Yet another aspect of the invention encompasses active in the intestinal epithelium (e.g. nucleotides -1178 to methods to identify microbial produced compounds that +28 of the rat intestinal fatty acid binding protein gene) modulate Fiaf transcription or activity and non microbial linked to Fiaf could be introduced into Fiaf-l-mice So the produced compounds that modulate Fiaf transcription or effects of Fiaf activation can be studied and additional activity. Generally speaking, methods generally known in targets for pharmacologic manipulation of Fiaf-related path the art, Such as those described in Section I, may be utilized ways that lead to reduced adiposity can be performed. to identify compounds that modulate Fiaf transcription or activity. In one embodiment, a method for Screening for a 0159. A variety of protocols for measuring Fiaf polypep compound that is effective in altering expression of a poly tides, including ELISAS and RIAS, and may be used in any nucleotide encoding a Fiaf polypeptide is provided, Such as of the Screening methods delineated above. in gnotobiotic Zebrafish as shown in Example 10. DEFINITIONS 0154) In one embodiment, a method for screening for a compound that is effective in altering expression of a poly 0160 Acc1 stands for acetyl-CoA carboxylase. nucleotide (gene) encoding a Fiaf polypeptide is provided. 0.161 The term “antagonist” refers to a molecule that Effective compounds may alter polynucleotide expression inhibits or attenuates the biological activity of a Fiaf by acting on transcriptional or translational regulators of polypeptide and in particular, the ability of Fiaf to inhibit Fiaf expression. LPL. Antagonists may include proteins Such as antibodies, 0.155. At least one, and up to a plurality, of test com nucleic acids, carbohydrates, Small molecules, or other com pounds may be Screened for effectiveness in altering expres pounds or compositions that modulate the activity of a Fiaf Sion of a specific Fiaf polynucleotide. A test compound may polypeptide either by directly interacting with the polypep be obtained by any method commonly known in the art, tide or by acting on components of the biological pathway including but not limited to Selection from an existing, in which Fiaf participates. commercially-available or proprietary library of naturally 0162 The term “agonist” refers to a molecule that occurring or non-natural chemical compounds, Selection enhances or increases the biological activity of a Fiaf from a library of chemical compounds created combinato polypeptide and in particular, the ability of Fiaf to inhibit rially or randomly, or purification from a natural product, LPL. Agonists may include ptoteins, peptides, nucleic acids, Such as extracts of gut microbes grown in Vitro or from carbohydrates, Small molecules (e.g., Such as metabolites), conditioned medium harvested after culture of a gut microbe or other compounds or compositions that modulate the or collection of gut microbes. Alterations in the expression activity of a Fiaf polypeptide either by directly interacting of a polynucleotide encoding a Fiaf polypeptide may be with the polypeptide or by acting on components of the assayed by a number of methods commonly known in the art biological pathway in which Fiaf participates. including but not limited to qRT-PCR, as described above. Detection of a change in the expression of a Fiaf polynucle 0163 The term “altering” as used in the phrase “altering otide, or its protein product, indicates that the test compound the microbiota population' is to be construed in its broadest is effective in altering Fiaf gene expression. Another interpretation to mean a change in the representation of embodiment is to observe changes in expression of a trans microbes in the gastrointestinal tract of a Subject. The gene containing Fiaf transcriptional regulatory elements change may be a decrease or an increase in the presence of responsive to microbial Signals, linked to an open reading a particular microbial Species. frame encoding a fluorescent protein reporter, in gnotobiotic 0.164 “BMI” as used herein is defined as a human Zebrafish. Subjects weight (in kilograms) divided by height (in meters) 0156 Another embodiment is to test the activity of Fiaf Squared. peptides, peptidomimetics or related compounds in germ 0165 CHREBP stands for carbohydrate response ele free Fiaf-l-mice to determine whether they reduce their high ment binding protein. fat content. 0166 CONV-D stands for conventionalization of germ O157 Another aspect of the invention encompasses the free animals with a gut microbiata harvested from conven use of a Fiaf polypeptide to Screen for compounds that tionally-raised donor animals. modulate the activity of the Fiaf polypeptide. Such com pounds may include agonists as detailed above. In one 0.167 CONV-R stands for conventionally raised, i.e., embodiment, an assay is performed under conditions per acquiring microbes beginning at birth. "Conservative amino missive for Fiaf polypeptide activity, wherein the Fiaf acid Substitutions are those Substitutions that are predicted polypeptide is combined with at least one test compound, to least interfere with the properties of the original protein, and the activity of the Subject polypeptide in the presence of i.e., the Structure and especially the function of the protein a test compound is compared with the activity of the Fiaf is conserved and not significantly changed by Such Substi polypeptide in the absence of the test compound. Activity tutions. could, for example, be defined as the capacity to inhibit 0168 A“detectable label” refers to a reporter molecule or LPL-catalyzed biochemical reactions in vitro. A change in enzyme that is capable of generating a measurable Signal the activity of Fiaf in the presence of the test compound is and is covalently or noncovalently joined to a polynucle indicative of a compound that modulates the activity of Fiaf otide or polypeptide. polypeptides. At least one and up to a plurality of test 0169. An “effective amount” is a therapeutically-effec compounds may be Screened. tive amount that is intended to qualify the amount of agent 0158. In another embodiment, a transgene consisting of that will achieve the goal of a decrease in body fat, or in transcriptional regulatory elements that are constitutively promoting weight loSS. Fas Stands for fatty acid Synthase. US 2005/02397O6 A1 Oct. 27, 2005 0170 Fiaf stands for fasting-induced adipocyte factor. contained in the above description and in the examples given 0171 A“gene” is a hereditary unit that has one or more below, shall be interpreted as illustrative and not in a Specific effects upon the phenotype of the organism, and that limiting Sense. can mutate to various allelic forms. EXAMPLES 0172 GF stands for germ free. 0.184 The following examples illustrate the invention. 0173 LPL stands for lipoprotein lipase. 0.174 A“nucleic acid” is a nucleotide polymer of DNA or 0185. Part I. Examples 1-4 correspond to section I of the RNA, it consists of purine or pyrimidine base, e.g. with detailed description. EXAMPLE 1. asSociated pentose Sugars, and phosphate groups. 0186 Age-matched groups of 7-15 week-old germ-free 0175 PPAR stands for peroxisome proliferator-activator NMRI/KI mice were maintained in plastic gnotobiotic iso receptor. lators on a 12 hour light cycle, and given free access to an autoclaved chow diet (B&K Universal). Males were inocu 0176 “Peptide' is defined as a compound formed of two lated with wild-type B. thetaiotaomicron (strain VPI-5482) or more amino acids, with an amino acid defined according (L. Hooper, et al. (1999) supra). Mice were sacrificed 10 to Standard definitions. days later, 2 hours after lights were turned on. The distal 1 0177. The term “pharmaceutically acceptable” is used cm of the Small intestine was used to define the number of adjectivally herein to mean that the modified noun is appro colony forming units per ml of extruded luminal contents. priate for use in a pharmaceutical product; that is the 0187 Ileal RNA was isolated from mice with >107 “pharmaceutically acceptable' material is relatively Safe colony forming units (CFU) of bacteria per ml of luminal and/or non-toxic, though not necessarily providing a sepa contents. Earlier studies had shown that 10 days was rable therapeutic benefit by itself. Pharmaceutically accept Sufficient to produce robust colonization of the ileum and able cations include metallic ions and organic ions. More that =107 CFU/ml were necessary for full induction of preferred metallic ions include, but are not limited to appro fucosylated glycan production in the ileal epithelium (L. priate alkali metal Salts, alkaline earth metal Salts and other physiologically acceptable metal ions. Exemplary ions Hooper, et al., (1999) supra; L. Bry, et al., Science 273, 1380 include aluminum, calcium, lithium, magnesium, potassium, (1996))). Sodium and Zinc in their usual valences. Preferred organic 0188 Total ileal RNA samples were prepared from the 3 ions include protonated tertiary amines and quaternary cm of intestine adjacent the distal 1 cm of the Small intestine ammonium cations, including in part, trimethylamine, of 4 mice from 3 independent colonizations, and from age diethylamine, N,N'-dibenzylethylenediamine, chlorop and gender-matched germ-free mice (n=8), using a RNA rocaine, choline, diethanolamine, ethylenediamine, meglu (Qiagen RNeasy kit). Ileal RNAS from each treatment group mine (N-methylglucamine) and procaine. Exemplary phar were pooled, in equal amounts, for generation of biotiny maceutically acceptable acids include without limitation lated cRNA targets. Two targets were prepared, indepen hydrochloric acid, hydrobromic acid, phosphoric acid, Sul dently, from 30 lug of each total cellular RNA pool, using the furic acid, methaneSulfonic acid, acetic acid, formic acid, method outlined by C. K. Lee, et al., Science 285, 1390 tartaric acid, maleic acid, malic acid, citric acid, isocitric (1999)). acid, Succinic acid, lactic acid, gluconic acid, glucuronic acid, pyruvic acid, oxalacetic acid, fumaric acid, propionic 0189 SYBR green-based real-time quantitative RT-PCR studies (N. Steuerwald, et al., Mol. Hum. Reprod., 5, 1034 acid, aspartic acid, glutamic acid, benzoic acid, and the like. (1999)) were performed using the gene-specific primers 0.178 A“polypeptide' is a polymer made up of less than listed in Table 3 above and DNAse-treated RNAS. Control 350 amino acids. experiments established that the Signal for each amplicon 0179) “Protein' is defined as a molecule composed of one was derived from cDNA and not from primer dimers or or more polypeptide chains, each composed of a linear chain genomic DNA. Signals were normalized to an internal of amino acids covalently linked by peptide bonds. Most reference mRNA (glyceraldehyde 3-phosphate dehydroge proteins have a mass between 10 and 100 kilodaltons. A nase). The normalized data were used to quantitate the levels protein is often Symbolized by its mass in kDa. of a given mRNA in germ-free and colonized ileums (AACT analysis; Bulletin #2, ABI Prism 7700 Sequence Detection 0180. SREBP-1 stands for sterol response element bind System). ing protein 1. 0190. Each cRNA was hybridized to Affymetrix Mu11 K 0181 “Subject” as used herein typically is a mammalian and Mu19K chip sets representing about -25,000 unique Species. Non-limiting examples of Subjects that may be mouse genes from Unigene Build 4 and the TIGR cluster treated by the methods of the invention include a human, a databases, according to Affymetrix protocols. Data collected dog, a cat, a cow, a horse, a rabbit, a pig, a sheep, a goat, as from each chip were Scaled So that the overall fluorescence well as non-mammalian species including an avian Species intensity across each chip was equivalent (target intensity and a fish Species. =150). Pairwise comparisons of germ-free versus 'colo 0182 A“vector” is a self-replication DNA molecule that nized expression levels were performed. transferS a DNA segment to a host cell. 0191) A 2-fold or more difference was recorded if three 0183 AS various changes could be made in the above criteria were met: the GeneChip software returned a differ compounds, products and methods without departing from ence call of “increased' or “decreased,” the mRNA was the Scope of the invention, it is intended that all matter called present by GeneChip software in either germ-free or US 2005/02397O6 A1 Oct. 27, 2005 colonized cRNA, and the difference was observed in dupli nella, an invasive gut pathogen (L. Eckmann, et al., J. Biol. cate microarray hybridizations. Chem. 275. 14084 (2000)). The lack of evidence for an 0.192 mRNAS represented by 118 probe sets changed by evoked in Vivo immuno-inflammatory response is consistent at least 2-fold with colonization, as defined by duplicate with the host's need to accommodate resident gut microbes, microarray hybridizations. Such as B. thetaiota Omicron, for its entire lifespan. 0193 It was found that transcripts represented by 95 EXAMPLE 2 probe-Sets were increased, while those lo represented by 23 probe-Sets were decreased. The genes represented by 84 of 0198 In a further analysis two techniques were com these probe sets (71 unique genes) were assigned to fumc bined. First, laser-capture microdissection (LCM) was used tional groups and these are set out in Table 1. In this table, to recover three cell populations from frozen Sections of results are presented as the fold-difference in mRNA levels ileum harvested immediately after Sacrifice of germ-free and between colonized and germ-free ileum and represent aver colonized mice. The three populations are (i) epithelium age values from duplicate microarray hybridizations. The present in crypts (the proliferative compartment of the average fold-changes for genes represented by 2 or more intestine containing undifferentiated cells as well as differ entiated members of the Paneth cell lineage); (ii) epithelium independent probe Sets are listed Separately. overlying Villi (containing post-mitotic, differentiated mem 0194 Importantly, a large of number of the genes iden bers of the intestine's other three lineages); and (iii) mes tified using these criteria are involved in modulating funda enchyme underlying crypt-villus units (FIG. 1). mental intestinal functions: 20 of the 71 genes (28%) were 0199 LCM was performed on groups of mice indepen grouped under nutrient uptake and metabolism. There was dent of those used to generate RNA for the microarray also a concerted rise in expression of Several components of analysis. 7 um-thick Sections were cut from frozen ileums the host's lipid absorption/export machinery, including pan and LCM conducted using the PixCell II system from creatic lipase-related protein-2 (PLRP-2), colipase, liver Arcturus (7.5 um diameter laser spot). RNA was prepared fatty acid binding protein (L-FABP), and apolipoprotein from dissected cell populations using the RNA Micro A-IV (Table 1). As noted above, there was a prominent Isolation Kit (Strategene) and Standard histochemical pro decrease in expression of Fiaf, a novel PPARy target known tocols. (LCM was carried out using conventional methods as to be induced with fasting (S. Kersten, et al., J. Biol. Chem. described by M. R. Emmert-Buck, et al., Science, 274, 998 275. 28488 (2000)). (1996) and R. F. Bonner, et al., Science, 278, 1203 (1997).) 0.195 Additionally, there were changes in expression of four genes involved in dietary metal absorption. A high 0200. The results are shown in FIG. 1. affinity epithelial copper transporter (CRTI) mRNA was 0201 Second, real-time RT-PCR was used to quantitate increased, while metallothionein-I, metallothionein-II, and levels of Specific mRNAS in the laser captured cell popula ferritin heavy chain mRNAs were decreased (Table 1). tions. The LCM/qRT-PCR analysis was performed using These changes Suggest that colonization engenders germ-free and colonized mice from three experiments that increased capacity to absorb heavy metals (e.g., via CRT1) were independent of those used for microarray profiling. and a concomitant decreased capacity to Sequester them within cells (MT-I/II, ferritin). This implies greater host 0202) Each sample was analyzed in triplicate in four demand for these compounds, either due to increased utili independent experiments. Mean values for the independent Zation by the hosts own metabolic pathways or to compe determinations +1 S. D. are shown in Table 2. tition with the microbe. The changes in SGLT1, colipase, 0203) Therefore, LCM and real-time RT-PCR analysis L-FABP, and MTI (plus 8 other mRNAs discussed below), were employed to delineate the cellular origins of its were independently validated by qRT-PCR (C.A. Heid, et response to B. thetaiota Omicron. al., Genome Res., 6,986 (1996) (Table 2). 0204. The results show that Sprr2a mRNA is confined to 0196. Of these, genes which were found to have a dif the epithelium where its concentration is 7-fold higher on ference in expression levels of 5-fold or more as a result of the villus compared to the crypt (FIG. 1B). B. thetaiotaomi B. thetaiota Omicron colonization were colipase, liver fatty cron elicits a 280-fold increase in the villus epithelium. This acid binding protein, fasting-induced adipose factor, metal value is in good agreement with the increase documented in lothionein I and metallothionein II, malate oxidoreductase, total ileal RNA (Table 2). The cellular origin of the Sprr2a Sprr2a, angiogenin-4, angiogenin-related protein, gelsolin, response Supports the hypothesis that it participates in gp106(TB2/DP1) and rac 2. Of these, colipase, Fiaf, angio fortifying the intestinal epithelial barrier in response to genin-4 and Sprr2a genes showed a difference in expression bacterial colonization. levels of 9-fold or more. 0205 Colipase is produced by the exocrine acinar cells of 0.197 Anotable feature of the host response to B. thetaio the pancreas. Expression in the intestine had not been taOmicron was the absence of detectable or changed expres reported previously. LCM/qRT-PCR revealed that colipase Sion of the many genes involved in immuno-inflammatory mRNA is also present in the ileal crypt epithelium, where it processes that are represented on the microarrayS. These increases 10-fold upon B. thetaiotaOmicron colonization include genes involved in the NF-KB-regulated processes (FIG. 1B). This accounts for the increase detected by that are critical regulators of host responses to invasive microarray and qRT-PCR analyses of total ileal RNA (Tables pathogens (D. Elewaut, et al., J. Immunol. 163, 1457 1, 2). Colipase plays a critical role in dietary lipid metabo (1999)). The absence of these responses can be contrasted to lism by Stimulating the activity of both pancreatic triglyc results obtained in a recent cDNA microarray analysis of the eride lipase and PLRP-2 (M. E. Lowe, etal., J. Biol. Chem. response of a human intestinal epithelial cell line to Salmo 273, 31215 (1998)). US 2005/02397O6 A1 Oct. 27, 2005 0206 LCM and qRT-PCR revealed that the crypt epithe 0219 qRT-PCR was used to compare ileal lactase mRNA lium is the predominant location of a gene, amplifiable using levels in each group (all animals had=107 CFU/ml ileal primers such as SEQ ID NO 12 and 25 (see Table 3 contents). The results are shown in FIG. 3. hereinbefore), which encodes a new protein, angiogenin-4 0220 Colonization with any of the three gram-negative (see example 4 below). However, LCM and real-time RT anerobes elicited an equivalent decline in lactase expression PCR analysis revealed that in colonized ileum, the levels of relative to germ-free controls (FIG.3). This decline was also this mRNA are highest in crypt epithelium (values in the observed after inoculation of a complete ileal/cecal flora. ileal villus epithelium and mesenchyme are 14- and 15-fold qRT-PCR of the same RNAS revealed that ileal expression lower, respectively; FIG. 2). of colipase and angiogenin-4 was induced after colonization 0207. The LCM/qRT-PCR studies of Sprr2a colipase and of all three organisms, and by the ileal/cecal flora (FIG. 3). angiogenin-4 establish the feasibility of assigning an in vivo host response to a particular cell population in a complex 0221) The levels of colipase and angiogenin-4 mRNAS tissue, and of describing the cellular response in quantitative achieved in the ileums of these ex-germ-free mice were terms. In recovering a responding cell population and comparable to those of age-matched mice that have been expressing its reaction to a microorganism in quantitative conventionally-raised since birth (FIG. 3). terms, the applicants results demonstrate how it is possible 0222. In contrast to these findings, the response of Sprr2a to move beyond in vitro models and use in Vivo Systems to to colonization was dependent upon the colonizing Species. Study the impact of a microbe on host cell gene expression. While B. thetaiota Omicron produced a pronounced rise in Sprr2a mRNA that recapitulates the response to a 10 day 0208 Colonization of germ-free mice with B. thetaio colonization with the ileal/cecal flora, colonization with B. taOmicron produces a decrease in ileal LPH mRNA levels infantis and E. coli produce only negligible increases in (Table 1, 2). Analysis of RNA isolated from laser-captured epithelial and mesenchymal cell populations established that mRNA levels (FIG. 3). the colonization-induced reduction in LPH mRNA levels 0223 Mdrla and glutathione-S-transferase, which act in occurs primarily within the villus epithelium (FIG. 2). concert to metabolize Xenobiotics and electrophiles, also exhibited Species-specific (and concerted) responses. Unlike 0209 Comparison of transcript levels between germ-free B. thetaiotaOmicron, which Suppresses expression, E. coli and B. thetaiota Omicron-associated mice revealed a coloni and B. infantis both elicit increases in these mRNAS. In Zation-associated increase in expression of angiogenin-4. contrast, the multi-component ileal/cecal flora did not pro duce a significant (i.e., =2-fold) change in levels of either EXAMPLE 3 mRNA when compared to germ-free controls, 0210. The concept that microbes such as B. thetaiotaomi 0224. The Mdr1 a/GST responses provide direct evi cron may help legislate changes in expression of a given dence that components of the normal microflora can modu gene in the intestine, raises the question of whether Some or late host genes involved in drug metabolism, and Suggest many components of the microbiota can elicit these changes. that variations in drug metabolism between individuals may arise, in part, from differences in their resident gut micro 0211. In order to examine this, age-matched groups biota. (n=4-8 mice/group) of 7-15 week-old germ-free NMR1/KI mice were maintained in plastic gnotobiotic isolators on a 12 EXAMPLE 4 hour light cycle, and given free access to an autoclaved chow diet (B&K Universal). Males were inoculated with 0225. Following the observation that a 10 d colonization one of the following groups. was associated with a 11-fold increase in ileal expression of a mRNA detected by an Affymetrix-designed probe-set 0212 (i) Nothing--Germ-free control, designed from the published Sequence of angiogenin-3, we 0213 (ii) B. thetaiotaomicron strain VPI-5482 (L. V. designed primerS Specific for the 3' and 5' ends of the mouse Hooper, et al., Proc. Natl, Acad. Sci. U.S.A. 96.9833 (1999)). angiogenin-3. They were: 0214) (iii) E. coli K12 which was originally recovered from a normal human fecal flora, ORF forward primer: (SEQ ID NO 27) 0215 (iv) Bifidobacterium infantis (ATCC 15697), a 5'-CCTTGGATCCATGGTGATGAGCCCAGGTTCTTTG prominent component of the pre-weaning human and mouse ileal flora and a commonly used probiotic. 0226 which incorporates a BamHI site at the 5' end; 0216 (v) a “complete ileal/cecal microbiota harvested from conventionally-raised mice (L. Bry, et al., Science 273, ewese primer (SEQ ID NO 28) 1380 (1996)). 5'-CCTTTCTAGACTACGGACTGATAAAAGACTCATCGAAG 0217. A further control group comprised mice conven tionally raised since birth. 0227 which incorporates an Xbal site at the 5' end. 0218 Mice were sacrificed 10 days later, 2 hours after 0228. These primers were used together with RT-PCR to lights were turned on. The distal 1 cm of the small intestine amplify a 438 bp sequence from RNA prepared from the was used to define CFU/ml ileal contents. The 3 cm of ileums of ex-germ-free NMRI mice. These mice had been intestine just proximal to this Segment was used to isolate colonized for 10 d with a complete ileal/cecal flora harvested total ileal RNA (Qiagen RNeasy kit). from conventionally-raised animals belonging to the same US 2005/02397O6 A1 Oct. 27, 2005 inbred strain. We subcloned the PCR product into BamHI/ genin-4-specific primers and qRT-PCR were used to Xbal digested pCEX-KG and Sequenced it using vector compare angiogenin-4 mRNA levels along the length of the Specific primers. small intestine of germ-free NMRI mice and germ-free mice colonized for 10 d with an ileal/cecal flora harvested from 0229 Surprisingly, the nucleotide sequence of the ORF conventionally raised NMRI animals. Pair-wise compari was only 90% identical to that of mouse angiogenin-3. Since Sons revealed that expression of angiogenin-4 is highest in the primer Sequences used in the PCR reaction (specific for the jejunum of colonized mice, and that conventionalization angiogenin-3) were incorporated into the product, we used induces up to a 17-fold increase in angiogenin-4 expression 5'- and 3'-RACE to (a) obtain accurate sequence at the 5' and in this region (FIG. 11). Mono-association of germ-free 3' ends of the ORF of this new angiogenin, and (b) charac NMRI mice with B. thetaiotaomicron for 10 d resulted in a terize the 5’- and 3' untranslated regions of its mRNA. The comparable induction of angiogenin-4 expression (data not results revealed only 88.3% nucleotide sequence identity shown). Regulation of Angiogenin-4 Expression During with angiogenin-3 mRNA. Postnatal Development is Consistent with its Microbial 0230. The nucleotide sequence that encodes the angio Regulation genin-4 protein, aligned with the angiogenin-3 Sequence is 0236. The developmental patterns of angiogenin-4 shown hereinafter in FIG. 4 as SEQ ID NO 29 and 30, expression in postnatal day 5 (P5) to P30 germ-free and respectively. conventionally raised NMRI mice (n=3 mice per time point 0231 Angiogenin-4 has 74 to 81% amino acid sequence per group) was then assessed (FIG. 9). Relative levels of the identity to the other 3 members of the mouse angiogenin angiogenin-4 transcript remained relatively low until P20 in family (FIG. 5). It was found that the 5' and 3'-untranslated both groups of mice. Expression rose slightly (2-3 fold) in regions of angiogenin-4 are closely related to the corre germ-free animals after this time point. In contrast, angio sponding regions of angiogenin-3 mRNA (FIG. 4). genin-4 expression increased more than 20-fold between P15 and P30 in conventionally-raised animals. These results 0232 Subsequently a comparative analysis of the tissue indicate that angiogenin-4 is induced during the Suckling/ distribution of the various mouse angiogenin mRNAS, was weaning transition -coincident with a major shift in the gut conducted. cDNA was synthesized from RNAS isolated microbiota. The lack of angiogenin-4 induction in postnatal from tissues harvested from conventionally raised adult germ-free mice is also consistent with the conclusion that (12-14 week old) male and female NMRI mice (25 tissues/ components of the microbiota play an important role in mouse). To quantitate relative levels of expression of each regulating angiogenin-4 expression. gene, we designed primer Sets Specific for each of the four mouse angiogenin family members (FIG. 6; Table 4 below) 0237 Cellular Localization of Angiogenin-4 and used them for SYBR-Green-based real-time quantitative 0238. The previous laser capture microdissection (LCM)/ RT-PCR (qRT-PCR) analyses. qRT-PCR Study of the cellular origins of angiogenin protein TABLE 4 SEQ ID Gene Primer NO. Sequence angiogenin- 4 forward 35 5' CTCTGGCTCAGAATGTAAGGTACGA ewese 36 5' GAAATCTTTAAAGGCTCGGTACCC angiogenin-3 forward 37 5' CTGGCTCAGGATAACTACAGGTACAT ewese 38 5' GCCTGGGAGACCCTCCTTT angiogenin-1 forward 39 5' AGCGAATGGAAGCCCTTACA ewese 40 5' CTCATCGAAGTGGACCGGCA angiogenin forward 41 5' GGTGAAAAGAAAGCTAACCTCTTTC related protein reverse 42 5 ' AGACTTGCTTATTCTTAAATCG 0233 Remarkably, angiogenin-4 mRNA was restricted expression (Example 2) used primers that recognize both the intestine where it is expressed from the duodenum to the angiogenin-3 and angiogenin-4, and RNAS that had been rectum (FIG. 7). In contrast, angiogenin-1 expression is isolated from captured crypt epithelium, Villus epithelium, highest in liver, lung, and pancreas (FIG. 8), while angio or mesenchymal populations from the Villus core. The genin-3 is expressed primarily in liver, lung, pancreas, and qRT-PCR analysis indicated that the microbially-regulated prostate (FIG. 9). Angiogenin-related protein mRNA was angiogenin was produced in epithelial cells located at the undetectable in all tissues Surveyed even after 40 cycles of base of crypts of Lieberkuhn (Hooper, et al., Science, 291, PCR (FIG. 10). 881 (2001); and Hooper, et al., Nature Immunol, 4, 269 0234 Thus, the highly restricted, intestine-specific pat (2003)). tern of angiogenin-4 expression makes it unique among 0239). To test the hypothesis that angiogenin-4 expression mouse angiogenin family members. occurs in Paneth cells, we used LCM to isolate cells located 0235. These findings indicated that there was microbial at the base of jejunal crypts from (a) germ-free adult (12 regulation of angiogenin-4 rather than angiogenin-3 expres week old) transgenic mice with an attenuated diphtheria Sion in the intestine. To test this hypothesis directly, angio toxin-A fragment (tox 176)-mediated Paneth cell lineage US 2005/02397O6 A1 Oct. 27, 2005 ablation (CR2-tox176 mice) (Garabedian, et al., J. Biol. 0248 Measurement of Total Body Fat Content and Meta Chem., 272, 23729 (1997), and (b) their age and gender bolic Rate (Oxygen Consumption) Total body fat content matched germ-free normal littermates. QRT-PCR using was determined 5 min after mice were anesthesized with an angiogenin-4-Specific primers revealed that angiogenin-4 i.p. injection of ketamine (10 mg/kg body weight) and mRNA levels are 10-fold higher in RNA purified from crypt Xylazine (10 mg/kg). The protocol used for dual-energy base epithelial cells of normal mice compared to CR2 X-ray absorptiometry (Lunar PIXImus Mouse, GE Medical tox176 littermates (FIG. 10). Systems, Waukesha, Wis.) has been described in C. Bernard 0240 A follow-up study was conducted using conven Mizrachi, et al., Arterioscler. Thromb. Vasc. Biol., 22, 961 tionally raised NMRI mice. Three cellular pools were har (2002). vested by LCM: Paneth cells alone; epithelial cells from the 0249. Oxygen consumption was determined in con upper crypt and Villus (a Paneth cell-minus fraction); and Scious, individually caged mice, in a fed State, by using mesenchyme retrieved from the Villus core and the peri open-circuit indirect calorimetry (single-chamber Small-ani cryptal region. The distribution of angiogenin-4 mRNA mal Oxymar System, Columbus Instruments, Columbus, closely paralleled the distribution of phospholipase A2-the OH). Animals were allowed to adapt to the metabolic product of the Mom-1 locus and a well-known Paneth chamber for 20 min before VO was measured every 30s for cell-specific gene product (data not shown). 1 h. 0241 Part II-Examples 5-13 correspond to section II of 0250) SYBR-Green-Based Real-Tie Quantitative RT-PCR (qRT-PCR). RNA was isolated as described in the the detailed description and utilize the following materials art and reverse-transcribed by using SuperScript II and dTs and methods: primers lo (Invitrogen). qRT-PCR assays were performed 0242) Materials and Methods 25-ul reactions that contained cDNA corresponding to 1 ng of total RNA and 900 nM gene-specific primers (Table 1). 0243 Animals. C57BL/6J (B6) WT and Ragl-/-mice All assays were performed in triplicate with an ABI Prism were purchased from The Jackson Laboratory. B6 peroxi 7700 Sequence Detector (Applied Biosystems). Data were Some proliferator-activator receptor-O. (Ppara) -/- mice were normalized to L32 RNA (AA analysis). kindly provided by F. J. Gonzales (National Institutes of Health, Bethesda). Fasting-induced adipocyte factor 0251 Analysis of Lipoprotein Lipase (LPL). LPL activ (Fiaj)+/- heterozygotes on a mixed B6: 129/Sv background ity in epididymal fat pads was determined according to P. H. were generated as described below, and Fiaf-/+, Fiaf +/-, Iverius and A. M. Ostlund-Lindquist Methods Entynol, 129, and Fiaf-f-littermates, obtained from crosses of Fiaff 691 (1986). heterozygotes were compared. Animals were genotyped by 0252 Statistically significant differences were deter using PCR in accordance with methods known in the art. mined by using Student's t tests. Comparisons between 0244 Conventionally raised (CONV-R) wild-type and more than two groups of mice were made by a one-way knockout mice were rederived as germ-free (GF) as ANOVA followed by Tukey's post hoc multiple comparison described (L. V. Hooper, et al., Methods in Microbiology, 31, teSt. 559 (2002)). GF animals were maintained in gnotobiotic EXAMPLE 5 isolators, under a strict 12-hlight cycle (lights on at 0600 0253) Comparisons of 8-10 week old male C57B1/6J hours), and fed an autoclaved chow diet (B & KUniversal, (B6) GF mice raised in the absence of any microorganisms East Yorkshire, U.K.) ad libitum. All manipulations of mice (germ-free; GF, with mice that harbored a microbiota begin were performed by using protocols approved by the Wash ning at birth revealed that the latter contain 42% more total ington University Animal Studies Committee. body fat, as defined by dual energy X-ray absorptiometry 0245 Colonization of GF Mice-The cecal contents of (DEXA, FIG. 14A). Epididymal fat pad weights were also each 8-week-old CONV-R mouse were resuspended in 10 significantly greater (47%; FIG. 14B). The increase in body ml of sterile PBS, and 2-ml aliquots were spread on the fur fat observed in CONV-R animals is intriguing given that of 7- to 10-week-old GF recipients. The resulting conven their daily consumption of a standard rodent chow diet (57% tionalized (CONV-D) mice were housed in gnotobiotic carbohydrates, 5% fat) was 29% less than their GF coun isolators for 10-28 d under the same conditions and fed the terparts (FIG. 14C). Same diet as their GF counterparts. 0254. A 14d colonization of 8-10 week old male GF B6 recipients with an unfractionated microbiota harvested from 0246 CONV-R animals were maintained in microisola the distal intestines (cecums) of adult CONV-R donors, a tor cages in a specified pathogen-free State in a barrier process known as 'conventionalization, produced a dra facility on the autoclaved B & K diet. They were transferred matic 57% increase in their total body fat content (FIG. to gnotobiotic isolators 2 weeks before they were killed at 14A), and a 61% increase in epididymal fat weight (FIG. 8-10 weeks of age to mimic the housing conditions of GF 14B). The increase in body fat was associated with a 7% and CONV-D mice. decrease in lean body mass resulting in no significant 0247 Eight- to 10-week-old GF mice were orally gav differences in total body weight between the two groups aged with 10 Bacteroides thetaiotaOmicron strain VPI (23.5+2.6 g (GF) versus 23.4+2.6 g (CONV-D); n=21; 5482. Colonization density in the distal intestine, cecum, p>0.05). Fasting serum triglyceride values were similar and colon ranged from 10 to 10' colony-forming units/ml (p>0.05) in both GF and CONVentionalized (CONV-D) luminal contents, as defined by culturing Samples of luminal mice (data not shown). contents on BHI blood agar for 2-3 d at 37 C. under 0255. A similar increase in total body fat content was anaerobic conditions. observed after a shorter, 10d conventionalization (66%; US 2005/02397O6 A1 Oct. 27, 2005 p>0.05 compared to 14d). A more prolonged conventional nization, CONV-D animals had 3-fold higher circulating ization (28d) did not produce further increments in total levels of leptin compared to their GF counterparts (FIG. body fat content, or in epididymal fat pad weight (data not 15A). This increase in leptin was proportional to the increase shown). The increased fat storage produced by a 14d con in body fat (r°=0.977), and provides one potential explana ventionalization also occurred in the face of decreased chow tion for the higher oxygen consumption and reduced food consumption (27% lower than GF; FIG. 14C). intake observed after a two-week colonization. 0256 These effects were not unique to males: CONV-D 0261) The increase in fat content was also accompanied B6 females exhibited increases in body fat (85%) and by Statistically significant elevations in fasting glucose and reductions in lean body mass (9%) that were not signifi insulin levels (FIG. 15A), and an insulin-resistant state, as cantly different from age- matched males (pa0.05). In addi defined by glucose- and insulin-tolerance tests (FIG. 15B, tion, the fat Storage phenotype was not limited to the C). C57B1/6J inbred strain: a 14d conventionalization of 8 week-old male NMRI mice produced a 90% increase in total EXAMPLE 7 body fat content (p<0.01) and a 31% decrease in chow consumption (p<0.05). 0262 Glucose and insulin are known to induce expres 0257 Sequence-based 16S r)NA enumeration studies of sion of lipogenic enzymes in the liver (H. C. Towle, Proc the cecal microbiota revealed great Similarities in the frac Natl AcadSci USA, 98, 13476 (2001)). A 14d convention tional representation of the predominant species in CONV-R alization of GF mice produced a 2.3-fold increase in liver donors and CONV-D B6 recipients (FIG. 19: Table S1). As triglyceride content (FIG. 16A, B), but no appreciable in many humans, Bacteroides and CloStridium were the changes in total liver free fatty acids or cholesterol (p-0.05; most prevalent genera. We colonized B6 mice for 2 weeks data not shown). qRT-PCR assays confirmed that conven with the sequenced B. thetaiotaOmicron strain (VPI-5482), tionalization was accompanied by Statistically significant to determine whether a single Saccharolytic bacterial Species elevations in liver mRNAS encoding two key enzymes in the could, by itself, effect host fat Storage. A two-week coloni de novo fatty acid biosynthetic pathway, acetyl-CoA car zation of the adult B6 GF mouse gut produced a statistically boxylase (AccI) and fatty acid synthase (Fas) (FIG. 16C). Significant increase in total body fat content, although the 0263 Sterol response element binding protein 1 magnitude of the increase was less than that obtained with (SREBP-1) and carbohydrate response element binding pro an unfractionated mouse cecal microbiota (23% versus 57%, tein (ChREBP), two basic helix-loop-helix/leucine zipper respectively; n=10 mice/group, p<0.01). transcription factors, mediate hepatocyte lipogenic responses to insulin and glucose, respectively, and appear to EXAMPLE 6 act synergistically (R. Dentin et al., J Biol Chem, 279, 20314 0258 Because the microbiota-mediated increase in body (2004)). Both Accl and Fas are known targets of ChREBP fat content was not due to increased chow consumption, and SREBP-1 (H. C. Towle, supra. qRT-PCR assays of liver open-circuit indirect calorimetry was performed to deter RNAS revealed that conventionalization increases liver mine whether it reflected decreased energy expenditure. This ChREBP mRNA, and to a lesser extent SREBP-1 mRNA explanation was excluded when we found that the leaner GF levels (FIG. 16C). mice had a metabolic rate (VO) that was 27% lower than 0264 ChREBP is translocated from the cytoplasm to the age- and gender-matched (male) B6 mice conventionalized nucleus after it is dephosphorylated by the Serine/threonine for 14d (p<0.01; FIG. 14D). CONV-D mice had VO. values phosphatase PP2A (H. Yamashita et al., Proc Natl Acad Sci that were not significantly different from age- and gender USA, 98,9116 (2001); T. Kawaguchi et al., Proc Natl Acad matched CONV-R animals (FIG. 14D). Sci USA, 98, 13710 (2001)). PP2A, in turn, is activated by 0259 The increase in VO observed with conventional xylulose-5-phosphate (Xu5P) (T. Kabashima et al., Proc ization could reflect increased metabolic rate in the host Natl AcadSci USA, 100,5107 (2003)), an intermediate in the and/or the metabolic contribution of their recently acquired hexose mono-phosphate shunt. Mice colonized with a microbial community. There are no available methods for microbiota had elevated levels of liver Xu5P compared to measuring the metabolic activity of the microbiota in Vivo. their GF counterparts (1.6+0.4 versus 2.6+0.3 umol/g wet However, microanalytic biochemical assays of freeze weight of liver, p<0.01), and more nuclear-localized clamped gastrocnemius muscle and liver revealed signifi ChREBP (FIG. 16D). cant increases in the Steady State levels of TCA cycle 0265. The applicants have obtained direct biochemical intermediates in CONV-D versus GF animals. Despite this evidence that the presence of the microbiota promotes evidence of increased cycle activity, there were no signifi increased monosaccharide uptake from the gut. GF mice and cant alterations in tissue high-energy phosphate stores (n=5 their conventionalized counterparts (n=4/group) were given animals/group). Increasing oxygen consumption without a single gavage of 100 ul of a mixture of 5 mM glucose and increasing high-energy phosphate Stores implies the pres 0.2 mM 2-deoxyglucose, Sacrificed 15 min later, and ence of futile cycles, a biochemical correlate of inefficient 2-deoxyglucose 6-phosphate levels were measured in the metabolism in the host. distal intestine. Levels were 2-fold higher in CONV-D mice 0260 Leptin is an adipocyte-derived hormone whose (1.15+0.013 versus 0.55+0.04 pmol/lg protein; p<0.001). expression correlates with adipocyte lipid content (M. Once taken up into the intestine, transfer of monosaccha Maffei et al., Proc Natl AcadSci USA, 92,6957 (1995)). rides to the portal circulation is facilitated through an Moreover, leptin is known to reduce food intake and additional effect of the microbiota: we have shown previ increase energy expenditure in mice (M. A. Pelleymounter, ously that conventionalization results in a doubling of the et al., Science, 269, 540 (1995)). Fourteen days after colo density of capillaries that underlie the Small intestinal Villus US 2005/02397O6 A1 Oct. 27, 2005 22 epithelium to levels equivalent to that of age-matched Suggested that Fiaf could provide a signal that links con CONV-R animals (T. S. Stappenbeck, et al., Proc Natl Acad ventionalization with a change in host fuel partitioning. Sci USA, 99, 15451 (2002)). 0271 qRT-PCR assays disclosed that conventionalization 0266 Together, these findings are consistent with an of adult GF mice Suppressed Fiaf expression in their small increase in processing of dietary polysaccharides by micro intestines (ileum), but not in their livers or white fat (FIG. bial glycosylhydrolases in CONV-D mice, increased deliv 17D). Follow-up qRT-PCR studies of laser capture micro ery of absorbed monosaccharides (and short chain fatty dissected intestinal crypt and Villus epithelium and the acids) to their livers, and increased trans-activation of lipo mesenchyme established that microbial Suppression of Fiaf genic enzymes by CHREBP and perhaps SREBP-1. occurs in differentiated villus epithelial cells. 0267 The increased hepatic triglyceride levels could not 0272. These findings suggest that the microbiota acts to be ascribed to increased delivery of lactate generated by the Stimulate hepatic triglyceride production through effects microbiota, Since Serum lactate levels were higher in GF mediated by transcription factors such as ChREBP, and to mice (9.22+1.61 mM; n=21) compared to their CONV-D promote LPL-directed incorporation of these triglycerides counterparts (5.74+1.66 mM, n=16 p<0.001), and there were into adipocytes through transcriptional Suppression of an no detectable changes in hepatic monocarboxylate trans intestinal epithelial gene encoding a circulating LPL inhibi tor. We tested this hypothesis by generating mice with a null porter-1 mRNA levels (data not shown). Fiaf allele (FIG. 17E) and re-deriving them as GF. EXAMPLE 8 0273 Eight week-old male GF Fiaf-l-mice have 67% higher epididymal fat pad LPL activity than GF littermates 0268. The DNA content of epididymal fat pads recovered containing the wild-type Fiaf allele (p<0.01), confirming from GF and CONV-D mice were not significantly different. that Fiaf is an important inhibitor of this lipase in vivo. This finding, together with histochemical Studies allowed Conventionalization of GF knockout mice did not produce the applicants to conclude that the microbiota-induced Significant changes in LPL activity in fat pads (or heart) increase in epididymal fat pad weight reflected adipocyte (p>0.05; n=10 animals). hypertrophy (FIG. 17A). q RT-PCR analyses of fat pad RNA revealed that neither biomarkers of lipogenesis (Acc1, Fas) 0274 GF Fiaf-l- animals have the same amount of total or adipogenesis (aP2, Ppar-Y) were significantly changed body fat as their age- and gender-matched CONV-D (Fiaf following conventionalization (FIG. 17B). Suppressed) wild-type littermates (12.8-t1.1 versus 14.2+1.9, p>0.05). Moreover, a 14d conventionalization of 0269 Lipoprotein lipase (LPL) is a key regulator of fatty already Fiaf-deficient GF knockout animals produced only acid release from triglyceride-rich lipoproteins in muscle, minor increases in total body fat (10+8% versus 55+16% in heart, and fat (K. Preiss-Landl, et al., Curr Opin Lipidol 13, wild-type littermates; FIG. 17F). Fiaf-/-heterozygotes had 471 (2002)). Increased adipocyte LPL activity leads to an intermediate increase (33+12%). These results establish increased cellular uptake of fatty acids and adipocyte trig the importance of Fiaf as a prominent mediator of microbial lyceride accumulation. In white fat, LPL is regulated post regulation of peripheral fat Storage. transcriptionally by nutritional Status: fasting reduces and re-feeding increases enzyme activity (M. Bergo, et al, Bio EXAMPLE 9 chem J 313, 893 (1996). Intriguingly, we found that a 14d conventionalization increased LPL activity 122% in epid 0275. The mechanisms by which the mammalian gut idymal fat pads (FIG. 17C). Moreover, the increase was not microbial community influences host biology and gene confined to fat: enzymatic assays of heart revealed a 99% expression, Such as the Suppression of Fiaf remain almost increase with conventionalization (FIG. 17C). Increased entirely unknown. The Zebrafish, Danio rerio, has Several insulin levels produce reductions in muscle LPL activity (H. unique features that make it an attractive model organism for Lithell, Atherosclerosis 30, 89 (1978)). Therefore, our find analyzing these pathways. First, Zebrafish larvae and their ings indicated that the microbiota induces the observed digestive tracts are transparent from the time of fertilization general increase in LPL through another mechanism. through early adulthood, allowing in Vivo observation of the developing gut (M. S. Pack, et al., Development, 123,321 0270) Fasting-induced adipose factor (Fiaf), also known (1996); S. A. Farber, et al., Science, 292, 1385 (2001)) and as angiopoietin-like protein 4, is produced by brown and its resident microorganisms (J. M. Davis, et al., Immunity, white fat, liver, as well as intestine (S. Kersten et al., J. Biol 17, 693 (2002); A. M. van der Sar, et al., Cell Microbiol.5, Chem 275,28488 (2000); J. C. Yoon et al., Mol Cell Biol 20, 601 (2003). Second, Zebrafish development occurs rapidly. 5343 (2000); L. V. Hooper et al., Science 291,881 (2001)). Larvae hatch from their chorions at ~3 days post-fertiliza This secreted protein is a potent inhibitor of LPL in vitro tion (dpf). By 5 dpf, the yolk is largely absorbed and gut (IC=200 nM; (K. Yoshida, et al., J Lipid Res, 43, 1770 morphogenesis has proceeded to a stage that Supports feed (2002)). RT-PCR analysis of intestinal Fiaf expression dur ing and digestion (M. Pack, et al., Supra; S. A. Farber, et al., ing postnatal period disclosed that the gene is induced in GF Supra). Third, the organization of the Zebrafish gut is similar mice during the Suckling-weaning transition. Induction does to that of mammals. AS in mice and humans, the intestinal not occur in CONV-R animals, producing Significantly epithelium undergoes renewal throughout life. A prolifera lower levels of Fiaf mRNA in adult CONV-R versus GF tive compartment, analogous to the mammalian crypt of intestine (FIG. 20). During the Suckling-weaning transition, Lieberkuhn, is located at the bases of intestinal villi (K. N. the diet Switches from lipid/lactose-rich mother's milk to Wallace, et al., Mech Dev, 122, 157 (2005)). Epithelial low fat/polysaccharide-rich chow, with coincident expan progenitors give rise to cell types encountered in other sion of the microbiota and a shift from facultative to obligate vertebrates, including absorptive enterocytes, mucus-pro anaerobes (e.g., Bacteroides). These developmental studies ducing goblet cells, and an enteroendocrine lineage (M. L. US 2005/02397O6 A1 Oct. 27, 2005 23 Pack, et al., Supra). Fourth, GF larvae of other fish species times with Sterile water at room temperature, immersed in have been produced by aseptically removing gametes from 0.003% sodium hypochlorite (Novel Wash Co.) for 20 min adults, and treating fertilized eggs with germicidal agents at room temperature, and Simultaneously transferred into while they develop in the axenic environment provided by plastic gnotobiotic isolators (Standard Safety Equipment). their protective chorions (J. A. Baker, et al., Proc. Soc. Exp. Once inside the gnotobiotic isolators, Zebrafish embryos Biol. Med., 51, 116 (1942); T. J. Trust, Appl. Microbiol., 28, were rinsed 3 times with Sterile water, and then reared in 340 (1974); R. Lesel, R., et al., Ann Hydrobiol, 7, 21 these isolators in a Static Solution of gnotobiotic Zebrafish (1976)). Finally, the capacity to perform forward genetic medium GZM; 0.3 g/L marine salt (Coralife); neutral pH analyses in a vertebrate that is transparent in the postem buffer (Bullseye 7.0, Wardley) at a density of 0.4 individu bryonic period has already led to the identification of als/mL GZM, in 400 mL glass beakers. Each day, 50% of the mutants with defects in gut development (M. L. Pack, et al., GZM in each beaker was replaced with fresh media. Water supra; A. N. Mayer, et al., Development, 130,3917 (2003)) temperature was maintained at 28 C. using an external and digestive physiology (S. A. Farber, et al., Supra). K-MOD 107 heating system (Allegiance Healthcare). Reverse genetic analysis using antisense morpholino oligo Beginning on 3 dpf, the Solution was Supplemented with nucleotides (A. Nasevicius, et al., Nat. Genet., 26, 216 dissolved autoclaved chow (ZMO00, ZM Ltd; 20 mg dry (2000) or target-selected mutagenesis (E. Wienholds, et al., weight/L). To insure that the isolators were free of contami Genome Res., 13, 2700 (2003)), as well as chemical screens nating bacteria or fungi, their inside Surfaces were routinely (R. T. Peterson, et al., Proc. Natl. AcadSci, USA, 97, 12965 Swabbed, and aliquots of GZM containing dissolved food (2000); S. M. Khersonsky, et al., J. Am. Chem. Soc., 125, were removed from beakers, and cultured aerobically and 11804 (2003)), provide additional means for identifying anaerobically at 28° C. and 37 C. in three different media molecular mediators of host-microbial interactions. The (nutrient broth, brain/heart infusion broth, and Sabouraud imminent completion of the Zebrafish genome will facilitate dextrose broth). many of these approaches (http://www.sanger.ac.uk/ Projects/D-rerio/). 0279. To generate conventionalized animals, water was collected from recirculating tanks in a conventional 0276 To investigate the impact of indigenous microbial Zebrafish aquaculture facility, and passed through a 5 um communities on Zebrafish biology, the applicants developed pore diameter filter (Millipore). Microbial density in the procedures for producing and rearing GF Zebrafish and for filtrate was defined by culture under aerobic and anaerobic conventionalizing them or colonizing them with Single com conditions at 28°C. on brain/heart infusion blood agar. 10' ponents of the normal Zebrafish or mammalian gut micro CFU of bacteria were added per mL GZM containing 3 dpf biota. GF Zebrafish. 0277 CONV-R zebrafish (C32 inbred strain) were reared 0280. In some experiments, GF animals were colonized through 14 dpf at a density of ~0.4 individuals per milliliter at 3 dpf with a single bacterial Species. Aeromonas hydro Static water that had been harvested from tanks in a recir phila (ATCC 35654) and Pseudomonas aeruginosa (strain culating Zebrafish acquaculture facility. Animals were Subse PAO1) were grown overnight under aerobic conditions in quently maintained at ~0.03 individuals/mL static water tryptic soy broth (TSB) at 30° C., and in nutrient broth at 37 through 28 dpf, and then moved to recirculating tanks. C., respectively, and then added to beakers containing 3 dpf CON-R zebrafish were fed rotifers (Aquatic Biosystems) GF Zebrafish at final concentrations of 10 CFU/mL GZM. beginning at 3 dpf, followed by brine shrimp (Acquafauna Bio-Marine) beginning at 14 dpf, and then advanced to a diet 0281 GF and CONV-R Zebrafish started to feed at 5 dpf of brine shrimp, TetraMin flakes (Tetra), and Hikari micro and were indistinguishable macroscopically through -8 dpf pellets (Hikari) at 28 dpf. (FIG. 24 A, B). At 9 dpf, GF animals began to develop a Stereotyped, rapidly progressive epidermal degeneration 0278. To generate and rear GF Zebrafish, adult male and phenotype manifested by epidermal opacity, loSS of epider female CONV-R zebrafish (C32 inbred strain) were col mal integrity, and sloughing of epidermal cells (FIG. 24 D, lected, euthanized in 3-amino benzoic acid ethyl ester E). Mortality was 100% by 20 dpf (n=824 zebrafish scored). (Sigma; final concentration 1 mg/mL, 10 min exposure), and The phenotype was rescued by exposing 3 dpf or 6 dpf GF then immersed in a bath of 10% polyvinylpyrrolidone (PSS animals to the microbiota contained in water obtained from Select) for 2 min at room temperature. After carefully a conventional Zebrafish aquaculture facility (FIG.24F, plus opening the abdominal walls of the males to avoid rupturing data not shown). This finding indicates that the degenerative their intestines, testes were removed, placed in a Sterile 1.5 changes observed in late larval Stage GF animals are not due mL Eppendorf tube containing 500 ul of sterile Hanks to irreversible insults acquired earlier in development. Our solution (4 C.), and dissociated with a sterile pestle. The observations that (i) animals conventionalized at 3 dpf and abdominal walls of gravid females were opened in a similar fed the same autoclaved diet can live to adulthood (242 fashion, ovaries were ruptured, and eggs removed from the dpf), and (ii) unfed GF animals do not develop this pheno body cavity with a sterile Pasteur pipette. Eggs were fertil type through 12 dpf (n=44 Scored) Suggest that this phe ized in vitro with the collected sperm insterile plastic 60 mm nomenon is due to deleterious effects of exposure to auto diameter Petri dishes (10 min incubation at room tempera claved chow that are ameliorated by the presence of the ture). Fertilized eggs were Subsequently washed three times microbiota. If activated carbon filters are included in the in Sterile water (3 min/cycle at room temperature), and Static rearing vessels, GF Zebrafish do not develop this incubated for 6 hat room temperature in ~10 mL of a sterile epidermal degeneration phenotype, and can be reared into solution of 0.3 mg/mL marine salt (Coralife), 100 lug/mL ampicillin, 5 lug/mL kanamycin, and 250 ng/mL amphoteri adult Stages. cin B. Embryos were then washed at room temperature in 0282 GF Zebrafish harvested at 6 dpf, and animals con 0.1% polyvinylpyrrolidone (PSS Select) for 2 min, rinsed 3 ventionalized at 3 dpf and sacrificed 3 days later (CONV-D), US 2005/02397O6 A1 Oct. 27, 2005 24 have a similar gross morphology (FIG. 24B, C). Addition DNA microarrays were utilized per comparison of two ally, GF Zebrafish at 6dpf exhibit no statistically significant treatment groups. Oligonucleotide elements that (i) received differences in their average body length compared to age “present calls in all four microarrays and (ii) displayed matched CONV-D and CONV-R larvae 4.06+0.11 mm >1.55 mean Signal-to-noise ratio acroSS both dye channels in (GF); 4.09+0.11 mm (CONV-D); and 4.02+0.15 mm all four microarray replicates, were identified and all others (CONV-R); P>0.3 for each comparison based on Students were excluded. The log ratio of median dye intensities for t-test). Given the phenotype observed in GF fish 29 dpf, the each remaining element was averaged acroSS all four analysis was focused of the effects of the microbiota on host microarrayS. To account for measurement variance among biology using 6 dpf animals. replicate microarrays within an experiment, Standard devia 0283 The Zebrafish is a stomachless teleost: its pharynx tions of the averaged log ratioS of all remaining elements is continuous with the proximal intestine (Segment 1), which were averaged to identify the Standard deviation for the is largely responsible for lipid absorption. Segment 2 of the experiment (SDE) (I. V. Yang, et al. (2002) Genome Biol. 3, intestine (FIG. 24A) is involved in absorption of other research0062). macromolecules, while a short distal domain (Segment 3) is 0288 When considering the results of an experiment, the postulated to participate in water and ion transport (H.W. applicants defined differentially expressed genes as those Stroband, et al., Cell Tissue Res, 187, 181 (1978); J. Noail displaying an average log ratio with an absolute value of lac-Depeyre, et al., Tissue Cell, 8, 511 (1976); H. W. greater than 2 SDE, providing ~95% confidence (GF versus Stroband, et al., Histochemistry, 64, 235 (1979)). CONV-D 1 SDE=0.501; GF versus CONV-R 1 SDE=0.566). 0284. The proximal intestine, liver, pancreas, and gall Differentially expressed genes identified in this manner are referred to by the names of their putative mouse or human bladder of GF and CONV-D animals were indistinguishable, homologs. Homologies were assigned using the following whether judged by examination of wholemount preparations methods: (i) previous Zebrafish gene name assignment, (ii) (FIG.24B, C), serial hematoxylin and eosin stained sections EST assembly homology (http://zfish.wustl.edu), (iii) Uni (e.g., FIG. 24 G, J, n=20-34 animals/treatment), or by gene homology (http://www.ncbi.nlm.nih.gov), or (iv) transmission EM (data not shown). Ensembl gene prediction homology based on corresponding 0285 GF mice have reduced rates of epithelial prolifera genomic sequence (http://www.sanger.ac.uk/Projects/D tion in their intestinal crypts of Lieberkuhn compared to rerio). Functional classification of genes was based in part their CONV-R or CONV-D counterparts. A similar situation on the Gene Ontology Consortium database (http://www occurs in zebrafish. Quantitative BrdU labeling studies geneontology.org). For microarray image files, Scan Array disclosed that the fractional representation of S-phase cells output files, and other MIAME information, see http:// in the intestinal epithelium was significantly greater in 6 dpf gordonlab.wustl.edu/. CONV-D and CONV-R zebrafish compared to GF animals (P<0.0001 in each case based on Student's t-test; ne12 0289. Using the criteria described above, the applicants animals/condition; FIG. 25A-C). No significant differences identified 212 genes that exhibited differential expression in were observed in the underlying mesenchyme/muscle (FIG. both GF versus CONV-D and GF versus CONV-R compari 25C). The increase in epithelial proliferation was not accom Sons. In addition, the applicants referenced Zebrafish genes panied by a Statistically significant change in apoptosis, as culled from comparisons of GF versus CONV-D and/or GF judged by TUNEL assays of epithelium and underlying versus CONV-R animals to our previous DNA microarray mesenchyme/muscle in the same animals (data not shown; datasets of genes differentially expressed in the GI tracts P>0.3 for all comparisons). (Small intestine, colon, or liver) of adult GF mice versus ex-GF mice colonized with components of the normal 0286 To gain additional insights about the mechanisms mouse intestinal microbiota. Sixty-six homologous genes underlying these microbiota-associated phenotypes, as well were identified as responsive to the microbiota in both fish as other aspects of host physiology affected by gut microbes, and mice. Expression of 54 of these changed in the same the applicants conducted a broad, functional genomics direction (up or down) in both species. Moreover, 59 of the based analysis of gene expression in the digestive tracts of 66 genes were identified in the applicant's analysis of the 6 dpf GF, CONV-D, and CONV-R Zebrafish. Comparisons response of the mouse intestine, and did not occur in mouse were performed using DNA microarrays containing 16,228 liver datasets. 65-mer oligonucleotides representing Zebrafish genes and ESTs (Sigma-Genosys Zebrafish Oligonucleotide Library). 0290 For example, the increased epithelial proliferation RNA was isolated from the pooled digestive tracts of 30 associated with the microbiota was manifested by the animals per treatment group. Two independently generated increased expression of 15 genes involved in DNA replica cohorts of animals were evaluated for each condition (i.e., a tion and cell division. They include thymidylate kinase total of 60 animals). These “biological duplicates”, together (Dtymk), four minichromosome maintenance genes (Mcm2, with Cy3- and Cy5-labeled probe dye Swap controls, pro Mcm3, Mcm3, Mcmó), plus origin recognition complex duced a total of four DNA microarray datasets for each of Subunit 4 (Orc41), proliferating cell nuclear antigen (Pcna), the two comparisons performed (i.e., CONV-D versus GF; and ribonucleotide reductase subunit M2 (Rrm2). Impor CONV-R versus GF). tantly, the Zebrafish ortholog of Fiaf was suppressed by the 0287 Each experiment consisted of pairwise competitive microbiota. hybridizations from two treatment groups (CONV-D versus 0291 While these studies reveal a wide range of con GF at 6dpf, CONV-R versus GF at 6 dpf, 6 dpf versus 10 Served responses of the Zebrafish digestive tract to the dpf CONV- R, or 10 dpf versus 20 dpf CONV-R), plus presence of a microbiota, the nature of this microbiota, and reciprocal dye-Swap replicates. Since biological duplicates its degree of Similarity to microbial communities that reside were generated for each treatment group, a total of four in the mouse or human gut, had not been previously defined. US 2005/02397O6 A1 Oct. 27, 2005 25 Therefore, the applicants generated and Sequenced libraries whether there was colonization with an unfractionated of bacterial 16S rDNA amplicons produced by PCR of DNA microbiota or with either of the two individual species (FIG. prepared from the microdissected digestive tracts of 26A plus data not shown). In contrast, C3 responded to the CONV-R 6dpf, 10 dpf, 20 dpf, 30 dpf and adult animals. presence of a normal microbiota and to A. hydrophila, but Since a number of variables can affect the composition of a microbiota (e.g., nutrient Supply, aquaculture conditions, as not to Paeruginosa (FIG.26B). Conversely, Fiafresponded well as developmental stage), we used our Sequence data to a normal microbiota and P. aeruginosa, but not to A. only to identify genus/species that can occur within the hydrophila (FIG. 26C). These findings indicate that, as in Zebrafish digestive tract. mice (L. V. Hooper, et al., Science, 291,881 (2001), at least 0292. The only genera found at all timepoints surveyed a Subset of Zebrafish genes are Sensitive to factors repre were Aeromonas and Pseudomonas. Vibrio and LactococcuS Sented in only a Subset of bacterial components of the gut S.Sp. were also commonly encountered. Comparisons of the microbiota. digestive tract microbiotas of 6 dpf CONV-D versus 0297 To facilitate translation of findings in the Zebrafish CONV-R Zebrafish indicated an enrichment of Aeromonas to mammalian models, the applicants have determined in the former (61% of all sequenced clones in CONV-D whether members of the human/mouse gut microbiota could versus 0.3% in CONV-R), and of Vibrio in the latter (57% colonize the Zebrafish intestine and elicit evolutionarily in CONV-R versus 12% in CONV-D). conserved host responses. They found that Escherichia coli 0293 Previous culture-based enumerations of the intes can colonize the 3 dpf GF Zebrafish gut at densities com tinal microbiotas of freshwater and marine fish identified parable to endogenous community memberS Such as A. Pseudomonas, Aeromonas, Vibrio, and Flavobacterium gen hydrophila or P. aeruginosa (i.e., 10"/gut at 6 dpf). Further era as the most common components, with good, albeit more, E. coli is capable of eliciting many of the principal lower, representation of Lactobacillus, Staphylococcus, host responses to the gut microbiota in Zebrafish (i.e., Acinetobacter, Streptococcus, and Leuconostoc spp. (B. intestinal epithelial cell proliferation, innate immune Spanggaard, et al., Environ. Microbiol. 3, 755 (2001); M. response, and promotion of nutrient metabolism). For M. Cahill, Microb. Ecol., 19, 21 (1990); E. Ringo, et al., example, colonization of GF Zebrafish at 3 dpf with E. coli Aquaculture Res., 26,773 (1995)). Our results also revealed results in downregulation of Fiaf by 6 dpf (FIG. 23B). Some similarities to the mammalian gut microbiota. For 0298 As noted above, 3 dpf GF Zebrafish were placed in example, the Zebrafish microbiota contained members of a trans-Well cell culture dish containing gnotobiotic Bacteroidetes (e.g., Flavobacterium and Flexibacter), a Zebrafish medium (GZM) and autoclaved chow (ZMO00, major phylum in mice, humans and other mammals (D. C. ZM Ltd; 20 mg dry weight per mL). Live E. coli MG 1655 Savage, Annu. Rev. Microbiol, 31, 107 (1997), components in GZM with a similar concentration of fish chow (initial of Ralstonia and Plesiomonas genera (N. H. Salzman, et al. concentration 10 CFU/mL) were placed in the transwell Microbiology, 148, 3651 (2002); T. Arai, et al., J Hyg. chamber separated from the zebrafish by a filter with 0.4 um (London) 84,203 (1980)), as well as a number of lactic acid diameter pores. qRT-PCR studies of digestive tract RNA bacteria (Lactococcus lactis, Lactobacillus fermentum, Leu indicated that by 6 dpf, these GF Zebrafish displayed Fiaf conostoc citreum, and Weissella confusa). mRNA levels similar to standard E. coli mono-associated 0294 To determine whether some of the observed evo animals raised in the same media conditions (FIG. 23D). lutionarily conserved host responses to the microbiota The same result was obtained when 3 dpf GF Zebrafish were exhibited microbial Species Specificity, the applicants colo immersed with heat-killed E. coli for 3 days (FIG. 23D). nized 3 dpf GF Zebrafish with individual components of the 0299 These methods can be used to identify factors that digestive tract microbiota for 3 days. Two culturable and mediate microbial regulation of Fiaf and host nutrient genetically manipulatable Gram-negative bacterial Species metabolism by generating transgenic Zebrafish that express were chosen for these monoassociation experiments as rep cyan fluorescent protein (CFP) from Zebrafish Fiaf regula resentative of the Aeromonas and Pseudomonas genera that tory Sequences. These fish can then be exposed to condi were consistently represented in the digestive tracts of 6dpf tioned media, or derived fractions, or microbial extracts, or to adult Zebrafish (i.e., A. hydrophila and P. aeruginosa). derived fractions, and the effect on host Fiaf gene expression 0295 RNA was isolated from the pooled digestive tracts monitored by monitoring changes in the fluorescent protein of 10 animals per condition at 6 dpf (n=2 groups/condition), reporter using fluorescence imaging methods. and host transcriptional responses were quantified using qRT-PCR. Two control RNAS were used as reference stan EXAMPLE 10 dards: 6 dpf GF and 6 dpf CONV-D digestive tracts (n=30/ 0300 Two methods were applied to identify conserved group; two independent groups/condition to generate bio regulatory elements in the 10 kb of DNA sequence 5' to the logical duplicates). Importantly, the average number of transcriptional Start site of human, mouse, rat, Zebrafish and viable organisms recovered from the digestive tracts of fugu Fiaf orthologs. First, we searched for novel motifs CONV-D or monoassociated animals was not significantly using PhyloCon (T. Wang, et al., Bioinformatics 19, 2369 different (4.4–8.3x10 CFU/digestive tract; P20.26). (2003)). Two statistically significant motifs were identified: 0296) The qRT-PCR results showed that the response of one overlaps with the peroxisome proliferator-activator Some genes-Saal, Mpo, Apob, and Arg2-was robust receptor (Ppar) binding site; the other is similar to the Heb US 2005/02397O6 A1 Oct. 27, 2005 26 binding site, which contains an E-box (panel A in FIG. 21). (FIG. 22). Finally, qRT-PCR assays of intestinal RNAs Second, we searched the TRANSFAC database (V. Matyset isolated from GF and CONV-D wild-type and Ppara-/-mice al., Nucleic Acids Res, 31, 374 (2003)) of 466 vertebrate indicated that the absence of Ppar-C did not prevent tran specific transcription factor scoring matrices with PATSER Scriptional Suppression of Fiaf upon conventionalization (G. Hertz and G. Stormo, unpublished, http://ural.wustl.edu) (FIG. 22). We concluded that the host fat storage response for high-scoring binding Sites that appear in all five Fiaf to the microbiota does not require Ppar-O. A comparable orthologs, and in conserved Sequence blocks between the analysis of the role of Ppar-Y could not be performed because human and mouse genes. Over 40 matrices Satisfied these Pparg-/-mice die at embryonic day 10. two selection criteria (Table 2S), including sites recognized by Several fork head domain-containing factors (e.g., HNF3, EXAMPLE 12 HNF4C, FKH8), as well as interferon-stimulated response element (ISRE) (FIG. 21). 0302 Finding a conserved ISRE element in the ortholo gous Fiaf genes was intriguing in light of our previous EXAMPLE 11 GeneChip analyses of intestinal RNAS which revealed that 0301 Fiaf was identified during a screen for Ppar-C. conventionalization of B6 GF mice regulates expression of targets in liver (J. F. Rawls, et al., Proc Natl AcadSci USA, a number of genes involved in B- and T-cell responses (J. F. 101, 4596 (2004)). Ppar-O. is an important regulator of Rawls, et al., Proc. Natl. Acad. Sci. USA, 101,4596 (2004)). energy metabolism in a variety of tissues including intestine, Therefore, we re-derived B6 Rag1-/-deficient mice as GF to liver, heart and kidney (O. Braissant, et al., Endocrinology determine whether the presence or absence of mature T- and 137, 354 (1996)). We found that Ppar-O. mRNA levels B-cells had an effect on the capacity of the microbiota to decrease modestly (1.7+0.2 fold) in the Small intestines of increase body fat content or modulate Fiaf. Rag1+/+ and CONV-D compared to GF animals, but remain unchanged in Rag1-/-littermates had equivalent increases in body fat their livers and fat pads (p<0.05; see FIG.22). To directly content after a 14d conventionalization (59:16% versus test the role of Ppar-C. in regulating the microbiota-directed 67+16%; p>0.05) and similar degrees of Fiaf suppression change in body fat content and Suppression of Fiaf, B6 Ppara (2.8+0.3 and 3.8+0.3-fold, respectively). Thus, it appears knockout mice were re-derived as GF. 8-10 week old male that these cellular components of the adaptive immune GF Ppara-/-mice had the same amount of total body fat as System are not required to process signals or metabolic their age- and gender-matched GF wild-type littermates products emanating from the gut microbiota that promote fat (FIG. 22). Moreover, Ppara-/-animals had no impairment Storage. Data from Examples 5-12, is depicted in Tables S1, in their microbiota-induced increase in body fat content S2 and S3. TABLE S1 Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total Total Total Closest RDP entry seq. Acetivibrio 1. Acetivibrio 1. cellulolyticus (T). Anaerophaga 4 13 3 7 11 Anaerophaga 4 13 3 7 11 thermohalophila: Fru22. Bacilius 2 2 3 Bacilius SilveStris. 2 2 3 Bacteroides 56 51 47 51 33 Bacteroides acidofaciens. 14 1. 4 1. 3 Bacteroides 3 acidofaciens; A24. US 2005/02397O6 A1 Oct. 27, 2005 27 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total : Total Total Closest RDP entry seq. seq. seq. Bacteroides caccae (T); ATCC 43185T. Bacteroides 12 12 capillosus; ATCC 29799. Bacteroides cf. forsythus 2 10 oral clone BUO63 Bacteroides diataSonis 12 (T). Bacteroides eggerthii (T). Bacteroides merdae (T); ATCC 43184T. Bacteroides putredinis (T). Bacteroides sp. AR20; 1. AR2O. Bacteroides sp. C46; C46. Bacteroides sp. CJ44; CJ44. Bacteroides sp. CJ47; CJ47. Bacteroides sp. CS21; CS21. Bacteroides sp. CS24; 5 CS24. Bacteroides sp. oral clone BUO45; BUO45. Bacteroides 4 splanchnicus (T). Bacteroides vulgatus (T). Butyrivibrio 1 Butyrivibrio crossotus; NCDO 2416. Butyrivibrio fibrisolvens; 1. O/10. Butyrivibrio hungatei; JK 615. Candidatus US 2005/02397O6 A1 Oct. 27, 2005 28 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total Total Total Closest RDP entry seq. Candidatus Arthromitus sp. SFB-Trout. Catonella Catonella morbi (T); ATCC 51271. Catonella sp. oral clone BRO63; BRO63. Catonella sp. oral clone FLO37: FLO37. Citrobacter Citrobacter Clostridium 53 74 8 5 Clostridium aerotolerans 2 (T); DSM 5434. CloStridium aff. innocuum CM970; CM970. Clostridium aminophilum 1. 2 (T): F. Clostridium aminovalericum. Clostridium bolteae: 6 5 type strain; 16351 Clostridium celerecrescens 2 (T); DSM 5628. Clostridium celerecrescens; IrT-JG1-12. Clostridium cellulolyticum 2 (T); ATCC 35319. Clostridium cellulosi. Clostridium 5 5 clostridioforme: 1-53. CloStridium disporicum 6 (T); DSM5521. Clostridium fimetarium (T); Z-2189 US 2005/02397O6 A1 Oct. 27, 2005 29 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total : Total Total Closest RDP entry seq. seq. Clostridium fusiformis; 2 CM973. Clostridium glycolicum 1. (T); DSM 1288(T). Clostridium hathewayi; 1. type strain: DSM 13479 Clostridium herbivorans 1. (T); 54408. Clostridium indolis (T); 1. DSM 755. Clostridium indolis: 2 CM971. Clostridium leptum; 753 2 Clostridium leptum; 10900. Clostridium methylpentosum (T); DSM5476. Clostridium nexile 1 (T): 1-11 Clostridium nexile 1. (T); DSM 1787. Clostridium orbiscindens 2 (T); DSM 6740. Clostridium phytofermentans; ATCC 700394. Clostridium 2 polysaccharolyticum (T) Clostridium populeti (T); ATCC 35295. Clostridium propionicum (T); DSM 1682. Clostridium ramosum. 1. Clostridium Saccharolyticum (T); DSM 2544. Clostridium scindens (T); DSM5676. Clostridium sp. ArC5; 1 ArC5. US 2005/02397O6 A1 Oct. 27, 2005 30 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total : Total Total Closest RDP entry seq. seq. Clostridium sp. 1. ASF356; ASF 356. Clostridium sp. 1. ASF502; ASF 502. Clostridium sp. CITR8; CITR8. Clostridium sp. CJ67; CJ67. Clostridium sp. CS29; CO12. Clostridium sp. HAAP-2; 1. HAAP-2. Clostridium sp. JC3; JC3. Clostridium sp. XB90; XB90. Clostridium sp.: DR6A. Clostridium sp.: DR7. Clostridium sp.; DSM 4 10643; Lip1. Clostridium sp.; DSM 6877(FS41). Clostridium sp.: formate. 1. Clostridium sp.: LIP5. 2 Clostridium symbiosum (T). Clostridium viride (T); 1. T2-7 (DSM 6836). Clostridium xylanolyticum 2 (T); ATCC 4963. Coprococcus Coprococcus catus; VP-C6-61. Cytophaga Cytophaga marinoflava; ANTS103. Cytophaga sp.: BD1-16. US 2005/02397O6 A1 Oct. 27, 2005 31 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total : Total Total Closest RDP entry seq. seq. Desulfitobacterium Desulfitobacterium hafniense: DP7. Desulfomicrobium 2 Desulfomicrobium maceSti 2 (T); DSM 4194 Desulfovibrio 1. Desulfovibrio oryzae; DDv. Desulfovibrio sp. (T); 1. STL1. Desulfovibrio sp. UNSW3caefatS Eggerthella Eggerthella lentha (T); ATCC25559. Erysipelothrix Erysipelothrix rhusiopathiae; 715. Escherichia Escherichia albertii; type strain: LMG 20976. Escherichia coli. Eubacterium 18 38 21 32 Eubacterium biforme 3 (T); ATCC 27806. Eubacterium cylindroides. Eubacterium desmolans 1 (T). Eubacterium 2 oxidoreducens; DAS110. Eubacterium 1. plexicaudatum; ASF 492. Eubacterium ramulus (T). 3 Eubacterium runninantium (T); GA195. Eubacterium Siraeum (T). 1 Eubacterium sp. CJ70; CJ70. US 2005/02397O6 A1 Oct. 27, 2005 32 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total Total Total Closest RDP entry seq. Eubacterium sp. oral 2 clone BSO91; BSO91. Eubacterium sp. oral 2 clone BUO14; BUO14. Eubacterium sp. oral 1. clone FXO33; FXO33. Eubacterium sp. oral clone JHO12; JHO12. Eubacterium sp. TW2; TW2. Eubacterium sp. VPI 12708; VPI 12708. Eubacterium ventriosum. 2 Faecalibacterium Faecalibacterium prausnitzil: 1-84. Firmicutes O Firmicutes sp. oral clone AO068: AO068. Firmicutes sp. oral clone BB124; BB124. Firmicutes sp. oral clone CKO30; CKO30. Firmicutes sp. oral clone FO58: FO58. Firmicutes sp. oral clone FMO46; FMO46. Firmicutes sp. oral strain FTB41; FTB41. Firmicutes str. C38; C38. Flavobacterium Flavobacterium mizutaii: DSM 11724.T. Herbaspirillum Herbaspirillum sp. Chnp3-5; Chnp3-5. Holdemania US 2005/02397O6 A1 Oct. 27, 2005 33 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total : Total Total Closest RDP entry seq. seq. seq. Holdemania filiformis (T); ATCC 51649. Hyphomonas 1. Hyphomonas johnsonii 1. (T); MHS-2. Lachnobacterium Lachnobacterium bovis (T); LRC 5382 Lachnospira 1. Lachnospira pectinoschiza; 1. 1-10. Lactobacillus 36 Lactobacillus acidophilus; 19 BMF 6Lb6. Lactobacillus murinus; 1. ASF 361. Lactobacillus reuteri 2 (T); DSM 20016 T. Lactobacillus sp. 8 ASF360; ASF 360. Lactobacillus sp. CLE-4: CLE-4. Lactobacillus sp. oral clone 2 CXO36; CXO36. Lactobacillus vitulinus (T). 4. Marinilabilia Marinilabilia salmonicolor (T). Oscillospira 6 Oscillospira guillermondii; OSC2. Oscillospira sp. A.; A. Oscillospira sp. F. F. Oscillospira sp. G; G. 1. Oscillospira sp. H.; H. 1. Oscillospira 1 guillermondii; OSC3. US 2005/02397O6 A1 Oct. 27, 2005 34 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised (CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total Total Total Closest RDP entry seq. seq. seq. Oscillospira 3. guillermondii; OSC5. Papillibacter 1. Papillibacter 1. cinnaminovorans (T); CN1 Peptococcus Peptococcus sp. oral clone MCE10 265 Porphyromonas 6 Porphyromonas canis; JCM 10100. Porphyromonas sp. oral clone AWO32; AWO32. Porphyromonas sp. oral clone BR037; BRO37. Porphyromonas sp. oral 2 clone BSO77; BSO77. Porphyromonas sp. oral 4 clone EP003; EP003. Prevotella 3. Prevotella genomosp. 1. C1; C3MKMO81. Prevotella ruminicola; 2 TC2-28. Prevotella sp. oral clone BSO41; BSO41. Prevotella sp. oral clone FO45; FO45. Prevotella sp. oral clone FLO19; FLO19. Roseburia 1. Roseburia intestinalis; L1-8151. Roseburia intestinalis; 1. L1-82. Ruminococcus 14 Ruminococcus albus; OR108. Ruminococcus bromii. 1 US 2005/02397O6 A1 Oct. 27, 2005 35 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised CONV-R) donor mouse and four conventionalized (CONV-D) C57B1/6J recipients. Total Total Total Total Total Closest RDP entry seq. Ruminococcus callidus 1. (T); ATCC 27760. Ruminococcus 2 flavefaciens. Ruminococcus gnavus 3 (T); ATCC 29149. Runninococcus hydrogenotrophicus (T); S5a36. Ruminococcus lactaris (T); ATCC 29176. Ruminococcus productus 4 (T); ATCC 27340. Ruminococcus schinkii (T); 3. B: CIP 105464. Ruminococcus sp. CJ63; CJ63. Ruminococcus sp. CO28; CO28. Ruminococcus torques (T); ATCC 27756. Selenomonas Selenomonas ruminantium (T); GA192. Selenomonas sp. oral clone GIO64; GIO64. Shewanella. 15 12 2O 19 Shewanella algae; 15 12 2O 19 ATCC 8073. Shigella Shigella flexneri 2a str. 2457T, 2457T. Syntrophococcus Syntrophococcus sucromutans; DSM 3224. Syntrophococcus Sucromutans; S195. Tannerella 3 Tannerella forsythensis; 3 KM3. Tannerella forsythensis; TR6. Thermoanaerobacter Thermoanaerobacter kivui (T). Turicibacter 29 Turicibacter Sanguinis. 29 US 2005/02397O6 A1 Oct. 27, 2005 36 TABLE S1-continued Bacterial genera and species identified in the cecums of a conventionally raised CONV-R) donor mouse and four conventionalized (CONV-D) C57BL/6J recipients. Total Total Total Total Total Closest RDP entry seq. Xiphinematobacter 1. Xiphinematobacter rivesi. 1. Xylophilus 1. Xylophilus ampelinus; 1. DSM 7250. Bacterial 16S rDNA Ribosomal Database Project (RDP) entries are organized by genus (bold type) with specific RDP entries listed below each genus heading (plain type). Total number of 16S rDNA clones that (i) passed the selection criteria described in Materials and Methods, and (ii) were homologous to the respective RDP entry with species or genus information. 16S clones that are defined as “unidentified (shaded columns) because their closest relative in RDP is either (i) an entry without species assignment, or (ii) an entry with species or genus assignment but with less than 98% identity to the respective rDNA sequence. These clones are listed in the table according to their closest relative in RDP with species or genus assignment. GenBank Accession numbers for the sequences are AY667702-AY668946. Further details of homology analyses are available at http://gordonlab.wustl.edu/. 0303) TABLE S2 Conserved transcription factor binding sites identified in Orthologous Fiaf genes. Number of Potential TRANSFAC Sites Matrices COSCSS H M R Z F CHN Notes AP1 O1 NNNTGAGTCAKCN 2 4 1 3 3 Ap1 site, activator protein 1 AP1 C NTGASTCAG 5 2. 1 6 2. Ap1 site, activator protein 1 AP4 O6 CWCAGCTGGN 4 2 2 2 4 Ap4 site, activator protein 4 CEBPGAMMA O6 CTBATTTCARAAW 1 1 5 9 4 CCAAT enhancer binding protein CREL O1 SGGRNTTTCC 2 3 4 2 2 C-Rel, overlap with NFkB DR1 O3 RGGNCAAAGGTCA 2 2 4 2 2 PPAR, HNF4, direct repeat E12 O6 RRCAGGTGNCV 3 3 2 4 6 E-box E2A O2 NCACCTGYYNCNKN 2 3 3 5 5 E-box ETS O4 ANNCACTTCCTG 3 3 3 4 4 C-Ets, T-cell, mesodermal cell development FAC1 O1 NNNCAMAACACRNA 2 1 5 9 2 Fac1 site, fetal Alz-50 clone 1 FOXD3 O1 NAWTGTTTRTTT 2 7 4 31 5 3 Fork head box D3 FOXO1 O1 NNNWAAAYAAAYANNNNN 3 S 4 22 14 2 Fork head box O1 FOXO4 O1 RWAAACAANNN 2 4 4 18, 9 Fork head box O4 FOX O2 KAWTGTTTRTTW 3 S 16 7 Fork head factor GC O1 NRGGGGCGGGGCNK 8 4 4 2 3 2 GC box GRO6 NNNNNNCNNTNTGTNCTNN 3 1 2 glucocorticoid receptor site HFHS 01 NNNTGTTTATNTR 5 5 15 8 HNF3, Fkh8 site HNF3ALPHA. O6 TRTTTGYTYWN 5 4 22 4 HNF3-alpha site HNF3 O6 NWRARYAAAYANN 6 3 28 7 HNF3 site HNF4ALPHA. O6 WTGAACTTTGMMB 2 2 5 3 HNF4-alpha site HSF O6 TTCCMGARGYTTC 3 3 1. 3 Heat shock factor site ICSBP O6 RAARTGAAACTG 3 4 9 3 ICSBP, Interferon factor binding site IRF7 O1 TNSGAAWNCGAAANTNNN 1. 6 2. interferon regulatory factor 7 IRF O6 BNCRSTTTCANTTYY 4 4 6 11 7 Interferon regulatory factors ISRE O1 CAGTTTCWCTTTYCC 2 1 2 4 Interferon stimulated response element LBP1 O6 CAGCTGS 2 3 4 5 8 2 TATA box repressor LDSPOLYA B NNNSTGTGTDYYCWTN 2 3 2 6 3 Lentiviral Poly A downstream element LFA1 O6 GGGSTCWR 2 2 1 3 AID1; HNF-2: LFA1 site LMO2COM O1 CNNCAGGTGBNN 2 2 3 2 10 LIM-only protein 2 site MEIS1 O1 NNNTGACAGNNN 2 2 5 5 2 myeloid ecotropic viral integration site 1 MYOD O6 NNCACCTGNY 2 3 2 7 6 myoblast determining factor site MYOGENIN O6 RGCAGSTG 2 4 4 8 7 Myogenin site NF1 O6 NNTTGGCNNNNNNCCNNN 2 3 1. 3 2 nuclear factor 1 site NFE2 O1 TGCTGAGTCAY 3 1 1 3 1 nuclear factor erythroid 2 p45 site PIT1 O6 NMTTCATAWWTATNNMNA 2 8 5 18 7 Pit1, POU1F1 binding site US 2005/02397O6 A1 Oct. 27, 2005 37 TABLE S2-continued Conserved transcription factor binding sites identified in Orthologous Fiaf genes. Number of Potential TRANSFAC Sites Matrices COSCSS H M R Z F CHN Notes POU1F1 O6 ATGAATAAWT 2 5 2. 15 3 1 POU1F1 binding site PPAR DR1 O2 TGACCTTTGNCCY 1 2 5 1 3 1 peroxisome proliferator-activated receptor binding site PU1 O6 WGAGGAAG 5 5 4 2 6 2 Pu.1 site, interfere with erythroblast differentiation SP1 O1 GGGGCGGGGT 4 1 2 O 2 1. Sp1 site, stimulating protein 1 SP1 O6 NGGGGGCGGGGYN 8 3 S 2 4 2 Sp1 site, stimulating protein 1 TAL1BETAE47 O1 NNNAACAGATGKTNNN 1 2 1 3 2 1. Tal-1beta/E47 heterodimer binding site 0304 TABLE S3 Gene-specific primers used for cRT-PCR as says: Sequence Accession amplicon size Gene name Abbrevation primer primer sequences ID No. Number (bp) Acetyl-CoA Acc 1 forward AAGTCCTTGGTCGGGAAGTATACA 43 XM 109883 126 carboxylase rewerse ACTCCCTCAAAGTCATCACAAACA 44 aP2 Ap2 forward TTAAAAACACCGAGATTTCCTTCAA 45 NM 0.24 406 102 rewerse GGGCCCCGCCATCTAG 46 Carbohydrate Chrebp forward CGGGACATGTTTGATGACTATGTC 47 AF156604 105 regulatory element rewerse CATCCCATTGAAGGATTCAAATAAA 48 binding protein Fasting-induced Fiaf forward CAATGCCAAATTGCTCCAATT 49 AF278699 82 adipose factor rewerse TGGCCGTGGGCTCAGT 5 O Fatty acid Fas forward TGGTGAATTGTCTCCGAAAAGA 51 AF127033 149 synthase rewerse CACGTTCATCACGAGGTCATG 52 L32 ribosomal L32 forward CCTCTGGTGAAGCCCAAGATC 53 NM 172086 102 protein reverse TCTGGGTTTCCGCCAGTTT 54 Peroxisome Ppar-C. forward CACCTTCCTCTCCCAAAGCT 55 X57638 105 proliferator reverse GCGTCGGACTCGGTCTTCT 56 activated receptor C. Peroxisome Ppar-Y forward ATGTCTCACAATGCCATCAGGTT 57 U10374 116 proliferator rewerse GCTCGCAGATCAGCAGACTCT 58 activated receptor y Sterol regulatory Srebp-1 forward GCATGCCATGGGCAAGTAC 59 NM 011480 125 element binding reverse CCACATAGATCTCTGCCAGTGTTG 60 protein 1 EXAMPLE 13 overlying mucus layer). Colonization density ranged from 0305 Adult germ-free male NMRI/KI mice were main 10:10 CFU/mL in the distal small intestine (ileum) to tained on a Standard autoclaved chow diet rich in plant 10-10' CFU/mL in the cecum and proximal colon. Scan polysaccharides. Gas chromatographic-mass spectrometric ning electron microscopic Studies revealed B. thetaiota Omi (GC-MS) analysis established that glucose, arabinose, cron attached to Small food particles and embedded in Xylose and galactose are the predominant neutral Sugars mucus (FIG. 27). present in this chow (mole ratio=10:8:5:1). Seven week-old 0306 The cecum is an anatomically distinct structure, mice were colonized with a Single inoculum of B. thetaio located between the distal Small intestine and proximal taOmicron and sacrificed 10 days later (a period that spans colon that is a site of great microbial density and diversity 2-3 cycles of turnover of the intestinal epithelium and its in conventionally-raised mice (F. Backhed, et al., Proc. Natl. US 2005/02397O6 A1 Oct. 27, 2005 38 Acad. Sci. USA, 101, 15718 (2004)). Nutrient use by B. from individual gnotobiotic mice (panel A, FIG. 31). A total thetaiota Omicron in the cecum was defined initially by of 1237 genes were defined as Significantly upregulated in whole genome transcriptional profiling. Cecal contents, vivo compared with their expression in MM-G. The finc including the mucus layer, were removed immediately after tions of these upregulated genes were classified by clusters sacrifice of non-fasted mice (n=6), and the RNA extracted. of orthologous groups (COG) analysis. The largest upregu The B. thetaiota Omicron transcriptome was characterized lated group belonged to the 'carbohydrate transport and using custom GeneChips containing probe pairs derived metabolism COG. In contrast, the largest group of genes from 4719 of the organism's 4779 predicted genes (Table down-regulated in Vivo belonged to the amino acid trans S4). The results were compared to transcriptional profiles port and metabolism COG (FIG. 32, A). obtained from B. thetaiotaomicron grown from early log to Stationary phase in a chemostat containing a minimal 0308 SusC and Sus) are components of a B. thetaio taOmicron Outer membrane protein complex involved in medium plus glucose as the Sole fermentable carbohydrate binding of Starch and malto-oligosaccharides during their source (MM-G; FIG. 30). digestion by outer membrane and periplasmic glycoside hydrolases (J. A. Shipman, et al., J. Bacteriol. 182, 5365 TABLE 54 (2000)). Thirty-seven SusC and 16 SusD paralogs are Features of the B. theta GeneChip upregulated 210-fold in Vivo by comparison to bacteria growing in MM-G (FIG. 33). Naming No. of genes No. of Average no. of prefix of (probesets) probe probe pairs 0309 The indigestibility of xylan, pectin, and arabinose Functional category probesets represented pairs per probeset containing polysaccharides in dietary fiber reflects the pau Control sequences AFFX 51 831 16.3 city of host enzymes required for their degradation. The Bt chromosomal BT 4719 61737 13 human genome contains only one putative glycoside hydro genes' lase represented in the nine families of enzymes known in Bt genes on p5482 p5482 38 494 13 nature with Xylanase, arabinosidase, pectinase, or pectate Bt tRNA genes tRNA 36 468 13 lyase activities, while the mouse genome has none (http:// Genbenk accession number AEO15928 afmb.cnrs-mrs.fr/CAZY/). In contrast, B. thetaiotaomicron 'Genbenk accession number AY171301 has 64 Such enzymes (Table S5; http://afmb.cnrs-mrs.fr/ CAZY/), many of which were selectively upregulated 10- to 0307 Unsupervised hierarchical clustering of the Gene 823-fold in Vivo. These included five secreted Xylanases, Chip datasets disclosed remarkable uniformity in the in vivo five Secreted arabinosidases, plus a Secreted pectate lyase transcriptional profiles of B. thetaiotaOmicron harvested (FIG. 28A-C plus FIG. 33, B). TABLE 55 All families of glycoside hydrolases and polysoccharide lyases containing arabinosidase, Xylanase, pectinase or pectate lyase activities with at least one representative in either the human, mouse, or B. theta genomes (http://afmbenrsfirCAZY/). Family Known Activities in Family Homo Sapiens Mus musculus B. theta Glycoside Hydrolase Family 43 B-xylosidase (EC 3.2.1.37) O O 31 C-L-arabinofuranosidase (EC 3.2.1.55) arabinanase (EC 3.2.1.99) xylanase (EC 3.2.1.8) Glycoside Hydrolase Family 3 f-glucosidase (EC 3.2.1.21) 1. O 1O xylan 1,4-B-xylosidase (EC 3.2.1.37) B-N-ocetylhexosaminidase (EC 3.2.1.52) glucan 1,3-f-glucosidase (EC 3.2.1.58) glucan 1,4-B-glucosidase (EC 3.2.1.74) exo-1,3-1,4-glucanase (EC 3.2.1.-) C-L-arabinofuranosidase (EC 3.2.1.55) Glycoside Hydrolase Family 28 polygalacturonase (EC 3.2.1.15) O O 9 exo-polygalacturonase (EC 3.2.1.67) exo-polygalacturonase (EC 3.2.1.82) rhamnogalacturonase (EC not defined) Polysaccharide Lyase Family 1 pectate lyase (EC 4.2.2.2) O O 5 pectin lyase (EC 4.2.2.10) Glycoside Hydrolase Family 51 C-L-arabinofuranosidase (EC 3.2.1.55) O O 4 endoglucanase (EC 3.2.1.4) Polysaccharide Lyase Family.9 pectate lyase (EC 4.2.2.2) O O 2 exopolygalacturonate lyase (EC 4.2.2.9) Glycoside Hydrolase Family 5 chitosanase (EC 3.2.1.132) O O 1. B-mannosidase (EC 3.2.1.25) cellulose (EC 3.2.1.4) glucan 1,3-f-glucosidase (EC 3.2.1.58) licheninase (EC 3.2.1.73) glucan endo-1,6-B-glucosidase (EC 3.2.1.75) mannan endo-1,4-B-manosidase (EC 3.2.1.78) endo-1,4-B-xylanase (EC 3.2.1.8) US 2005/02397O6 A1 Oct. 27, 2005 39 TABLE 55-continued All families of glycoside hydrolases and polysoccharide lyases containing arabinosidase, Xylanase, pectinase or pectate lyase activities with at least one representative in either the human, mouse, or B. theta genomes (http://afnbenrsfrcAZY/). Family Known Activities in Family Homo Sapiens Mus musculus B. theta cellulose 1,4-f-cellobiosidase (EC 3.2.1.91) endo-1,6-B-galactanase (EC 3.2.1.-) B-1,3-mannanase (EC 3.2.1.-) Glycoside Hydrolase Family 93 exo-arabinanase (EC 3.2.1.55) O O 1. Polysaccharide Lyase Family 10 pectate lyase (EC 4.2.2.2) 0310 GC-MS analysis of total cecal contents harvested in the ceca of two groups of age- and gender-matched adult from fed germin-free mice revealed that Xylose, galactose, gnotobiotic mice. One group received the Standard polysac arabinose, and glucose were the most abundant monosac charide-rich chow diet from weaning to the time of Sacrifice. charide components (FIG. 28D). After 10 days of coloni The other group was Switched to a diet devoid of ferment Zation by B. thetaiotaOmicron, Significant reductions in cecal able polysaccharides but rich in simple Sugars (35% glucose; concentrations of three prominent hexoses (glucose, galac 35% sucrose) 14 days prior to colonization. All mice were tose, and mannose) were observed. There were no signifi colonized with B. thetaiotaOmicron for 10 days and bacterial cant decreases in pentose or amino-Sugars (FIG. 28D). The gene expression was defined in each of their ceca at the time selective depletion of hexoses likely reflects the combined of Sacrifice. effects of microbial and host utilization. B. thetaiota Omicron colonization increased host expression of the principal 0313 The presence or absence of polysaccharides in the Sodium/glucose transporter, Sglt1, in the intestinal epithe diet did not produce a significant effect on the density of lium, reflecting an enhancement of host utilization of liber cecal colonization (data not shown). Using the transcrip ated monosaccharides (Example 1 and Table 1). Morover, of tional profiles of 98 B. thetaiotaomicron genes from the the 1237 bacterial genes upregulated in vivo, 310 were “replication, recombination and repair COG as biomarkers, assignable to enzyme classification numbers in metabolic the cecal bacterial populations clustered most closely to cells maps in the Kyoto Encyclopedia of Genes and Genomes undergoing log phase growth in vitro, irrespective of the diet (KEGG; http://www.genome.adp/). The results of this (FIG. 31, B; Table S6). metabolic reconstruction were consistent with active deliv ery of mannose, galactose and glucose to the glycolytic TABLE 56 pathway, and arabinose and Xylose to the pentose phosphate pathway (FIG. 34; see http://gordonlab.wustl.edu/meta B. theta genes in the Replication Recombination and Repair COG used for hierarchical clustering of view/bt). GeneChip data shown in panel B of FIG. 25 0311 Host mucus provides a consistent endogenous Source of glycans in the cecal habitat that could offer Gene Annotation alternative nutrients to the microbiota during periods of BTO026 putative transposase change in the host's diet. B. thetaiotaOmicron embeds itself BTO069 conserved hypothetical protein BTO070 conserved hypothetical protein in this mucus layer (FIG.27D). GeneChip analysis provided BTO078 putative DNA repair protein evidence that the bacterium harvests glycans from mucus. BTO244 putative exonuclease For example, in Vivo, B. thetaiotaOmicron exhibited Signifi BTO245 ATP-dependent exonuclease abcC cant upregulation (2-10-fold; p<0.05) of (i) an operon BTO252 transcription-repair coupling factor BTO280 transposase for insertion sequence element 15RM3 (BTO455-BTO461) that encodes a sialidase, sialic acid BTO358 tranposase Specific 9-O-acetyl esterase, mannosidase, and three b-hex BTO419 putative endonuclease osaminidases (FIG. 28A), (ii) a mucin-desulfating sulfatase BTO570 excinuclease ABC Subunit B (BT3051), and (iii) a chondroitin lyase (BT3350). Fucose in BTO578 excinuclease ABC subunit A BTO625 DNA helicase host glycans is an attractive Source of food: it typically BTO630 exodecoxyribonudease occupies a terminal-linked position and is constitutively BTO657 ATP-dependent DNA helicase produced in the cecal mucosa of NMRI mice (L. Bry, et al., BTO721 DNA repair and recombination protein putative helicase Science, 273, 1380 (1996)). In B. thetaioatomicron we found BTO831 ATP-dependent RNA helicase BTO894 DNA ligase that two secreted a-fucosidases (BT1842, BT3665) and a BTO899 DNA gyrase subunit A five-component fucose utilization operon (BT1272 BT1054 ATP-dependent helicase BT1277) were also induced (FIG. 28A). Operon induction, BT1081 recombination protein recR which occurs through the interaction of L-fucose with a BT1154 ATP-independent RNA helicase BT1205 putative ATPase AAA family repressor encoded by its first open reading frame (L. V. BT1756 transposase invertase Hooper, et al., Proc. Natl. Acad. Sci. USA, 96, 9833 (1999)), BT1361 DNA repair protein recN (Recombination protein N) is indicative of bacterial import and utilization of this BT1364 DNA polymerase III beta chain hexose. BT1411 methylated-DNA-protein-cysteine methyltransferase BT1497 single-strand binding protein (SSB) 0312 To determine whether the absence of fermentable BT1498 AVG-specific adenine glycosylase polysaccharides in the diet increases foraging on mucus BT1499 DNA-binding protein HU glycans, B. thetaiotaomicron gene expression was compared US 2005/02397O6 A1 Oct. 27, 2005 40 Subsets of B. thetaiotaomicron's genome are dedicated to TABLE 56-continued retrieving either host or dietary polysaccharides, depending upon their availability, although it appears that when both B. theta genes in the Replication Recombination and Repair COG used for hierarchical clustering of Sources are available, harvesting energy from the diet is GeneChip data shown in panel B of FIG. 25 preferred. Gene Annotation 0317 Diet-associated changes in glycan foraging behav BT1516 replicative DNA helicase ior were accompanied by changes in the expression of B. BT1544 NADH pyrophosphatase, Mutl family hydrolase thetaiota Omicron's capsular polysaccharide Synthesis (CPS) BT1610 DNA polymerase III subunit gammaltau. loci (FIG.37). Compared with growth in MM-G, CPS3 was BT1664 crossover junction endodeoxyribonuclease ruvC BT1671 endonuclease III down-regulated in vivo irrespective of host diet, CPS4 was BT1739 excinuclease ABC subunit A upregulated in the ceca of mice fed a polysaccharide-rich BT1821 transposase diet, while CPS5 was upregulated with a high sugar diet BT1848 ATP-dependent DNA helicase reco BT1885 putative ATP-dependent RNA helicase (FIG. 37). The other five CPS loci did not manifest signifi BT1978 Holiday junction DNA helicase ruvA cant differences in their expression during growth in Vitro BT198O transposase Versus in Vivo, or with diet manipulation. These findings BT2056 conserved hypothetical protein BT2073 putative helicase Suggest that B. thetaiota Omicron is able to change its Surface BT2089 DNA topoisomerase II carbohydrates depending upon the nutrient glycan environ BT2130 uracil-DNA glycosylase ment that it is accessing and perhaps also for evasion of the BT2137 transposase host's adaptive immune response. BT2143 chromosomal replication initiator protein dnaA BT2230 DNA polymerase III alpha subunit BT2297 putative reverse transcriptase 0318 FIG.38 presents a schematic overview of how B. BT2355 site-specific DNA-methyltransferance thetaiotomicron might Scavenge for carbohydrates in the BT2400 DNA-3-methyladenine glycosylase I distal intestine. Groups of bacteria assemble on undigested BT2615 reverse transcriptase BT2617 reverse transcriptase or partially digested food particles, elements of the mucus BT2644 DNA topoisomerase I gel layer, and/or exfoliated epithelial cells. Bacterial attach BT2697 DNA mismatch repair protein mut5 ment to these nutrient reservoirs is directed by glycan Specific outer membrane binding proteins (exemplified by SuSC/D paralogs) that are opportunistically deployed 0314. The simple sugar diet evoked a B. thetaiota Omicron depending upon the glycan environment encountered by the tanscriptional response predominated by genes in the car bacterium. Attachment helps oppose bacterial washout from bohydrate transport and metabolism COG (FIG. 32, B). the intestinal bioreactor, promotes harvest of oligo- and Glycoside hydrolase and polysaccharide lyase genes monosaccharides by an adaptively expressed repertoire of upregulated 22.5-fold in mice compated with MM-G cul tures Segregated into distinct groups after unsupervised bacterial glycoside hydrolases, and facilitates Sharing of the hierarchical clustering (FIGS. 29). The group of 24 genes products of digestion with other microbial members whose most highly expressed on the Simple Sugar diet encoded nutritional niche overlaps that of B. thetaiotaOmicron. In this enzymes required for degradation of host glycans (e.g., eight Scheme, microbial nutrient metabolism along the length of hexosaminidases, two-fucosidases, plus a Sialidase), and did the intestine is a Summation of myriad Selfish and Syntrophic not include any plant polysaccharide-directed arabinosi relationships expressed by inhabitants of these micro-habi dases or pectin lyases. tats. Micro-habitat diversity and mutualistic cooperation among component species (including the degree to which 0315. In addition, all components of the fucose utilization Sanctions must be applied against cheats), are reflections of operon (BT1272-BT1277) were expressed at greater levels a dynamic interplay between the available nutrient founda in mice fed the Simple Sugar diet compared to those fed the tion, and the degree of flexible foraging (niche breadth) polysaccharide-rich diet (average induction compared to expressed by micro-habitat residents. Members of Bacteroi MM-G: 12-fold versus 6-fold). The sialylated glycan deg des with broad nutritional niches, Such as B. thetaiotaomi radation operon (BTO455-BTO461) exhibited a comparable cron, contribute to diversity and stability by adaptively augmentation of expression on the Simple Sugar diet. directing their glycan foraging behavior to the mucus when 0316 A similar cluster analysis revealed two distinct polysaccharide availability from the diet is reduced. Mucus groups of genes encoding carbohydrate binding/importing glycans, in turn, represent a point where host genotype and SuSC/SuSD paralogs: a group of 61 expressed at highest diet intersect to regulate the stability of the microbiota. The levels in B. thetaiota Omicron from the ceca of mice fed a highly variable outer chain Structures of mucus and epithe polysaccharide-rich diet, and a group of 21 expressed at lial cell Surface glycans are influenced by host genotype, and highest levels with a simple sugar diet (FIG. 35). Thirteen by microbial regulation of host glycosyltransferase gene of the upregulated SuSC/D paralogs from B. thetaiota Omi expression. Co-evolution of glycan Structural diversity in the cron in mice fed a polysaccharide-rich diet are components host, and an elaborate repertoire of nutrient-regulated gly of predicted operons that also contain ORFs Specifying coside hydrolase genes in gut Symbionts, endows the System glycoside hydrolases and polysaccharide lyases. Five pairs with flexibility in adapting to changes in diet. While the of the SuSC/D paralogs expressed at highest levels on a present Study has focused on the glycan foraging behavior of Simple Sugar diet are part of predicted operons. No SuSC/D B. thetaiota Omicron in mono-associated germ-free mice, paralogs from one diet group were found in operons con Similar analyses can now be used to assess the impact of taining upregulated glycoside hydrolase genes from the other members of the gut microbiota on B. thetaiotaomicron other diet group (FIG. 36). Together, the data indicate that and on one another. US 2005/02397O6 A1 Oct. 27, 2005 41 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 64 <210> SEQ ID NO 1 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 1 cagaga.cccc attactggag aca 23 <210> SEQ ID NO 2 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 2 tgacac catc ctdggcatt 19 <210> SEQ ID NO 3 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 3 citcc.ggcaag taccalattgc 20 <210> SEQ ID NO 4 &2 11s LENGTH 18 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 4 atgtgcc.cag ggctgttgt 18 <210 SEQ ID NO 5 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 5 cittcccitcct gtcctdagag git 22 <210> SEQ ID NO 6 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 6 caa.cccaggg tacaggctag to 22 US 2005/02397O6 A1 Oct. 27, 2005 42 -continued SEQ ID NO 7 LENGTH 18 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer <400 SEQUENCE: 7 ccttgtc.citc cccaag.cg 18 SEQ ID NO 8 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer <400 SEQUENCE: 8 gcc.gcttctt coaaagttcta ca 22 SEQ ID NO 9 LENGTH 22 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer <400 SEQUENCE: 9 catccagotc citagaa.gc.ca tt 22 SEQ ID NO 10 LENGTH 18 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer <400 SEQUENCE: 10 ttgaatgggc cacaggct 18 SEQ ID NO 11 LENGTH 21 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer <400 SEQUENCE: 11 gc gcagtaaa gaatgg catt c 21 SEQ ID NO 12 LENGTH 21 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer <400 SEQUENCE: 12 to gatticoag gtcaccactt g 21 SEQ ID NO 13 LENGTH 22 TYPE DNA US 2005/02397O6 A1 Oct. 27, 2005 43 -continued <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 13 tggcaaagtg gagattgttg cc 22 <210> SEQ ID NO 14 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 14 togttgcaca atgacctgat c 21 <210 SEQ ID NO 15 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 15 acaccggtag taaatcc.cat aaagg 25 <210> SEQ ID NO 16 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 16 tgtc.ctitccc tittctggatg ag 22 <210 SEQ ID NO 17 &2 11s LENGTH 26 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 17 aacagggtgg aactgtatag galagac 26 <210> SEQ ID NO 18 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 18 ggcgtaacta ggc.caggctt 20 <210 SEQ ID NO 19 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer US 2005/02397O6 A1 Oct. 27, 2005 44 -continued <400 SEQUENCE: 19 ggtggctotg gacaatgitat titc 23 <210> SEQ ID NO 20 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 20 aggg catgtt gacitgc cat 19 <210> SEQ ID NO 21 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 21 cgtgtctota citcccggittt co 22 <210> SEQ ID NO 22 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 22 gggttgcagg aacttcttaa ttgta 25 <210> SEQ ID NO 23 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 23 agcggacitat ggaggcgtag 20 <210> SEQ ID NO 24 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 24 citgtc.ttgag gatgtccaca gc 22 <210> SEQ ID NO 25 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 25 cacaggcaat aacaatatat citgaaatct 29 US 2005/02397O6 A1 Oct. 27, 2005 45 -continued <210> SEQ ID NO 26 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 26 aagatggtga tigggct tccc g 21 <210 SEQ ID NO 27 &2 11s LENGTH 34 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 27 ccittggat.cc atggtgatga gcc caggttc tittg 34 <210> SEQ ID NO 28 &2 11s LENGTH 38 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400> SEQUENCE: 28 cctttctaga citacgg acto ataaaag act catcgaag 38 <210 SEQ ID NO 29 &2 11s LENGTH 22 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 29 gagcttgaca cc.gaagg acc citgtc.tc.cag gag cacacag citagacitcgt coccagttgg 60 aggaaagctg gcc agctttg gaatcactgt tdgaagagat gacaatgagc ccatgtc.citt 120 tgttgttggit citt.cgtgcto ggtotggttg tdatticcitcc aactctggct cagaatgaaa 18O ggtacgaaaa atticcitacgt cagdactato atgccaagcc aaagg gcc.gg gacgacagat 240 actgttgaaag tatgatgaag gaaagaaag.c talaccitc.gcc ttgcaaagat gttcaa.cacct 3OO ttatccatgg caccaagaaa alacatcaggg ccatctgtgg aaagaaagga agcc.cittatg 360 gagaaaactt cagaataagc aattic tocct tccagat cac cacttgtacg cactcaagag 420 ggtotc.cctg gccitccatgc gggtaccgag cctittaaaga titt cagatat attgttattg 480 cctgttgaaga tiggctggcct gtc. cactitcg atgagtc.ttt tat cagtc.cg tag acagoag 540 gcc.cctggca cag accitagg totgttittct ttittatctoc cotcacagoc atgatcactg 600 gttcaccgtt cactgtcacg ggccagaaaa taattatct gaaatatact tct cotcatt 660 tataatgcac agaaataaag atatotcaaa amc cataaaa aaaaaaaaaa aaaaaaaaaa 720 a.a. 722 <210 SEQ ID NO 30 &2 11s LENGTH 708 &212> TYPE DNA <213> ORGANISM: Mus sp. US 2005/02397O6 A1 Oct. 27, 2005 46 -continued <400 SEQUENCE: 30 citctagottc acaccgcagg accotgtc.to caggagcacg aagctagaca catcc.ccc.gt 60 tggaggaaag citggcc agct ttggaatcto tdttggaaga gatggtgatg agcc.caggitt 120 citttgttgtt gotcitttittg citgagtctgg atgtgatccc toccactctg gotcaggata 18O actacaggta cataaaattic citgacitcago act at gatgc caa.gc.caact gg.ccgggatt 240 acagatact g c gaaagtatg atgaagaaaa gaaagctaac citc.gc.cittgc aaagaagttca 3OO acaccittt at tdatgacacc aagaacaa.ca toaaggc.cat citgtggagag aatggaaggc 360 cittatggagt aaactittaga ataag caatt citcgatticca ggtoaccact tocacgcaca 420 aaggaggg to tcc caggcct coatgccagt acaatgc citt taaagatttic agatatattg 480 ttattgcc to tdaagatggc tiggcctgtcc acttic gatga gtc.ttittatc agtcc.gtaga 540 cago aggc.cc ctdgcacaga cctaggtotg ttittctttitt atctocc ctic acago catga 600 to acto gttc agcattcact gtcagtggcc agaaaatgaa ttatctgaaa tatacttct c 660 citgatttata atgcacagaa ataaagatat citcaaaaacc aaaaaaaa 708 <210> SEQ ID NO 31 <211& LENGTH: 144 &212> TYPE PRT <213> ORGANISM: Mus sp. <400> SEQUENCE: 31 Met Thr Met Ser Pro Cys Pro Leu Leu Leu Val Phe Val Leu Gly Leu 1 5 10 15 Val Val Ile Pro Pro Thr Leu Ala Glin Asn Glu Arg Tyr Glu Lys Phe 2O 25 30 Leu Arg Glu His Tyr Asp Ala Lys Pro Lys Gly Arg Asp Asp Arg Tyr 35 40 45 Cys Glu Ser Met Met Lys Glu Arg Lys Lieu. Thir Ser Pro Cys Lys Asp 50 55 60 Val Asn. Thir Phe Ile His Gly Thr Lys Lys Asn. Ile Arg Ala Ile Cys 65 70 75 8O Gly Lys Lys Gly Ser Pro Tyr Gly Glu Asn. Phe Arg Ile Ser Asn. Ser 85 90 95 Pro Phe Glin Ile Thr Thr Cys Thr His Ser Arg Gly Ser Pro Trp Pro 100 105 110 Pro Cys Gly Tyr Arg Ala Phe Lys Asp Phe Arg Tyr Ile Val Ile Ala 115 120 125 Cys Glu Asp Gly Trp Pro Val His Phe Asp Glu Ser Phe Ile Ser Pro 130 135 1 4 0 <210> SEQ ID NO 32 &2 11s LENGTH 145 &212> TYPE PRT <213> ORGANISM: Mus sp. <400 SEQUENCE: 32 Met Ala Ile Ser Pro Gly Pro Leu Phe Leu Ile Phe Val Leu Gly Leu 1 5 10 15 Val Val Ile Pro Pro Thr Leu Ala Glin Asp Asp Ser Arg Tyr Thr Lys 2O 25 30 Phe Lieu. Thr Glin His His Asp Ala Lys Pro Lys Gly Arg Asp Asp Arg US 2005/02397O6 A1 Oct. 27, 2005 47 -continued 35 40 45 Tyr Cys Glu Arg Met Met Lys Arg Arg Ser Lieu. Thir Ser Pro Cys Lys 50 55 60 Asp Wall Asn. Thir Phe Ile His Gly Asn Lys Ser Asn. Ile Lys Ala Ile 65 70 75 8O Cys Gly Ala Asn Gly Ser Pro Tyr Arg Glu Asn Lieu Arg Met Ser Lys 85 90 95 Ser Pro Phe Glin Val Thir Thr Cys Lys His Thr Gly Gly Ser Pro Arg 100 105 110 Pro Pro Cys Glin Tyr Arg Ala Ser Ala Gly Phe Arg His Val Val Ile 115 120 125 Ala Cys Glu Asn Gly Leu Pro Val His Phe Asp Glu Ser Phe Phe Ser 130 135 1 4 0 Teu 145 <210 SEQ ID NO 33 &2 11s LENGTH 145 &212> TYPE PRT <213> ORGANISM: Mus sp. <400 SEQUENCE: 33 Met Val Met Ser Pro Gly Ser Leu Leu Leu Val Phe Leu Leu Ser Leu 1 5 10 15 Asp Val Ile Pro Pro Thr Lieu Ala Glin Asp Asn Tyr Arg Tyr Ile Lys 2O 25 30 Phe Lieu. Thr Glin His Tyr Asp Ala Lys Pro Thr Gly Arg Asp Tyr Arg 35 40 45 Tyr Cys Glu Ser Met Met Lys Lys Arg Lys Lieu. Thir Ser Pro Cys Lys 50 55 60 Glu Val Asn. Thir Phe Ile His Asp Thr Lys Asn. Asn. Ile Lys Ala Ile 65 70 75 8O Cys Gly Glu Asn Gly Arg Pro Tyr Gly Val Asn. Phe Arg Ile Ser Asn 85 90 95 Ser Arg Phe Glin Val Thir Thr Cys Thr His Lys Gly Gly Ser Pro Arg 100 105 110 Pro Pro Cys Glin Tyr Asn Ala Phe Lys Asp Phe Arg Tyr Ile Val Ile 115 120 125 Ala Cys Glu Asp Gly Trp Pro Val His Phe Asp Glu Ser Phe Ile Ser 130 135 1 4 0 Pro 145 <210> SEQ ID NO 34 &2 11s LENGTH 145 &212> TYPE PRT <213> ORGANISM: Mus sp. <400 SEQUENCE: 34 Met Ala Met Ser Pro Gly Pro Leu Phe Leu Val Phe Leu Leu Gly Leu 1 5 10 15 Val Val Ile Pro Pro Thr Leu Ser Glin Asp Asp Ser Arg Tyr Thr Lys 2O 25 30 Phe Lieu. Thr Glin His Tyr Asp Ala Lys Pro Lys Gly Arg Asp Asp Arg US 2005/02397O6 A1 Oct. 27, 2005 48 -continued 35 40 45 Tyr Cys Glu Ser Met Met Val Lys Arg Lys Leu Thir Ser Phe Cys Lys 50 55 60 Asp Wall Asn. Thir Phe Ile His Asp Thir Lys Asn. Asn. Ile Lys Ala Ile 65 70 75 8O Cys Gly Lys Lys Gly Ser Pro Tyr Gly Arg Asn Lieu Arg Ile Ser Lys 85 90 95 Ser Arg Phe Glin Val Thir Thr Cys Thr His Lys Gly Arg Ser Pro Arg 100 105 110 Pro Pro Cys Arg Tyr Arg Ala Ser Lys Gly Phe Arg Tyr Ile Ile Ile 115 120 125 Gly Cys Glu Asn Gly Trp Pro Val His Phe Asp Glu Ser Phe Ile Ser 130 135 1 4 0 Pro 145 <210 SEQ ID NO 35 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 35 citctggctica gaatgtaagg tacga 25 <210 SEQ ID NO 36 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 36 gaaatctitta aaggctic ggit acco 24 <210 SEQ ID NO 37 &2 11s LENGTH 26 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 37 citggcticagg ataactacag gtacat 26 <210 SEQ ID NO 38 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 38 gcctgg gaga cccitcottt 19 <210 SEQ ID NO 39 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 39 agcgaatgga agcc.cittaca 20 <210> SEQ ID NO 40 &2 11s LENGTH 2.0 US 2005/02397O6 A1 Oct. 27, 2005 49 -continued &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 40 citcatcgaag togg accggca 20 <210> SEQ ID NO 41 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 41 ggtgaaaaga aagcta acct cittitc 25 <210> SEQ ID NO 42 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 42 agacittgctt attcttaaat titcg 24 <210> SEQ ID NO 43 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 43 aagticcittgg togggaagta taca 24 <210> SEQ ID NO 44 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 44 actcccitcaa agtcatcaca aaca 24 <210> SEQ ID NO 45 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 45 ttaaaaacac cq agattitcc titcaa 25 <210> SEQ ID NO 46 &2 11s LENGTH 16 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 46 gggc.ccc.gcc atctag 16 US 2005/02397O6 A1 Oct. 27, 2005 50 -continued <210> SEQ ID NO 47 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 47 cgggacatgt ttgatgacta totc 24 <210> SEQ ID NO 48 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 48 catcc.cattg aaggattcaa ataaa 25 <210 SEQ ID NO 49 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 49 caatgccaaa ttgctccaat it 21 <210 SEQ ID NO 50 &2 11s LENGTH 16 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 50 tggcc.gtggg citcagt 16 <210 SEQ ID NO 51 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 51 tggtgaattg totcc.gaaaa ga 22 <210> SEQ ID NO 52 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 52 cacgttcatc acgagg to at g 21 <210 SEQ ID NO 53 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence US 2005/02397O6 A1 Oct. 27, 2005 51 -continued &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 53 ccitctggtga agcc.caagat c 21 <210> SEQ ID NO 54 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 54 totgggitttc cqccagttt 19 <210 SEQ ID NO 55 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 55 caccittccitc titcccaaag.c t 21 <210> SEQ ID NO 56 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 56 gc gtcggact c ggtottct 19 <210 SEQ ID NO 57 &2 11s LENGTH 23 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 57 atgtct caca atgccatcag gtt 23 <210 SEQ ID NO 58 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 58 gctogcagat cagoagacitc t 21 <210 SEQ ID NO 59 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Primer <400 SEQUENCE: 59 US 2005/02397O6 A1 Oct. 27, 2005 53 -continued to accotgca aagatgtcaa caccitttatc catggcaa.ca agagcaa.cat caaggccatc 240 tgtggagciga atggaa.gc.cc ttacagagaa aacttaagaa tagcaagtc. tcc ctitccag 3OO gtoaccactt gcaa.gcacac aggagggtct coccggc citc catgc.cagta Cogagccitct 360 gcagggttca gacatgttgt tattgcctgt gagaatggct toccggtoca citt.cgatgag 420 toatttittca gtctatag 4.38 <210> SEQ ID NO 64 &2 11s LENGTH 438 &212> TYPE DNA <213> ORGANISM: Mus sp. <400 SEQUENCE: 64 atgg.cgatga gcc caggtoc tttgttcttg gtottcc tot toggtotggit totgatc.cct 60 cc cactctgt citcaggatga citcCagg tac acaaaattcc toacticago a citatgatgcc 120 aa.gc.caaaag gocgggacga cagatact gc gaaagtatga tiggtgaaaag aaagcta acc 18O totttctgca aagatgtcaa caccitttatc catgacacca agaacaa.cat caaggccatc 240 tgtggaaaga aaggaa.gc.cc titatggacga aatttaagaa taagcaagtc. tcgct tccag 3OO gtoaccactt gcacacacaa aggaaggtot coccggc citc catgcaggta Cogagccitct 360 aaagggttca gatatatt at tattggctgt gagaatggct ggcctgtc.ca citttgatgag 420 tottittatca gtccatag 4.38 What is claimed is: 9. The method of claim 8, wherein the amount of or the 1. A method for treating obesity or an obesity-related activity of the Fiaf polypeptide is increased by administering disorder, the method comprising: (a) diagnosing a Subject in a PPAR agonist to the subject. need of treatment for obesity or an obesity-related disorder; 10. A method for decreasing body fat or for promoting and weight loSS in a Subject, the method comprising altering the (b) increasing either the amount of or the activity of a Fiaf microbiota population in the Subject's gastrointestinal tract polypeptide in the Subject. Such that at least one microbial-mediated Signaling pathway 2. The method of claim 1, wherein the amount of Fiaf in the Subject that regulates energy Storage is either Stimu polypeptide is increased in the Subject by administering an lated or substantially inhibited, whereby stimulating or effective amount of Fiaf polypeptide to the subject. inhibiting the Signaling pathway causes a decrease in body 3. The method of claim 2, wherein the subject is selected fat or promotes weight loSS in the Subject. from the group consisting of a human, a dog, a cat, a cow, 11. The method of claim 10, wherein the microbiota a horse, a rabbit, a pig, a sheep, a goat, an avian Species and population is altered by decreasing the presence of at least a fish species. one genera of Saccharolytic microbe. 4. The method of claim 3, wherein the obesity related 12. The method of claim 10, wherein the microbiota disorder is Selected from the group consisting of metabolic population is altered by decreasing the presence of B. Syndrome, type II diabetes, hypertension, cardiovascular thetaiota Omicron. disease, and nonalcoholic fatty liver disease. 5. The method of claim 4, wherein the amount of or the 13. The method of claim 11, wherein the presence of a activity of the Fiaf polypeptide is increased by administering microbe genera is decreased by administering a probiotic a PPAR agonist to the subject. Selected from the group consisting of Lactobacillus, Acido 6. A method for decreasing body fat or for promoting philus, Bifidobacteria and other components of the gut weight loSS in a Subject, the method comprising increasing microbiota. either the amount of or the activity of a Fiaf polypeptide in 14. The method of claim 10, wherein the signaling path the Subject. way regulates hepatic lipogenesis and is Substantially inhib 7. The method of claim 6, wherein the amount of Fiaf ited, thereby resulting in a decrease of triglyceride Storage in polypeptide is increased in the Subject by administering an the adipocytes of the Subject. effective amount of a Fiaf polypeptide to the Subject. 15. The method of claim 14, wherein the amount of at 8. The method of claim 7, wherein the subject is selected least one compound Selected from the group consisting of from the group consisting of a human, a dog, a cat, a cow, acetyl-CoA carboxylase, fatty acid Synthase, Sterol response a horse, a rabbit, a pig, a sheep, a goat, an avian Species and element binding protein 1 and carbohydrate response ele a fish species. ment binding protein is decreased in the Subject. US 2005/02397O6 A1 Oct. 27, 2005 54 16. The method of claim 14, wherein hepatic lipogenesis (b) altering the microbiota population in the Subjects is Substantially inhibited as a result of a decrease in micro gastrointestinal tract Such that microbial-mediated tran bial processing of dietary polysaccharides. Scriptional Suppression of a lipoprotein lipase inhibitor 17. The method of claim 14, wherein the signaling path in the Subject is decreased. way Substantially decreases lipoprotein lipase activity and 31. The method of claim 30, wherein the lipoprotein results in a decrease of triglyceride Storage in the adipocytes lipase inhibitor is a Fiaf polypeptide. of the subject. 32. The method of claim 31, wherein microbial-mediated 18. The method of claim 17, wherein lipoprotein lipase transcriptional Suppression of the Fiaf polypeptide occurs activity is Substantially decreased as a result of microbial only in the gastrointestinal tract of the Subject. mediated transcriptional Suppression of a Fiaf polypeptide. 33. The method of claim 30, wherein the microbiota 19. The method of claim 18, wherein microbial-mediated population is altered by decreasing the presence of at least transcriptional Suppression of the Fiaf polypeptide occurs one genera of Saccharolytic microbe. only in the gastrointestinal tract of the Subject. 34. The method of claim 30, wherein the microbiota 20. The method of claim 10, wherein the subject is population is altered by decreasing the presence of B. Selected from the group consisting of a human, a dog, a cat, thetaiota Omicron. a cow, a horse, a rabbit, a pig, a sheep, a goat, an avian 35. The method of claim 33, wherein the presence of a Species and a fish species. microbe genera is decreased by administering a probiotic 21. The method of claim 10, further comprising admin Selected from the group consisting of Lactobacillus, Acido istering to the Subject an effective amount of a Fiaf polypep philus, Bifidobacteria and other components of the gut tide. microbiota. 22. A method for decreasing body fat or for promoting 36. The method of claim 30, wherein the subject is weight loSS in a Subject, the method comprising altering the Selected from the group consisting of a human, a dog, a cat, microbiota population in the Subject's gastrointestinal tract a cow, a horse, a rabbit, a pig, a sheep, a goat, an avian Such that microbial-mediated transcriptional Suppression of Species and a fish species. a lipoprotein lipase inhibitor in the Subject is decreased. 37. The method of claim 30, wherein the obesity related 23. The method of claim 22, wherein the lipoprotein disorder is Selected from the group consisting of metabolic lipase inhibitor is a Fiaf polypeptide. Syndrome, type II diabetes, hypertension, cardiovascular 24. The method of claim 23, wherein microbial-mediated disease, and nonalcoholic fatty liver disease. transcriptional Suppression of the Fiaf polypeptide occurs 38. The method of claim 30, further comprising admin only in the gastrointestinal tract of the Subject. istering to the Subject an effective amount of a Fiaf polypep 25. The method of claim 22, wherein the microbiota tide. population is altered by decreasing the presence of at least 39. A composition for decreasing body fat or for promot one genera of Saccharolytic microbe. ing weight loSS, the composition comprising a Fiaf polypep 26. The method of claim 22, wherein the microbiota tide and an agent that alters the microbiota population in a population is altered by decreasing the presence of B. Subject's gastrointestinal tract Such that microbial-mediated thetaiota Omicron. transcriptional Suppression of a lipoprotein lipase inhibitor 27. The method of claim 25, wherein the presence of a in the Subject is decreased. microbe is decreased by administering a probiotic Selected 40. The composition of claim 39, wherein the agent is a from the group consisting of Lactobacillus, Acidophilus, probiotic Selected from the group consisting of Lactobacil Bifidobacteria and other components of the gut microbiota. lus, Acidophilus, Bifidobacteria and other components of the 28. The method of claim 22, wherein the subject is gut microbiota. Selected from the group consisting of a human, a dog, a cat, 41. The composition of claim 39, wherein the composi a cow, a horse, a rabbit, a pig, a sheep, a goat, an avian tion further comprises a compound Selected from the group Species and a fish species. consisting of acarbose, Xenical, orlistat, an amphetamine 29. The method of claim 22, further comprising admin and Sibutramine. istering to the Subject an effective amount of a Fiaf polypep 42. Abiomarker for use in predicting whether a Subject is tide. at risk for becoming obese or Suffering from an obesity 30. A method for treating obesity or an obesity-related related condition, the biomarker comprising the amount of disorder, the method comprising: (a) diagnosing a Subject in circulating Fiaf polypeptide. need of treatment for obesity or an obesity-related disorder; and k k k k k