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Linking genes to proLinking genes to pro-- and prebiotics:and prebiotics:
A bifidobacterial perspectiveA bifidobacterial perspective
Douwe van Sinderen,
Alimentary Pharmabiotic Centre,
University College Cork, Ireland
10th Meeting of the International Scientific Association
for Probiotics and Prebiotics
October 1-3, 2012
Cork, Ireland
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BifidobacteriaBifidobacteria
B. longum is dominant, B. bifidum is
second most prevalent
Discovered in 1900 by Henry Tissier as dominant
bacteria in breast-fed infants
Uncultured_Bifidobacterium
Bifidobacterium sp.
Turroni et al., 2012, PLoS One, e36957
Sim et al., 2012, PLoS One, e32543
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Do we benefit from bifidobacteria?
�Prevention of diarrhoea/constipation
�Amelioration of lactose intolerance
�Immunomodulation
�Development of immune functions
�CLA production�Coakley et al., JAM 2003
�Treatment of IBD/IBS� IBS, O’Mahony et al., Gastroenterology, 2005
�Reduction of serum cholesterol
�Reduction of microbial infections�Acetic acid; Fukuda et al., Nature, 2011
�EPS; Fanning et al., PNAS 2012
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Do we benefit from bifidobacteria?
�Prevention of diarrhoea/constipation
�Amelioration of lactose intolerance
�Immunomodulation
�Development of immune functions
�CLA production�Coakley et al., JAM 2003
�Treatment of IBD/IBS� IBS, O’Mahony et al., Gastroenterology, 2005
�Reduction of serum cholesterol
�Reduction of microbial infections�Acetic acid; Fukuda et al., Nature, 2011
�EPS; Fanning et al., PNAS 2012
Which genes support these activities
(or other abilities that allow gut life)???
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Oct 2002 First bifidobacterial genome published, Schell et al, PNAS
Oct 2012 65 bifidobacterial genomes projects on NCBI (11 species)
Bifidobacterial genes and genomes
How many genes would this represent?
Bottacini et al., 2010
Microbiology
14 genomes
2.0 - 2.8 Mbp
Core genome ~950 genes
Pangenome ~5000 genes
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Oct 2002 First bifidobacterial genome published, Schell et al, PNAS
Oct 2012 65 bifidobacterial genomes projects on NCBI (11 species)
Bifidobacterial genes and genomes
How many genes would this represent?
B. asteroides
can respire?!
Bottacini et al., 2012,
PLoS One, e44229
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B. breve UCC2003 as the bifidobacterial prototype
• Human-derived (nursling stool of a breast-fed baby)
• Site-directed mutagenesis
• Genome sequenced & custom-made arrays
• Stably colonizes mice
B. breve UCC2003 persistence
1.00E+001.00E+011.00E+021.00E+031.00E+041.00E+051.00E+061.00E+07
(Fee
d) 2
(Fee
d) 3 5 10 12 15 17 19 22 24 26 29 31 33
Time (days)
Log
cfu/
g
Test feces
Cronin et al., 2008, BMC Microbiol., 8: 161
O’Connell-Motherway et al.,
Microbial Biotech 2009 & PNAS 2011
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BifidobacterialBifidobacterial hosthost--colonization factors?colonization factors?
• Little known about molecular mechanisms required for colonization (B.
bifidum BopA: Guglielmetti et al., AEM 2008)
• In vitro tests reveal variable adhesion properties (e.g. B. breve UCC2003
adheres very poorly to Caco-2 or other cell lines)
• Perhaps genes involved in adhesion are not expressed under in vitro
conditions, but specifically in the gut?
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Genenumber
gene (protein function) Fold upregulated on
invivo array qRTPCR
Bbr_0132 tadZ- MinD) 3.7 25.1
Bbr_0133 tadA-(ATP-ase) 4.5 56.2
Bbr_0134 tadB 6.9 48.6
Bbr_0135 tadC 8.9 52.8
Bbr_0136 Pilin 5.3 nd
Bbr_0137 tadE (pseudopilin) 6.6 44.1
Bbr_0138 tadG (pseudopilin) 4.7 62.1
Analysis of in vivo gene expression data
Tad or type IVb pili
biosynthesis
O’Connell Motherway et al., PNAS, 2011
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Type IVb pili
[Immunogold labelling using antibodies raised against a
20 aa epitope of flp, encoding the pre-pilin precursor]
Univ of
Helsinki
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2
3
4
5
6
7
8
9
10
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
UCC2003-non-germ-free
UCC2003-monoassociated
UCC2003::tadA-non-germ-free
UCC2003::tadA-monoassociated
Colonisation of germ-free mice
Small
intestine
Caecum Large
intestine
* * * * * * * * * * ** *
Non-germ-free
inoculum
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Does EPS play a role in Does EPS play a role in bifidobacterialbifidobacterial
colonization?colonization?
Fanning, Hall et al., PNAS, 2012Fanning, Hall et al., PNAS, 2012
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EPS2AUCC2003
EPS2BUCC2003 Inv
Deletion
- +
The genetic structure of the The genetic structure of the B. B. brevebreve UCC2003 UCC2003 epseps clustercluster
eps2eps1
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EPS provides acid & bile resistance to B. breve UCC2003
and also affects persistence in the gutand also affects persistence in the gut
caecum
1000
10000
100000
1000000
10000000
CFU
/org
an
** *
colon
1000
10000
100000
* *
EPS+ UCC2003EPS- UCC2003-EPSdel
EPS- UCC2003::Bbr_0430
C
0 5 10 15 20 25 30 35104
105
106
107
108
Day
CFU
/g
Bp < 0.05 – 0.001
D
EPS+ UCC2003
EPS- UCC2003-EPSdelEPS- UCC2003::Bbr_0430
acid (pH5)
0 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.8
time (h)
OD
600n
m
0.3% bile
0 2 4 6 8 10 12 14 16 18 20 22 240.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
time (h)
OD
600n
m
p < 0.05 - 0.001p < 0.05 - 0.01
A
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na ïve
UCC2003
UCC2003 - EPSdelMice fed with:
Ab titre
feacal IgA
0
25
50
75
100
end-
poin
t tit
re
† †
feacal IgA
0
25
50
75
100
end-
poin
t tit
re
† †
UCC2003
UCC2003 - EPSdel
total Ig
0
2500
5000
7500
10000
12500
†† ††† †
Mice fed with EPSMice fed with EPS-- B. B. brevebreve have higher levels of B cells have higher levels of B cells
anti-UCC2003 serum
anti-UCC2003
-EPSdelserum
UCC2003
UCC2003-EPS2del
Agglutination test shows antibody specificity
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na ïve
UCC2003
UCC2003 - EPSdelMice fed with:
Ab titre
feacal IgA
0
25
50
75
100
end-
poin
t tit
re
† †
feacal IgA
0
25
50
75
100
end-
poin
t tit
re
† †
UCC2003
UCC2003 - EPSdel
total Ig
0
2500
5000
7500
10000
12500
†† ††† †
Mice fed with EPSMice fed with EPS-- B. B. brevebreve have higher levels of B cells have higher levels of B cells
anti-UCC2003 serum
anti-UCC2003
-EPSdelserum
UCC2003
UCC2003-EPS2del
Agglutination test shows antibody specificity
BifidobacterialBifidobacterial EPS allows EPS allows evasion of B cellevasion of B cell--mediated mediated adaptive immune responseadaptive immune response
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C. rodentium
C. rodentium
B. breve EPS-
C. rodentium
B. breve EPS+
EPSEPS--producing producing B. B. brevebreve also provides also provides protection against a murine pathogenprotection against a murine pathogen
Day 1 Day 6 Day 8 Day 14
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Role of bifidobacterial surface EPS
1)1) Bile and acid resistanceBile and acid resistance
2)2) Immune evasion Immune evasion
3)3) Pathogen protection (competitive exclusion/biofilm?)Pathogen protection (competitive exclusion/biofilm?)
4)4) Abrogation of cytotoxicity (Abrogation of cytotoxicity (RuasRuas--MadiedoMadiedo et al., JAM, 2010) et al., JAM, 2010)
Efficient colonization Efficient colonization
and persistenceand persistence
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Starch/glycogen
Cellodextrin
Palatinose
Palatinose
Ribose
Fructose
Raffinose/melibiose
Galactan/GOS
Sucrose
Carbohydrate/prebiotic metabolism
B. breve UCC2003
UCC2003 is specialized to degrade complex plant-derived oligo/polysaccharides
Reviewed by
Pokusaeva et al.,
Genes Nutr. 2011
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Galacto-oligosaccharide (GOS)
metabolism by B. breve
galAgalRgalGgalEgalDgalC
lacZlacS
galT galK
lacI
gosCgosD gosE gosGgosR
O’Connell Motherway et al., Microb. Biotech, 2011 & 2012, in revision
Ge
ne
s u
pre
gu
late
d w
he
n B
. b
reve
UC
C2
00
3 is
gro
wn
on
pG
OS
Galactan (βGal 1-4 βGal)
Lactose (βGal 1-4 βGlu)
Galactose metabolism
Other βGal links??
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Growth on galactan
WT
galA mut
Compl galA mut
Growth on pGOS
WT
galA mut
Compl galA mut
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0
500
1000
1500
2000
2500
3000
3500
4000
DP1 DP 2 DP 3 DP 4 DP 5 DP 6 DP 7 DP 8 DP 9 DP 10
UCC2003-galA T0
UCC2003-galA T24
GOS contains a lot of different oligosaccharides
of varying chain length
The GalA enzyme is responsible for metabolism of high DP GOS
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Growth of various B. breve strains on pGOS
galA positive strains
galA negative strains
Prebiotics may cause very subtle microbiota changes
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B. breve UCC2003 can utilise pullulan, many other bacteria lack this ability
MalGMal
F
Mal
E
Uptake system
lacI
glucosidases
α-1,4 α−1,6
Pullulan metabolism
Cell wall-associated amylopullulanase
apuB
pullulan
Reducing glucoseglucoseα-1-4 glycosidic bondα-1-6 glycosidic bond
O’Connell Motherway et al., AEM 2008 & unpublished
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0
0.5
1
1.5
2
2.5
3
starch amylopectin glycogen pullulan glucose
B. breve UCC2003
B. breve UCC2003::apuB
Carbohydrate source
Opt
ical
den
sity
OD
600
nm
apuB mutation causes growth defects of B. breveon starch, amylopectin, glycogen and pullulan
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What will the next ten years bring?
�From genes to molecules to mechanisms:
discovery of ‘probiotic’ genes
�Genome exploration and animal models,
coupled to NGS to reveal novel prebiotics
�Prebiotics with high strain/species specificity
(infant vs adult/elderly)
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Acknowledgements
Gerald FitzgeraldPaul O’TooleColin HillFergus Shanahan
University of Parma, Italy
Marco VenturaFrancesca Turroni
Francesca Bottacini
David Goulding & Gordon Dougan
Sanger Institute, Cambridge, UK
• Mary O’Connell-Motherway
• Saranna Fanning
• Lindsay Hall
• Aldert Zomer
• Michelle Cronin
• Sinead Leahy
• Marcus Claesson
• Karina Pokusaeva
• Pat Casey
• Aileen Houston
• Ken Nally
• John MacSharry
• Jose Moreno-Munoz
Willem de Vos, Airi Palva,
Justus Reunanen,
Univ Helsinki, Finland
Abelardo Margolles
IPLA, Oviedo, Spain
Julian Marchesi,
Cardiff Univ, UK