1

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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)???

3

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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

8

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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

http://apc.ucc.ie

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