Nutrition and the infant gut microbiota: Health and disease in the first 1000 days

David A. Mills

Peter J. Shields Endowed Chair Dept. Food Science & Technology

Dept. Viticulture & Enology UC Davis

Which food has evolved to benefit

the health of the consumer?

MILK!

Both consumers…infants and the

infant gut microbiota

MILK!

Time

Infant Optima

B<C

Benefits Costs

B<(r)C

Maternal Optima

Milk is the result of 200 M years of evolution constrained by parent – offspring conflict

Human milk composition

Lactose Water

Macro- and micronutrients HMOs

Proteins

Lipids

Milk Macro-/Micronutrients

Protein Immunoglobulins Lysozyme Lactoferrin Lactoferricin Triglycerides Free fatty acids

Microbiota shaping

Food!

Newburg 2005, 2009

Garrido et al Microbiology In Press

Human Milk Glycans

Lactose

HMOs Proteins

Lipids

Protein

Human milk oligosaccharides

Lactose

HMOs Proteins

Lipids

HMOs

Nature 468 S5-S7 (23 December 2010)

Chain

Length

4

5

6

7

8

8

9

10

Other HMOs of

longer lengths

Protein

Garrido et al Microbiology (In Press)

• Human indigestible and

highly variable

• Higher proportion of

fucosylated (40-70%) than

sialyated (4-38%)

• Nearly 200 species in pooled

human milk

Carlito Lebrilla UCD Chemistry

Why Make Glycoconjugates/Free Glycans? (if they are not consumed by the infant)

Immune development

Pathogen deflection

Neural development

Enrich specific microbes

Bode 2009

Breast milk enriches bifidobacterial populations

FISH 531 infants

Fallani et al Microbiology 2011

Grzeskowiak et al JPGN 2012 South Eastern African and Northern European

Malawi N=44

Finland N=31

Bifidobacterium

Penders et al. Pediatrics 2006

Quantitative PCR

FISH

Breast milk enriches

bifidobacterial populations

“Most shotgun and 16S rRNA V4 sequences (75 ± 20%) in all babies mapped to members

of the Bifidobacterium genus.”

N= 531 (16S) N=110 (metagenome)

•n=110 babies •1 month of age •Measured by qPCR

High Bifs

Low Bifs

Mikami et al. Pediatrics Research 2009

Why bifidobacteria?

Mouse survival after O157 infection

BA = B. animales BL = B. longum

Nature 2010

If bifidobacteria can grow well on a targeted sugar in situ, growth and

accompanying production of acetate is protective

Nature 2010

Do different mother’s milk glycan types

influence different microbiota populations?

Secretor vs non-secretor

FUT2 (“Secretor” gene) • Produces the 2’

fucosylated precursor to the A, B, H, and Lewis b antigens in secretions, including breast milk

• 20% of U.S. population are non-secretors

Secretor vs. non-secretor milk protects differently

Incidence of diarrhea per 100 child-months

Morrow et al J. Pediatrics 2004

We need to be cautious on associations

Which bifidobacterial species grow on HMO?

HMOs as “prebiotics” for

Bifidobacteria

Bifidobacteria vs Bacteroides in situ

Marcobal… & Sonnenburg CHM 2011

Lacto-N-neotetrose supplementation of gnotobiotic mice with Bifidobacterium infantis and Bacteroides thetaiotaomicron

Bifidobacterial HMO Glycoprofiling

OD (600 nm)

Several small MW

oligosaccharides

consumed by B. infantis

B. infantis

B. breve

B. longum

Locascio et al JAFC 2007

Time(hours)

0

5

1 0

1 5

2 0

2 5

% H

MO

ab

un

da

nce

in

bre

as

t m

ilk

HMO m/z

HMO abundance in pooled breast milk

100

50

0

50

100

% H

MO

Co

nsu

me

d

HMO m/z

B. infantis

consumption

Single HMO composition

consumed by other

bifidobacteria

.

0.5

1

1.5

2

2.5

0

New 1 43 % ±4

MFLNH III 81 % ±2

MFLNH I 17 % ±4

IFLNH III 31 % ±1

IFLNH I 46 % ±2

m/z 611.2387

NanoLC separation of individual HMO compositions

Carlito Lebrilla UCD Chemistry

Lewis x

Lewis x

Lewis x,a

4120a 43 % ±4

MFLNH III 81 % ±2

MFLNH I 17 % ±4

IFLNH III 31 % ±1

IFLNH I 46 % ±2

DFLNO I 68 % ±1

DFLNnO II 79 % ±1

5230a 10 % ±5

DFLNnO I or DFLNO II 54 % ±2

5230b 79 % ±5

DFpLNH II 42 % ±12

DFLNH b 67 % ±1

DFLNH a 51 % ±3

TBA 62 % ±13

5130a 79 % ±6

5130b 67 % ±1

F-LNO 37 % ±10

5130c 65 % ±2

LNH 56 % ±0.5

LNnH 5 % ±4

p-LNH 44 % ±5

TFiLNO 66 % ±5

5330a 77 % ±3

β1-4

β1-3

β1-4

β1-4 β1-3

Quantification of isomer-specific consumption of human milk

oligosaccharides by B. infantis at the mid-exponential growth phase

β1-3

β1-3

β1-3

Strum et al Anal Biochem 2012

Do bifidobacteria consume

sialyated HMOs?

What genome features are

required to utilize human milk

oligosaccharides?

HMO utilization by Bifidobacteria

B. longum subsp.

infantis

ATCC15697

2,832,748 bp

Comparative Bifidobacterium Genomics

PNAS 2008

B. longum subsp.

longum

DJO10A

2,389,526 bp

BMC Genomics 2008

Adult derived strain Infant derived strain

What’s Needed to Deconstruct HMOs?

Glc

Gal

GlcNAc

Neu5Ac

Fuc Sialidase Galactosidase

Fucosidase Hexosaminidase

• Transport systems for oligo & monosaccharides • Glycosyl hydrolases

B. infantis HMO cluster

0 40 kb 30 kb 20 kb 10 kb

? - ESB ? - galactosidase fucosidase sialidase hexosaminidase

permease permease permease

ATPase

SBP SBP SBP SBP SBP

SBP

- HMOs are bound by SBP lipoproteins proximal to permeases

ATP ADP

cytoplasm

Extracelluar Solute binding protein (SBP)

permease

ATPase

- ATP hydrolysis prompts transport of oligosaccharides across membrane

Glycolytic enzymes - Intracellular glycolytic enzymes

deconstruct oligosaccharide

All 4 glycosyl hydrolases Array of oligosaccharide transporters

Sela PNAS 2008

HMO GOS FOS / Inulin

Blon_2061 Blon_2414 Blon_2347 Blon_2344

Leloir pathway

Bifidus shunt

&

glycolysis HexNAc catabolism

Blon_2348: -sialidase

Blon_2335: -hexosaminidase

Blon_2056: Exo-inulinase

Blon_0787: Exo-inulinase

Blon_0128: Suc phosphorylase

Blon_2336: -fucosidase

Blon_0732: -hexosaminidase

Blon_2016: -galactosidase

Blon_2334: -galactosidase

Blon_2416: -galactosidase

Blon_1740: GH, family 13

Blon_2453: GH, family13

Blon_2416: -galactosidase

Blon_0268: -galactosidase

Blon_2460: -galactosidase

HMO GOS FOS/Inulin

Whole cell proteomics of B. infantis grown on

different prebiotic sugars

Jae Han Kim

Genes unique to milk-associated bifidobacteria are uniquely expressed

during growth on milk sugars

PLoS One 2013

Sialidase Galactosidase

Fucosidase Hexosaminidase

Characterization of the glycosidases from B. infantis?

(5)

(5) (3)

(2)

Sialidase - ------------------ Sela et al JBC 2011 Fucosidase - --------------- Sela et al AEM 2012 Hexosaminidase - -------- Garrido et al Anaerobe 2012 Galactosidase - ----------- Garrido et al Food Micro 2012

Garrido, et al. Advances in Nutrition (2012)

What about bifidobacterial growth on milk glycoproteins?

20 kDa

15 kDa

C SC81 SC95 SC139 SC154

C KA179 15700 15701 15698 JCM7016 JCM7017 JCM7019 JCM7020 S17-c S46

C SC91 SC116 SC156 SC558 DJO10A SC618 SC630 SC706

SC143 C SC142 17930 15702 7007 25692 7009 701 1 11346

20 kDa

15 kDa

20 kDa

15 kDa

20 kDa

15 kDa

20 kDa

B. longum

B. infan s

B. breve

B. breve

B. breve

N-linked exp. Lactoferrin Immunoglobulins

O-linked exp. Caseins (Κ)

What about bifidobacterial growth on milk glycoproteins?

RNaseB as proxy N-linked glycan

Garrido et al Molecular Cellular Proteomics 2012

GH18a (EndoBI-1)

GH18b (EndoBI-2)

GH85 (EndoBB)

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

B. longum SC706

B. longum SC116

B. longum SC630

B. breve SC559

B. infantis SC142

B. infantis SC143

EndoD

B. breve UCC2003

B. breve JCM1273

B. longum DJO10A

B. breve JCM7019

B. breve JCM7020

B. breve KA179

B. breve SC139

B. breve SC506

B. breve SC568

B. breve SC95

0 . 2

Endoglycosidase genes

in bifidobacteria

Endo-beta-N-acetylglucosaminidases

EndoBI-1 and EndoBI-2 active on all N-linked milk glycoproteins

Garrido et al Molecular Cellular Proteomics 2012

Model for bifidobacterial enrichment in the infant GIT

Enteroendocrine cells

soluble factors

soluble factors

Model for bifidobacteria enrichment in the infant GIT

Complex milk glycans enhance efficacy of specific bifidobacteria

Can this knowledge be translated?

Time (hours)

OD600

Will synbiotic feeding HMO+ B. infantis with HMOs help establish bifidobacteria?

OD

Mark Underwood

HMO+ B. infantis

HMO- B. lactis

UCD Med School Neonatology

Milk processing enzymes

Translation

Prebiotic milk oligosaccharides

Milk-enhanced Probiotics

(bifidobacteria)

Translation

Human milk research

Bovine milk research

Milk Bioactives Project activities

Glycoproteins, glycopeptides,

glycolipids

Translation

GOS DP 3

N-acetyl-glucosaminyl-lactose

3-Sialyl-lactose

GOS DP 5

PIs: Carlito Lebrilla, J. Bruce German, Xi Chen, Mark Underwood, Chuck Bevins, Helen Raybould Students/Postdocs: David Sela, Maciej Chichlowski, Karen Kalanetra, Santiago

Ruiz-Moyano, Milady Ninonuevo, Riccardo LoCascio, Yanhong Lin, Larry Lerno, Jae

Han Kim, Mariana Barboza, Scott Kronewitter, Richard Siepert, Aaron Adamson, Daniel Garrido, Angela Marcobal, Robert Ward and Samara Freeman

Acknowledgements