Nutrition and the infant gut microbiota: Health and...
Transcript of Nutrition and the infant gut microbiota: Health and...
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
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
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
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
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?
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
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
Enteroendocrine cells
soluble factors
soluble factors
Model for bifidobacteria enrichment in the infant GIT
Complex milk glycans enhance efficacy of specific bifidobacteria
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
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