Production of Bioactives by Intestinal bacteria:

Production of
Bioactives by
Intestinal bacteria:
As a basis for explaining
probiotic mechanisms
FOOD MICRO, August 31 st , 2010

Gut bacteria produce an almost limitless set of metabolites
Understanding these may provide the key to unlocking many
heath-promoting functions of gut bacteria/probiotics
Fergus Shanahan

1.
Bacteriocin production
Production of Salivaricin by
Lactobacillus salivarius
Discovery of Thuricin: a
bacteriocin which specifically
kills Clostridium dificile
Microbial Production of Bioactives
Probiotics versus our resident flora
HOW PROBIOTICS WORK?
Bifidobacterium breve
2.
Fatty acid production
Production of Conjugated
linoleic acid (CLA),
Production of
DHA and EPA

Probiotics
• Probiotics have mainly
transient effects –
“tourists”
• Anti-infectiveImmunomodulatory
effects
• Out-compete pathogens
Role of Resident flora
• Anti-infective/Immunomodulatory
effects
• Key long term role in digestion
• Production of essential
nutrients
• Production of bioactive
substances e.g. antimicrobials,
fatty acids, neurotransmitors,
ACE inhibitors etc.

1.
Bacteriocin production
Production of Salivaricin by
Lactobacillus salivarius
Discovery of Thuricin: a
bacteriocin which specifically
kills Clostridium dificile
Microbial Production of Bioactives
Probiotics versus our resident flora
HOW PROBIOTICS WORK?
Bifidobacterium breve
2.
Fatty acid production
Production of Conjugated
linoleic acid (CLA),
Production of
DHA and EPA

Colin Hill
Many gut bacteria
produce Bacteriocins
Antimicrobial peptides produced by
one bacterium which can kill other
bacteria
Bacteriocins are heat stable, active at
nanomolar range, producers are
immune to their own bacteriocin
Bacteriocin production is widespread
among bacteria, including gut
bacteria

Class 2
Mode of Action
Lipid II
Class 1
Class 3
Bacteriocins: developing innate immunity for food
Cotter el al. 2005

Bacteriocin Production
as a Probiotic Trait
1. Inhibition of pathogens
2. Contribute to dominance
Lactobacillus salivarius strains from different sources commonly produce salivaricins
(O’Shea, FEMS Microbial Lett, 2009)
Salivaricins are two-component bacteriocins which kill Listeria and lactobacilli

Corr S. C. et.al. PNAS 2007;104:7617-7621
Bacteriocin production mediates Lb. salivarius UCC118 protection against L. monocytogenes
infection of A/J mice
Placebo Bac +
Lux tagged
Listeria
Corr S. C. et.al. PNAS 2007;104:7617-7621
Bac -

Probiotic for Salmonella Reduction
Lactobacillus pentosus
DPC6004
Lactobacillus salivarius
DPC6005
LIVE5
Lactobacillus murinus
DPC6003
Pediococcus pentosaceus
DPC6006
Lactobacillus murinus
DPC6002

11.6kg
Control
11.7kg
Probiotic 1
(Live 5 suspension)
11.7kg
Probiotic 2
(Live 5 fermentate)
Salmonella challenge
Day post-infection
1 2 3 4 5 6 7
28.6kg
34.1kg
33.2kg

Reduce Salmonella shedding in pigs deliberately infected with S. Typhimurium
Salmonella shedding
(cfu/g faeces)
10 6
10 5
10 4
10 3
10 2
10 1
Salmonella Typhimurium infection
1000 fold
reduction
0 5 10 15 20 25
Control
Live 5 suspension
Live 5 fermentate
Time (days)
Gardiner et al. Appl. Environ. Microbiol 2004
Casey et al. Appl. Environm. Microbiol 2007
Walsh et al, FEMS Microbiol Ecol, 2008

Mean Live5 count in ileum
= 1.3 x 10 5 CFU/g
Live5 detection in small intestine of pigs

Intraspecies Diversity: Lactobacillus salivarius
6027
6502
6189
6005
118
6196
7.3
6488

Salivaricin P, 13 kb
Natural Variants from Gut Strains
AP118
ORF 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Sln1
Sln2
SlnIM
SlnIP
SlnK SlnR SlnT SlnD
Production/
immunity
Regulation Transport
UCC118 KNGYGGSGNRWVHCGAGIVGGALIGAIGGPWSAVAGGISGGFTSCR
DPC6196 KNGYGGSGNRWVHCGAGIVGGALIGTIGGPWSAVAGGISGGFISCR
DPC6005 KNGYGGSGNRWVHCGAGIVGGALIGAIGGPWSAVAGGISGGFASCH

Trypsin Resistant Variants of Salivaricin
Pepti
de Amino acid sequence
Lengt
h (aa) MIC50 *
(nM)
Sln1 K R GPNCVGNFLGGLFAGAAAGVPLGPAGIVGGANLGMVGGALTCL 45 50
Sln1-1 R GPNCVGNFLGGLFAGAAAGVPLGPAGIVGGANLGMVGGALTCL 44 80
Sln1-2 H GPNCVGNFLGGLFAGAAAGVPLGPAGIVGGANLGMVGGALTCL 44 200
Sln1-3 KPH GPNCVGNFLGGLFAGAAAGVPLGPAGIVGGANLGMVGGALTCL 46 100
Sln1-4 HRPGPNCVGNFLGGLFAGAAAGVPLGPAGIVGGANLGMVGGALTCL 46 130
Sln1-5 KPR PNCVGNFLGGLFAGAAAGVPLGPAGIVGGANLGMVGGALTCL 45 80
Sln1-6 K R GPNCVGNFLGGLFAGAAAGVPLGPAGIVGGANLGLVGGALTCL 45 120
Sln2 K NGYGGSGNR WVHCGAGIVGGALIGAIGGPWSAVAGGISGGFASCH 46 50
Sln2-1 K NGYGGSGNH WVHCGAGIVGGALIGAIGGPWSAVAGGISGGFASCH 46 200
Sln2-2 KPNGYGGSGNH WVHCGAGIVGGALIGAIGGPWSAVAGGISGGFASCH 47 200
Sln2-3 H NGYGGSGNH WVHCGAGIVGGALIGAIGGPWSAVAGGISGGFASCH 46 120
Sln2-4 K NGYGGSGNR 10 -
Sln2-5 RPWVHCGAGIVGGALIGAIGGPWSAVAGGISGGFASCH 38 250
* MIC 50 of bacterioicn variant combined with wild type complementary
peptide
The carboxyl side of lysine (K) and arginine (R) represent trypsin specific cleavage sites.
MIC = 80nM
MIC = 300nM
O’Shea et al. AEM in press

Abp118 is encoded on megaplasmid pMP118 (242kb)
Lb. salivarius UCC118 genome, 2.3 Mb
Claesson et al, PNAS (2006)

242 kb megaplasmid
1.83 Mb circular chromosome
Microarray based identification of novel Lb. salivarius bacteriocins
pSF118_20
Bacteriocin operon
SalivaricinT Paul O’Toole
6488

Salivaricin T genes
4433.90 Da 5655.57 Da 5269.0 Da
MALDI-TOF MS analysis of cell free supernatant of Lb. salivarius
DPC6488 revealed all 3 potential bacteriocin genes are expressed

Antimicrobial activity of salivaricin 6488 component peptides, Sln3 and Sln4
ORF1 2 3 4 5 6 7 8 9 10
sal B
sln3
sal IM
sln4 sal IP
sal IM
sal K
Sln3
Sln3
Sln4
Sln3 + Sln4
Sln3 M----MKEFTILTECELAKVDGGYTPKNCAMAVGGGMLSGAIRGGMSGTVFGVGTGNLAGAFAGAHIGLVAGGLACIGGYLGS--H !
ThmA MNTITICKFDVLDAELLSTVEGGYSGKDCLKDMGGYALAGAGSGALWGAPAG-GVGALPGAFVGAHVGAIAGGFACMGGMIGNKFN !
* : :* :* *:.*:***: *:* :** *:** *.: *: * *.* *.***.***:* :***:**:** :*. : !
Leader sequence Mature peptide (61aa; 5655 Da)
Sln4 M----SYEKLNNEELSKILGGNGINWGAVAGSCASGAVIGAAFGNPL---TGCVANSAFSFSWQAFKNRPRPKKIA!
ThmB MKQYNGFEVLHELDLANVTGGQ-INWGSVVGHCIGGAIIGGAFSGGAAAGVGCLVGSGKAII-----------NGL !
* .:* *:: :*::: **: ****:*.* * .**:**.**.. .**:..*. :: : !
Leader sequence Mature peptide (52aa; 5269 Da)
Sln4

Microbial Production of Bioactives
1.
Bacteriocin production as
A probiotic trait
Production of Salivaricin by
Lactobacillus salivarius
Discovery of Thuricin: a
bacteriocin which specifically
kills C. dificile
HOW PROBIOTICS WORK?
Bifidobacterium breve
2.
Fatty acid production
as a probiotic trait?
Production of Conjugated
linoleic acid (CLA),
Production of
DHA and EPA

Why C. difficile as target?
• Major GI infectious agent
• Sensitive to metronidazole and vancomycin.
• Increasingly associated with GI disorders
• Causes 15-25% of all antibiotic associated diarrhoea
• Toxin producer which can be fatal in the elderly
• Incidence is on the increase

Mary Rea
Bacillus thuringiensis
Rea et al., unpublished
Thuricin CD
Thuricin CD; a two
component
bacteriocin
Overlaid with Clostridium difficile
30,000 sporeformers
Rea et al. PNAS 2010 (1)

Thuricin Spectrum
bacilli
clostridia
listeria
Rea et al. PNAS 2010 (1)

Thuricin contains unusual post-translational modifications
8#+2*44#.?##
@>.A+.B:./,/-#
*/CB0+1.D0*2#
:,:C6,4E#
!&#"%*(#+%
!"#)%*(#+%
!"#$"%$#&'($)*+),&-./"0$

Trn!#
Trn"#
Vederas Group
Rea et al. PNAS 2010 (1)

! # s s s
"# s s s
Rea et al. PNAS 2010 (2)

Distal Colon Model
control
Thuricin CD
(90uM)
20% human faecal slurry
10 6 Clostridium
difficile
control
Vancomycin
(90uM)
Metranidazole
(90uM)
24h 24h 24h 24h 24h
Total DNA purified, amplified V4 region of 16S rRNA, pyrosequencing, MEGAN
Rea et al. PNAS 2010 (2)

C. diff
log
8
7
6
5
4
3
Faecal fermentations
Thuricin CD (90 uM) Vancomycin (90 uM) Metranidazole (90 uM)
0 4 8 12 16 20 24 h
C. diff
log
8
7
6
5
4
3
0 4 8 12 16 20 24 h
0 4 8 12 16 20 24 h 0 4 8 12 16 20 24 h 0 4 8 12 16 20 24 h
C. diff
log
8
7
6
5
4
3
0 4 8 12 16 20 24 h
Rea et al. PNAS 2010 (2)

Collateral
damage
16S profiling
Phylum
Rea et al. PNAS 2010 (2)

Microbial Production of Bioactives
1.
Bacteriocin production as
A probiotic trait
Production of Salivaricin by
Lactobacillus salivarius
Discovery of Thuricin: a
bacteriocin which specifically
kills C. dificile
HOW PROBIOTICS WORK?
Bifidobacterium breve
2.
Fatty acid production
as a probiotic trait?
Production of Conjugated
linoleic acid (CLA),
Effect on fatty acid
composition

The Importance of the Microbiota
Obesity
“Our results indicate that the obese microbiome has an increased
capacity to harvest energy from the diet. Furthermore, this trait is
transmissible”:
“Our findings indicate that obesity has a microbial component,
which might have potential therapeutic implications.”
IBD
Obesity
NAFLD

• Gut microbiota proposed to exert a quantitative influence
on host fat
• Qualitative changes in host fat have not been extensively
studied
• Since some microbial-derived fatty acids are bioactive,
eg conjugated linoleic acid (CLA), we used this as a
marker of fat composition
Does the microbiota affect the composition
of fat as well as its quantity?
Catherine Stanton

Recombinant Lactobacillus paracasei producing
t10,c12 CLA in vivo -
Lactoacillus paracasei
Expressing CLA isomerase (P. acnes)
Intensity (mV)
100
90
80
70
60
50
40
30
20
10
LA
GLC
t 10, c 12 CLA
0
30 32 34 36 38 40
Retention Time (min)
Increased t10, c12 CLA content of adipose tissue following dietary intervention
g/100 g FAME
0.03
0.02
0.01
0.00
Vector
Control
***
CLA
Producing
n = 8
8 weeks

%
Conversion
to CLA
90
80
70
60
50
49
30
20
10
Production of Bioactive Lipids - CLA
Bifidobacterium breve
LA
GLC
CLA
Bifidobacterium longum
Barret E Applied Environ. Microbiol 2007
Hennessy at al. J. Appl. Micro 2009

Linoleic acid is metabolised by B. breve to c9, t11 CLA
in the murine and porcine GIT
Linoleic acid (g/100g FAME)
Linoleic acid (LA) and c9t11 CLA in faeces
r= -0.863
c9, t11 CLA (g/100g FAME)
Linoleic acid + B. breve
Linoleic acid
8 weeks

***
LA + B. breve
LA
BALB/c mice SCID mice
*
*
Pigs
LA + B. breve
Control

LPLs
Anti-inflammatory effect mediated by B. breve
NCIMB 702258 in pigs
LA + B. breve
Control
Wall et al. Am. J. Clin. Nutr. 2009

Compositional change is not limited to CLA
EPA (Eicosapentaenoic Acid)
Liver Adipose tissue
n = 8 per group
* = p

Increased EPA content of liver following supplementation with !linolenic
acid and B. breve compared to !-linolenic acid alone
n = 8 per group
* = p

Increased DHA content of brain following supplementation with !linolenic
acid and B. breve compared to !-linolenic acid alone
Wall et al. Lipids 2010

Liver
Intestine
Luminal production by
gut microbiota
Bacteria changing food into
Healthy bioactive substances
Adipose
Brain
Wall et al. Am. J. Clin. Nutr. 2009

Conclusions
• Gut flora/probiotics produce a wide
range of bioactive substances which may
positively affect human health
• Production of bacteriocins may facilitate
probiotic dominance and pathogen
inhibition
• Certain bifidobacteria can influence fatty
acid composition at different sites in the
body including the liver and the brain
Bifidobacterium breve

FATTY ACIDS
Catherine Stanton
Ger Fitzgerald
Fergus Shanahan
Barry Kiely
Rebecca Wall
Mairead Coakley
Eoin Barrett
Eva Rosberg
Dr. Liam O’Mahony
Seamus Aherne
Katie O’Mahony
Alan Hennessey
Thanks
ANTIMICROBIALS
Colin Hill
Paul Cotter
John Vederas
Mary Rea
Eileen O’Shea
Paula O’Connor
Orla O’Sullivan
Gillian Gardiner
Sinead Corr
Evelyn Clayton
Alleson Dobson
Fiona Crispie
Sheila Morgan

Trn-!
Trn-!
T 0h
T 5h
Trn-! and ! treated with Pepsin
G N A A C V I G C i G S C V I S E G I G S L V G T A F T L G
G W V A C V G A C G T V C L A S G G V G T E F A A A S Y F L
! !
! !
0h 1h
!+!
!+!
T/0h
T/1h

Trn-!
Trn-!
0h
Trn-! +! treated with !-Chymotrypsin
G N A A C V I G C i G S C V I S E G I G S L V G T A F T L G
G W V A C V G A C G T V C L A S G G V G T E F A A A S Y F L
! !
!+!
0h 0h
5h
5h
! !
!+!
1h