• Probiotic derived antibacterial substances (microcins)
• Competitive inhibition of pathogens
• Modulation of inflammatory responses:
• Increasing antiinflammatory cytokine production
• Suppressing proinflammatory cytokine production
• Modulation of TLR´s
• Induction of barrier effector molecules
Probiotic bacteria: mechanisms of action?
The mucus barrier: defensins + mucins
Intestinal Lumen1014 (an)aerobic bacteria
Outer mucus layer (700 um)mucins (-) and
antibacterial peptides (+)
(defensins, cathelicidines)
Inner mucus layer (100 um)
adherent, rich in
antibacterial peptides
Mucosa (200 um)
epithelial cell barrier
secretion of mucus
and defensins etc.
Normal
A. Swidsinski
Crohn´s diseaseFewmicrobes
Sterile
Constitutive
HBD-1
Permanent war
at host interface
alpha and beta-Defensins
Cathelicidins
Elafin / Antiproteases
BPI
Lysozyme
A Plethora of Antibacterial Peptides
Binding of antimicrobial peptides to mucins in mucus
while retaining functional activity
Mucus (mucins) binding HBD-2
HBD-2
E. coli ATCC 29522
Untreated control
E. coli ATCC 29522
Incubated with
colonic mucus
Staining with SYTO 9®
(living bacteria green) and
Propidiumiodide (dead
bacteria red)
Dr. Sabine Nuding
First Case Report on the Treatment of Colitis with E. coli
Nissle 1917 (MUTAFLOR®)A. Nissle (1918). Die antagonistische Behandlung chronischer Darmstörungen mit Colibakterien. Med. Klinik No 2:29-33.
Rembacken et al., Lancet 1999
Kruis et al., Alimentary Pharmacol Ther 1997
Kruis et al., Gut 2005
Treatment with the non pathogenic E. coli Nissle is
equivalent to mesalazine in maintaining remission in
ulcerative colitis
Probiotic E. coli in IBD: Background
Current consensus:
Probiotic E. coli Nissle (and other Probiotic strains) seem to be
effective in maintaining remission of ulcerative colitis,
rather no effect in Crohn´s disease
Alfred Nissle
0
20
40
60
80
Kruis I Rembacken Kruis II
Mesalazin
E. coli
3 Mon
12 Mon12 Mon
Defensin expression in IBD
Hypothesis:
The benefit from E. coli Nissle in maintenance of remission may be due to induction of defensins
Lack of induction of the
human beta defensin-2 (hBD-2)
in inflamed Crohn´s disease
and in noninflamed UC
Defensins = antimicrobial peptides
Spectrum: gram-positive and gram-negative bacteria, viruses
Wehkamp et al, IBD 2003:
unst
imul
ated
E. c
oli N
issle
1917
EPEC
K12
DSM
498
UPE
C
PZ 8
65
PZ 8
60
PZ 8
62
PZ 8
66
PZ 9
15
PZ 8
61
PZ 8
64
PZ 8
68
PZ 8
70
PZ 8
63
PZ 8
67
PZ 8
35
PZ 8
300
5
10
15
20
25 ***
E. coli strains
rela
tive t
ran
scri
pt
level
HB
D-2
Induction of the human β-defensin-2
in intestinal epithelial cells by E. coli Nissle
* p < 0,001
** p < 0,0001
EcN K12 Unst.
* p < 0,001
** p < 0,0001
E. coli Nissle is quite unique among E. coli´sWehkamp et al. Infection and Immunity 2004
30 more E. coli isolates
tested: all negative.
Mechanism?
+1AP-1
-121/-127-186/-205-572/-596
NF-BNF-B
hBD-2-2338-luc Exon 1
+1AP-1
-121/-127-186/-205-572/-596
NF-BNF-B
NF-B-mut1+2-luc Exon 1
+1AP-1
-121/-127-186/-205-572/-596
NF-BNF-B
AP-1-mut-luc Exon 1
+1AP-1
-121/-127-186/-205-572/-596
NF-BNF-B
AP-1+NF-B-mut-luc Exon 1
+1AP-1
-121/-127-186/-205-572/-596
NF-BNF-B
NF-B-mut1-luc Exon 1
+1AP-1
-121/-127-186/-205-572/-596
NF-BNF-B
NF-B-mut2-luc Exon 1
hBD-2- 2338-luc
NF-B-mut1-luc
AP-1-mut-luc
Relative hBD-2 promoter activity
Fold increase
3.6
-
9.4
-
0 100 200 300 400 500 600 700 800
NF-B-mut2-luc
NF-B-mut1+2-luc
NF-B-mut1+2+AP-1-luc
1.7
8.9
control
Nissle 1917
The relative importance of the NF-κB and AP-1
binding sites in the hBD-2 promoter
Wehkamp et al. Infection and Immunity 2004
Probiotic induction of defensins (HBD2) is
NF-κB and AP-1 dependent
Flagellae of H1 type
chromosome
rfb
STOP
K5 capsule
kpsfla
csg
foc
fimmcmmch
Fe3+
Fe3+Fe3+
Fe3+
ent
ybt
aer
chu
cit
5 kb
3 kb
Fimbriae
MicrocinsIron
acquisition
systems
Characteristics of E. coli Nissle 1917
Special LPS of O6 typeSpecial LPS of O6 type
O6 : K5 : H1 serotypeO6 : K5 : H1 serotype
Small cryptic plasmids
cbtFe3+
Fe3+
p
Induction of defensins: what about other
probiotics?
E. c
oli N
issle
191
7K1
2
L. g
asse
ri PZ
116
0
L. a
c. P
Z 10
30
L. a
c. P
Z 11
29
L. a
c. P
Z 11
30
L. fe
rm. P
Z 11
62
Leuc
onos
toc
L. p
arac
asei
L. p
lant
arum
Pedi
ococ
cus
Cock
tail
0
50
100
150
hB
D-2
mR
NA
in
du
ctio
n
in %
* * * *Wehkamp et al. Infection and Immunity 2004
Schlee et al. Clin and Exp. Immun 2008
The effect is not specifc to E. coli Nissle but can be found in
other probiotic bacteria
2 in vitro
E. Coli probiotic (Symbioflor) treatment induces intestinal
secretion of HBD-2 peptide in man
a
b
a
b
c
1 in vivo
Fecal HBD-2 peptide
concentration in volunteers
HBD-2 expression in
cell culture
Antimicrobial Activity against Probiotics
• Suicidal effect of probiotic stimulation?
Explanation for need of daily delivery?
…is associated with thinner mucus layerPullan et. al. Gut 1994
…mucus depletion is associated with intestinal inflammationJohannson et al PNAS 2008 ahead of print
Ulcerative colitis?
Some probiotics affect
the barrier
• Inducible defensin formation
• Mucin (Muc 1) synthesis
• The combination of both
