Human Milk and its role in shaping the infant intestinal microbiota

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1 Human Milk and its role in shaping the infant intestinal microbiota David A. Mills Peter J. Shields Endowed Chair Dept. Food Science & Technology Dept. Viticulture & Enology UC Davis

ecosystems, diversity drives stability, results in few open niches Low

2 Birth and Nursing Early microbiota in humans is less complex, more unstable, and increases in complexity over the first two years of life In ecosystems, diversity drives stability, results in few open niches Low diversity until weaning, then increases in diversity >3 yrs coincides with adult diet

3 C-section Impact?

4 The Diet Drives Microbiota Specifically, carbon sources Experiments from the ecosystem to genomic to mechanistic level point to this

5 Diet Drives Microbiota MILK!

6 Milk is the result of 200 M years of evolution constrained by parent offspring conflict Maternal Optima Benefits B<C Costs Time Infant Optima B<(r)C

7 Human milk composition Milk Macro-/Micronutrients Macro- and micronutrients HMOs Proteins Lipids Water Lactose Newburg 2005, 2009 Bode 2012

8 Human milk composition Milk Macro- and micronutrients Water Macro-/Micronutrients HMOs Proteins Lipids Lactose Microbiota shaping Immunoglobulins Lysozyme Lactoferrin Lactoferricin Triglycerides Free fatty acids Food! Newburg 2005, 2009

9 Multiple Functions of Components: Common Theme in Milk

10 Multiple Functions of Components: Common Theme in Milk

11 Human Milk Glycans HMOs Proteins Lipids Lactose Garrido et al Microbiology 2013

12 Human milk composition Milk Macro-/Micronutrients HMOs Macro- and micronutrients HMOs Proteins Chain Length 4 5 Lipids Water Lactose Other HMOs of longer lengths Nature 468 S5-S7 (23 December 2010)

14 Associations with Milk Oligosaccharides and Infant Health Lower amounts of HMOs delivered were associated with children who had developed poorly (stunted) Cell 164, , 2016

16 Human Milk Glycans Inhibit Pathogen Binding Newburg Ann Rev Nutr 2005

Probiotics & prebiotics Definitions and misperceptions Probiotics live microorganisms that when administered in adequate amounts confer a health benefit on the host (UNFAO/WHO 2001).

17 Probiotics & prebiotics Definitions and misperceptions Probiotics live microorganisms that when administered in adequate amounts confer a health benefit on the host (UNFAO/WHO 2001). Prebiotics a prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora, that confer benefits upon host well-being and health (J. Nutr :830S-837S) Synbiotics combinations of prebiotics and probiotics

18 HMO Structural Diversity FOS/GOS lacto-n-biose lactose b1-4 a1-2 a1-3 a1-4 a2-3 a2-6 b1-3 b1-4 N-acetyllactosamine b1-3 b1-6 n=0-15 b1-4 b1-3 b1-3 b1-4 Lacto-N-tetraose (LNT) (type 1 chain) b1-4 b1-3 b1-4 Lacto-N-neotetraose (LNnT) (type 2 chain) b1-6 b1-4 b1-3 b1-3 Lacto-N-hexaose (LNH) b1-4 b1-4 a1-2 a fucosyllactose (2 FL) a2-3 b1-4 3-fucosyllactose (3FL) a2-6 b1-4 b1-3 b1-3 b1-4 b1-6 b1-4 b1-3 b1-3 b1-3 b1-4 b1-4 b1-6 b1-4 b1-3 b1-3 b1-3 b1-3 b1-4 b1-3 b1-4 b1-3 b sialyllactose (3 SL) 6 -sialyllactose (6 SL) iso-lacto-n-octaose iso-lacto-n-neooctaose para-lacto-n-octaose b1-4 b1-4 b1-4 b1-4 b1-3 a1-4 b1-3 b1-4 b1-3 b1-3 a1-3 b1-3 b1-3 a1-3 b1-3 a1-2 Lacto-N-fucopentaose I (LNFP I) Lacto-N-fucopentaose II (LNFP II) Lacto-N-fucopentaose III (LNFP III) Lacto-N-fucopentaose V (LNFP V) b1-4 b1-4 b1-4 b1-4 a2-3 b1-3 b1-3 a2-6 b1-3 b1-3 a2-6 b1-4 b1-3 a2-3 a2-6 b1-3 b1-3 LS-Tetrasaccharide a (LST a) LS-Tetrasaccharide b (LST b) LS-Tetrasaccharide c (LST c) Disialyllacto-N-tetraose (DSLNT) Glucose (Glc) Galactose (Gal) N-acetylglucosamine (GlcNAc) Fucose (Fuc) N-acetylneuraminic acid (NeuAc) different HMO Courtesy of Lars Bode

19 In what way is the infant microbiota modulated by milk?

Breast milk enriches bifidobacterial populations Culture based methods Non-culture based methods FISH flow cytometry 531 infants Fallani et al

20 Breast milk enriches bifidobacterial populations Culture based methods Non-culture based methods FISH flow cytometry 531 infants Fallani et al Microbiology 2011 South Eastern African and Northern European Grzeskowiak et al JPGN 2012 Malawi Biavati and Mattarelli 2006 Finland Bifidobacterium

21 Breast milk enriches bifidobacterial populations N= 531 (16S) N=110 (metagenome) Most shotgun and 16S rrna V4 sequences (75 ± 20%) in all babies mapped to members of the Bifidobacterium genus.

22 Bifidobacterium species Taxonomy Bifido shunt

23 Milk Oriented Microbiota Bifidobacteria MOM WHY? Protection from harm

24 Mouse survival after O157 infection Nature 2010 BA = B. adolescentis BL = B. longum

25 Mouse survival after O157 infection Nature 2010 BA = B. adolescentis BL = B. longum

26 Nature 2010

% Actinobacteria 82 Bifidobacteriales 80

longum longum 95 68 32 International Center

27 B. infantis benefit? Bangladesh Infant Vitamin A Study NGS analysis % Actinobacteria 82 Bifidobacteriales 80 Bifidobacteriaceae 80 Bifidobacterium 68 Bif-TRFLP analysis B. longum B. longum infantis B. longum longum International Center for Diarrhoeal Disease Research (Bangladesh) Charles Stephenson Western Human Nutrition Research Center (Davis, CA) Pediatrics 2014

28 Positive associations of Actinobacteria genera and Bifidobacterium species/subsp with vaccine responses Vaccine OPV PPD TT HBV SEB T-cell Prolif. subsp longum subsp infantis subsp infantis subsp infantis subsp infantis T-cell Cytokine ALS IgG DTH Skin Test Bifidobacterium n/a subsp longum-th2 n/a Bifidobacterium, Rothia subsp infantis- IL-2/Th2/17 B. longum-th2/17 subsp infantis-th17 Actinomyces Rothia n/a n/a subsp longum-th2 n/a n/a Huda et al Pediatrics 2014

29 Are HMOs natural prebiotics for Bifidobacteria? Are there differences in growth on HMOs of Bifidobacteria vs. other GIT bacteria? Do Bifidobacterial species isolated from different GIT environments grow differently on HMO (i.e. adult vs. infant-borne)?

30 Select gut microbes What bacteria grow well with HMOs as a sole carbon source? Clostridial sp. Lactobacillus sp. Eubacterium sp. Streptococcus sp. Escherichia coli Enterococcus sp. Bacteriodes sp. Bifidobacterial sp. Yes No Weakly No No No No Yes Ward et al. AEM 2006, Marcobal JAFC 2010, Marcobal CHM 2011

31 What bifidobacteria grow to high cell density with HMOs as a sole carbon source? Infants Adults B. infantis Most B. bifidum Most B. breve Some B. longum Some B. animales None B. adolescentis None Ward et al. AEM 2006, LoCascio MicroBiotech 2009.

32 Growth of different intestinal microbes on 2 fucosyl-lactose Glycobiology vol. 23 no. 11 pp , 2013

33 2 fucosyl-lactose Glycobiology vol. 23 no. 11 pp , 2013 Drop in ph Lactate SCFA Fucosidase

34 Glycobiology vol. 23 no. 11 pp , 2013 Lactodifucotetraose (LDFT) OD Drop in ph Lactate SCFA Fucosidase

35 In Vitro Fecal enrichment with HMO and FOS Glycobiology vol. 23 no. 2 pp , 2013

36 HMOs as prebiotics for Bifidobacteria Which oligosaccharides are being consumed?

37 Bifidobacterial HMO Glycoprofiling % HMO Consumed % HMO abundance in breast milk 2.5 B. infantis HMO abundance in pooled breast milk OD (600 nm) 10 1 B. breve Time(hours) B. longum 100 HMO m/z B. infantis consumption Several small MW oligosaccharides consumed by B. infantis Single HMO composition consumed by other bifidobacteria Locascio et al JAFC HMO m/z

38 HMO structures consumed by B. infantis CH 2 Lacto-N-Tetraose [M+ Na] + =732.3 CH 2 O CH 2 O CH 2 O O O O CH 2 Lacto-N-Neohexose CH 2 O CH 2 O CH 2 [M+ Na] + = O O O CH 2 CH 2 O NHAc O CH 2 O O O CH 2 NHAc NHAc Isomeric fucosylated Lacto-N-Hexose [M+ Na] + = CH 2 CH 2 O CH 2 O CH 2 O O O O O H 3 C CH 2 O O O NHAc CH 2 NHAc O CH 2 Difucosyllacto-N-Hexaose CH 2 O CH 2 O O [M+ Na] + = O CH CH 2 NHAc 2 O O O H O CH 3C 2 CH 2 O O CH 3 O O NHAc O O H 3C

39 Which neutral/fucosylated oligosaccharides are consumed by B. infantis over time? Sela, Garrido AEM 2012

% Consumed Sela JBC 2011 80 70 60 50 Which sialyated oligosaccharides are

40 % Consumed Sela JBC Which sialyated oligosaccharides are consumed by B. infantis over time? m/z m/z OD Early exponential I Early exponential II Mid-exponential Late exponential Early stationary -10 Time course over growth 0

In Vitro Isomer Specific Consumption B. infantis LNH 56 % ±0.5 LNnH 5 % ±4 p-lnh 44 % ±5 Carlito Lebrilla UCD Chemistry m/z 538.

41 In Vitro Isomer Specific Consumption B. infantis LNH 56 % ±0.5 LNnH 5 % ±4 p-lnh 44 % ±5 Carlito Lebrilla UCD Chemistry m/z New 1 43 % ±4 MFLNH III 81 % ±2 MFLNH I 17 % ±4 IFLNH III 31 % ±1 IFLNH I 46 % ±2 m/z Strum et al Anal. Chem. 2013

42 Percent Consumption of HMO Structures Consumption array of B. infantis ATCC Strum, Lee, Mills Anal. Chem Bacteria can be characterized by their HMO consumption profile. Orange circles are sized proportionally to the percent consumption.

43 HMOs as prebiotics for bifidobacteria in situ? Do bifidobacteria successfully compete for these oligosaccharides?

44 Bacteroides sp. growth on HMO

45 Marcobal & Sonnenburg Cell Host Microbe 2011 Bifidobacteria vs Bacteroides in situ Lacto-N-neotetrose supplementation of gnotobiotic mice with Bifidobacterium infantis and Bacteroides thetaiotaomicron

46 What genome features are required Do bifidobacteria to utilize human consume milk oligosaccharides? sialyated HMOs?

47 HMO utilization by Bifidobacteria

48 Comparative Bifidobacterium Genomics B. longum subsp. longum DJO10A 2,389,526 bp B. longum subsp. infantis ATCC ,832,748 bp Adult derived strain BMC Genomics 2008 Infant derived strain PNAS 2008

49 What genes are required to utilize HMO? - Hydrolytic enzymes active on specific glycolytic linkages - Transport systems for oligosaccharides and/or monosaccharides Fucosidase Hexosaminidase Sialidase Galactosidase

50 HMO cluster 1 All 4 glycosyl hydrolases Array of oligosaccharide transporters 0 10 kb 20 kb 30 kb 40 kb permease permease SBP permease ESB?- galactosidase fucosidase SBP SBPsialidase SBP SBP SBP hexosaminidase ATPas e - HMOs are bound by SBP lipoproteins proximal to permeases Extracelluar Solute binding protein (SBP) - ATP hydrolysis prompts transport of oligosaccharides across membrane - Intracellular glycolytic enzymes deconstruct oligosaccharide permease ATP ADP ATPas e cytoplasm Glycolytic enzymes Sela PNAS 2008

51 Are HMO related genes, growth and glycoprofile conserved in other Bifidobacteria?

52 1.40 Distribution of the HMO growth phenotype in bifidobacteria Growth Curves Bl. infantis OD 600 OD (600 nm) Bl. longum & other Bifs DJ OD :00:00 4:48:00 9:36:00 14:24:00 19:12:00 24:00:00 28:48:00 33:36:00 38:24:00 T (hrs) Time (hours) LoCascio et al Microbial Biotech 2009

53 Distribution of HMO glycoprofiles in Bifidobacteria lon LoCascio et al Microbial Biotech 2009

54 Main HMO cluster permease permease SBP permease ESB?- galactosidase fucosidase SBP SBP sialidase SBP SBP SBP hexosaminidase pfam00449 pfam01120 pfam01547 pfam02012 Genome urease, alpha fucosidase sbp fam 1 sialidase Bifidobacterium longum infantis ATCC Bifidobacterium longum infantis UCD Bifidobacterium longum infantis UCD Bifidobacterium longum infantis UCD Bifidobacterium longum infantis UCD Bifidobacterium longum infantis UCD Bifidobacterium longum infantis UCD Bifidobacterium longum infantis UCD Bifidobacterium longum DJO10A Bifidobacterium longum infantis ATCC Bifidobacterium longum infantis CCUG Bifidobacterium longum longum UCD Bifidobacterium longum longum UCD Bifidobacterium longum longum UCD Bifidobacterium longum longum UCD Bifidobacterium longum longum UCD Bifidobacterium longum longum UCD Bifidobacterium longum suis UCD Bifidobacterium longum suis UCD Bifidobacterium longum suis UCD Bifidobacterium longum suis UCD ATPas e Unpublished

55 Many B. infantis probiotics incorrectly labeled B. longum subsp. infantis, subp. longum and subsp. suis confirmed as subspecies International Journal of Systematic and Evolutionary Microbiology (2008), 58, B. longum subsp. infantis genome sequenced (Sela et al PNAS, 2008) confirming infantis/longum subspecies split IDed HMO consumption genes as linked to infantis subspecies (urease too) 2016 Many probiotics not up to date on current taxonomy Lewis, Z. T Validating bifidobacterial species and subspecies identity in commercial probiotic products. Pediatrics Research 79:

56 Oligosaccharide Degradation Plant-derived oligosaccharide catabolic genes Capacity Mammal-derived oligosaccharide catabolic genes Transketolase 1 3 Beta-glucosidase 2 4 Alpha-glucosidase Evolved to 0 6 Beta-fructosidase 2 5 Beta-xylosidase consume 1 6 Alpha-L-arabinofuranosidase 1 5 xylose transporter (periplasmic) 0 2 plant sugars xylose transporter (permease) 0 1 xylose isomerase 0 1 B. longum DJO10A Beta-galactosidase 5 3 N-acetyl-beta-hexosaminidase 4 1 Alpha-L-fucosidase 3 0 Evolved to Fucose dissimilation protein 2 0 Sialidase 2 0 N-acetylmannosamine-6-P consume epi. 2 0 Sialate O-Acetylesterase 1 0 N-Acetylneuraminate milk sugars Lyase 1 0 Endo-_-N-acetylglucosaminidase 1 0 B. infantis UCD272 Adult derived strain Infant derived strain

57 B. longum sp infantis B. longum sp. longum ABC AA transporter urease Urease γ α uree ref ureg ured HMO Clusters Regulatory HisK MFS 1 fucosidase Regulatory MFS 1 fucosidase Regulatory nane Sugar kinase? sialidase nana satabcd (sialic acid ABC transporter) ABC transporter (LNB) LNB phosphorylase nahk galt gale kb 10 kb 20 kb 30 kb 40 kb 1 0 M M P P P P ESB?- P P A β-galactosidase fucosidase sialidase β-hexosaminidase (Blon_2334) (Blon_2336) (Blon_2348) (Blon_2355) JCM1272 JCM11346 LoCascio et al AEM 76:

# Reads Aligned 1.40 Growth phenotype Illumina sequencing of UCD299 Growth Curves eads ned 1.20 2620457-2628032 1.

longum 272 264 265 301 306 315 316 263 DJ 298 299 300 302 303 304 305 0.

58 # Reads Aligned 1.40 Growth phenotype Illumina sequencing of UCD299 Growth Curves eads ned OD (600 nm) ssp. infantis ssp. longum DJ :00:00 4:48:00 9:36:00 14:24:00 19:12:00 24:00:00 28:48:00 33:36:00 38:24:00 T (hrs) ESB? IS Perm Perm 2344 SBP 2347 SBP Perm Perm 10 kb 20 kb 30 kb 40 kb HMO Cluster 2350 SBP 2351 SBP 2352 SBP 2354 SBP LoCascio et al Applied and Environmental Microbiology 76:

59 permease permease SBP permease ESB?- galactosidase fucosidase SBP SBP sialidase SBP SBP SBP hexosaminidase ATPas e Do the functional characteristics of the HMO cluster genes correspond to HMO metabolism?

H + H + H + H + Transporter symbols fucose EIIBC Glc EIIBC NAG glucose galactose EIIA Glc ~P EIIA Glc ATP ADP ATP ADP ATP ADP EIIA NAG ~P EIIA NAG ATP ADP ATP ADP oligosaccharide HPr~P HPr HPr~P HPr

60 H + H + H + H + Transporter symbols fucose EIIBC Glc EIIBC NAG glucose galactose EIIA Glc ~P EIIA Glc ATP ADP ATP ADP ATP ADP EIIA NAG ~P EIIA NAG ATP ADP ATP ADP oligosaccharide HPr~P HPr HPr~P HPr LNB EI~P EI Pyruvate PEP Intracellular HMO hydrolysis EI~P Pyruvate EI PEP GlcNAc Neu5Ac Glycomic symbols Glc Gal Lactaldehyde NADH NAD + L-fucose L-fuculose ATP ADP 22 1,2 propanediol fucosidase β-galactosidase LNB phosphorylase β-hexosaminidase sialidase?? Fuculose 1P? DHAP 23 2 ATP 2 ADP Galactose 2 Glucose F6P 3 GA-3P Ribose 5P 5 Ribulose 5P Leloir pathway 1 F6P 4 P i 2 NH 3 Erythrose 4P Sedoheptulose 7P 6 21 Xylulose 5P H 2 O glucosamine 6P 7 Acetate H 2 O Acetyl-P 8 3 Acetate 2 Acetyl-P 3 ADP 3 ATP 2 ADP 2 GA-3P Pyruvate 2 NAD + 2 NADH N-acetylglucosamine ATP ADP N-acetylglucosamine 1P 20 2 ATP NAG 6P 17 2 NADH 2 NAD + 2 L-Lactate Sialic acid N-acetylmannosamine ATP ADP N-acetylmannosamine 6P GlcNAc Fuc Neu5Ac Lac LNB Intracellular GH activity LNT Jae-Han Kim HMO Metabolic symbols Glycosidic bond cleavage

61 Whole cell proteomics of B. infantis grown on different prebiotic sugars HMO GOS GOS FOS/Inulin / Blon_2344 Blon_2347 Blon_2414 Blon_2061 Blon_2348: a-sialidase Blon_2335: b-hexosaminidase Blon_2416: b-galactosidase Blon_0268: b-galactosidase Blon_2460: a-galactosidase Blon_2453: GH, family13 Blon_2336: a-fucosidase Blon_0732: b-hexosaminidase Blon_2016: b-galactosidase Blon_2334: b-galactosidase Blon_2416: b-galactosidase Blon_1740: GH, family 13 Leloir pathway Blon_2056: Exo-inulinase Blon_0787: Exo-inulinase Blon_0128: Suc phosphorylase HexNAc catabolism Bifidus shunt & glycolysis PLoS One 2013 Jae Han Kim

62 What about the glycosidases in B. infantis? (3) (5) Fucosidase Hexosaminidase (2) Sialidase Galactosidase (5)

63 Sialidase expression permease permease SBP permease ESB?- galactosidase fucosidase SBP SBP sialidase SBP SBP SBP hexosaminidase NanH2 ATPas e Sela JBC 2011

64 B. infantis sialidase activities NanH1 Only NanH2 is expressed on NanH2 during growth on HMO NanH2 has 175 fold higher turnover rate than NanH1 Only NanH2 cleaves HMO in vitro HMO Commercial Sialidase NanH2 NanH1 Xi Chen Chemistry UC Davis C. Lebrilla Chemistry UC Davis

65 α-fucosidase gene expression Lacto-N-tetraose (LNT) Lacto-N-neotetraose (LNnT) Fucosidase genes Sela, Garrido et al, AEM 2012

66 Blon_2335 is an α1-2 fucosidase 2 Fucosyl lactose 3 Fucosyl lactose Lewis a Blood group H Sela, Garrido et al, AEM 2012

67 Blon_2336 is an α1-3/4 fucosidase Fucose Lactose 3 FL Fucosyl lactose Lewis a Sela, Garrido et al, AEM 2012

β-hexosaminidases and β-galactosidases Gal GlcNAc β-hexosaminidases: Blon_0459 Blon_0732 Blon_2355 Lacto-N-tetraose β-galactosidases active on HMO

68 β-hexosaminidases and β-galactosidases Gal GlcNAc β-hexosaminidases: Blon_0459 Blon_0732 Blon_2355 Lacto-N-tetraose β-galactosidases active on HMO Blon_2016 (Galβ1-3GlcNAc) Blon_2334 (Galβ1-4GlcNAc) Yoshida et al Glycobiology 2012 Garrido et al Food Microbiology 2013 Garrido et al, Anaerobe 2012

2013 Bacteria can be characterized by their HMO consumption

69 Percent Consumption of HMO Structures Consumption array of B. infantis ATCC Strum, Lee, Mills Anal. Chem Bacteria can be characterized by their HMO consumption profile. Orange circles are sized proportionally to the percent consumption.

β-hexosaminidases active on HMO Gal; Glc Gal-Glc Lac;

Active on linear and branched HMO Blon_2355: Limited

70 β-hexosaminidases active on HMO Gal; Glc Gal-Glc Lac; LNB Lactose Gal; Glc Gal-Glc-GlcNAc Lac; Lactose LNB trimer Triose LNT LNT LNT LNH β-hexosaminidases: Blon_0459 and Blon_0732 Active on linear and branched HMO Blon_2355: Limited to linear HMO Lacto-N-hexaose Garrido et al, Anaerobe 2012

71 HMO cluster SBPs Family 1 (oligosaccharide binding) Extracellular Solute binding protein (SBP) permease cytoplasm HMO cluster Garrido PLoS One 2011

72 Garrido PLoS One 2011 SBP expression in B. infantis during growth on HMO permease permease SBP permease ESB?- galactosidase fucosidase SBP SBP sialidase SBP SBP SBP hexosaminidase ATPas e Extracelluar Solute binding protein (SBP)

73 Garrido PLoS One 2011 HMO cluster SBP Blon2347

74 Current consumption model

75 Garrido, et al. Advances in Nutrition (2012)

76 What about bifidobacterial growth on milk glycoproteins? N-linked exp. Lactoferrin Immunoglobulins O-linked exp. Caseins (Κ)

77 What about bifidobacterial growth on milk glycoproteins? RNaseB as proxy N-linked glycan Garrido et al Molecular and Cellular Proteomics 2012

78 What about bifidobacterial growth on milk glycoproteins? Yeast mannoprotein used as proxy N-linked glycan Strains vary in growth response Garrido et al Molecular and Cellular Proteomics 2012

79 Endoglycosidase genes in bifidobacteria GH18a (EndoBI-1) B. infantis JCM11346 B. infantis JCM7007 B. infantis ATCC17930 B. infantis ATCC15702 B. infantis ATCC15697 B. infantis JCM7009 B. infantis JCM7011 EndoE(alpha subunit) B. infantis 157F GH18b (EndoBI-2) B. longum SC706 B. longum SC116 B. longum SC630 B. breve SC559 B. infantis SC142 B. infantis SC143 B. breve UCC2003 B. breve JCM1273 EndoD Endo-beta-N-acetylglucosaminidases EndoBI-1 and EndoBI-2 active on all N-linked milk glycoproteins Garrido et al Molecular and Cellular Proteomics GH85 (EndoBB) B. longum DJO10A B. breve JCM7019 B. breve JCM7020 B. breve KA179 B. breve SC139 B. breve SC506 B. breve SC568 B. breve SC95

80 Bifidobacterial consumption of milk glycoproteins Oligosaccharides released from whey proteins using EndoBI-1. Intact whey proteins (not treated) Deglycosylated whey proteins Karav et al. AEM 2016

81 Bifidobacterial consumption of milk glycoproteins EndoBI-1 gene expression Karav et al. AEM 2016

82 We ve characterized consumption of HMO and glycoproteins in vitro Does consumption of milk glycans encourage colonization behaviors by bifidobacteria?

83 Garrido PLoS One 2011 SBP Blon2347 binding to Caco2 cells Extracellular Solute binding protein (SBP) permease cytoplasm Caco-2 + Blon2347 GalNAca1-3(Fuca1-2)Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAc 74. 1%

84 Growth on milk oligosaccharides helps some bifidobacteria bind intestinal cells Caco-2 HMO vs Lac grown cells: Induce TJ proteins Induce antiinflammatory cytokines (IL-10) HT-29 Chichlowski et al JPGN 2012

85 Binding of HMO-grown B. infantis to intestinal cells reduces inflammation-related genes Wickramasinghe et al. BMC Microbiology (2015) 15:172

86 Model for bifidobacterial enrichment in the infant GIT

87 Model for bifidobacteria enrichment in the infant GIT Complex milk glycans enhance efficacy of specific bifidobacteria

88 Take home points. Milk provides a model of a food which expressly modulates the microbiota in a healthful way Specificity of that modulation is driven in part by glycan complexity and cognate bacterial catabolism B. longum subsp. infantis (B. infantis) is well suited for rapid consumption of HMOs and colonization of the infant GIT Enzymatic repertoire in B. infantis enables consumption of a many types of HMOs (complementing enzyme specificities)

An evolutionary link between bifidobacterial probiotics and milk. David Mills

An evolutionary link between bifidobacterial probiotics and milk David Mills damills@ucdavis.edu Probiotics are historically linked to dairy products, however Many controlled studies have documented effects