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Bacterial lectins and infection – from structural
glycobiology to antiadhesive strategies
e-Learning in Glycobiology and Glycochemistry
Lisbonn
8 october 2012 Anne Imberty
CERMAV-CNRS
Grenoble, France
)
Lectins
-Ubiquitous- > 500 crystal structures
(www.cermav.cnrs/lectines)
-Multivalency achieved by -Tandem repeat
-Oligomerisation
-Surface presentation
-Affinity and avidity
-Low affinity for monosaccharides
-Avidity for multivalent ligands
)
)
- Lectins : distribution and role in infection
- Structure, specificity, multivalency of lectins
- Lectins as drug targets against infection
Learning objectives
)
New Lectin-3D database(http://glyco3d.cermav.cnrs.fr/glyco3d/)
> 1000 entries
)
Lectins in mammalsQuality control, Cargo-Traffic, Cell-cell recognition, Pathogen opsonisation
Drickamer &Taylor, Trends Biochem. Sci., 1998
)
Sinularia lochmodes
Lectins(SLL1 to SLL3)
Symbiosis with zooxanthella Symbiodinium
(Koiki et al., Biol. Bull. 2004)
Helix pomatia
Protection of eggsfrom pathogens
AgglutininHPA
.
(Uhlenbruck & Prokop Vox Sang. 1966) (Barondes et al., J. Cell Biol. 1983)
Dictyostelium discoideum
Discoidins(DiscI, DiscII)
Development (Sanchez et al, J. Biol. Chem. 2006)
Lectins in invertebratesSelf / non-self recognition
)
Lectin in plant and fungiDefense - symbiosis
Schubert et al. PLoS Pathog. 2012
CCL2 from Coprinopsis cinereaGlcNAcβ1,4[Fucα1,3]GlcNAc
Liu et al. Science 2012
LysM domain of chitin receptorfrom Arabidopsis /chitotetraose
Fluorescence microscopy of C.
elegans feeding on E. coli expressing
a dTomato-CCL2 fusion protein
)
Dendritic cells Fluids
Macrophages
DC-SIGN
SP-DMBP Mannose-
receptor
Soluble lectins Toxins
Adhesins
Bacterial GBPs
Host GBPs
+
+ -
-
)
Imberty & Varrot, Curr. Opin. Struct. Biol. 2008
Lectin in bacteria and viruses
Common in freshwater:
“Hot Tub” folliculitis
Acute diffuse otitis externa (swimmer’s ear)
Pseudomonas “hot-foot” syndrome
Most common pathogen isolated from patients hospitalized longer than one week
A major hospital pathogen:
10-15% nosocomial infection world while (mortality rate 40 to 60%)
high endemic potential in intensive care units: 18 %
Pseudomonas aeruginosa
The leading killer of cystic fibrosis patients
0
10
20
30
40
50
60
70
80
90
100
0 - 4 5 - 9 10 - 14 15 - 19 20 - 24 25 - 29 29 - 30 35 ou +
P. aeruginosa
S. aureus méti S
S. aureus méti R
H. influenzae
S. maltophilia
Classes d’âge
% Patients
0
10
20
30
40
50
60
70
80
90
100
0 - 4 5 - 9 10 - 14 15 - 19 20 - 24 25 - 29 29 - 30 35 ou +
P. aeruginosa
S. aureus méti S
S. aureus méti R
H. influenzae
S. maltophilia
Classes d’âge
% Patients
)
LecA
LecB
Type IV pili / asialo oligo
Type 1 and type p pili
FliD / Sialyl Lewis X ?
Pseudomonas aeruginosa glyco-strategy
Soluble lectins
)
Bacterial material
PAO1 PAO1 TnA PAO1 TnB
P. aeruginosa
PAO1 TnAR PAO1 TnBR
Wild type Mutant LecA Mutant LecB
Recomplementation
1 In vitro studies
� Lung epithelial cells (A549)
� Cytotoxicity de P. aeruginosa
2. In vivo studies
� Acute pneumonia experimental model
�P. aeruginosa pathogenicity
- Mortality
- Lung lesion (albumin 125I-albumin)
- Bacterial load in lungs
- Translocation from lungs
BalB/c
Endotracheal instillation
Role of LecA and LecB in P. aeruginosa pathogenicity
(coll. B. Guery & S. de Bentzmann)
)
H6 H1610 4
10 5
10 6
10 7
10 8
10 9
1010
PAO1
PAO1 TnA
PAO1 TnB
***
**
Temps après infection
UFC / poumons
Bacterial load in lungs
Inoculum = 5×106 UFC/souris
n = 10/groupe. * p < 0.05, *** p < 0.001 vs PAO1
Chemani et al. (2009) Infect .Immun.
CTR
PAO1
PAO1
TnA
PAO1
TnA R
PAO1
TnB
PAO1
TnB R
0
1
2
3
4
5
125- I-albumine (%)
B
H16
***
*
Pulmonary lesions from P. aeruginosa
Role of LecA and LecB in mice model infection
Strain ∆∆∆∆LecAStrain ∆∆∆∆LecB
)
0 1 2 3 4 5 6 70
20
40
60
80
100
PA
PA + Me-αααα-Fuc
PA + Gal-Nac
PA + Me-αααα-Gal
**
Temps (jours après infection)
Survie (%)
Protective effect of monosaccharides on P. aeruginosa
infection
n = 20/groupe. * p < 0.05 vs PAO1
Inoculum = 5××××107 UFCM/mice
�Experimental model of acute pneumoniae in mice
�
BalB/c
Co-administration endotracheal PA + sugars (15mM)
Survival (days)
Chemani et al. (2009) Infect .Immun.
)
- Lectins : distribution and role in infection
- Structure, specificity, multivalency of lectins
- Lectins as drug targets against infection
Learning objectives
-Lectins are ubiquitous and interprete the glycocode
- Many lectins are involved in host-pathogen interactions
- LecA and LecB from Pseudomonas aeruginosa are virulence factors
Characterisation of lectin/glycan interactions
Glycan arrayCFG
Specificity
Titration calorimetry
(ITC)
Affinity
Thermodynamics
n, Ka, ∆∆∆∆H
Affinity
Kinetics
Kon, Koff
Structure
Surface Plasmon Resonance
(SPR)
CrystallographyModeling
)
LecB from P. aeruginosa
PA-IIL / LecB
�114 amino acids / pI 3.88
�Specific for L-fucose / D-mannose
� Affinity in the micromolar range for fucose
� Tetrameric
� Binding properties dependent on divalent cations
Gilboa-Garber,Meth. Enzymol. 1992
� Recombinant in E. coli
� Purified in 1 step on mannose/agarose column
)
Consortium for Functional Glycomics : http://www.functionalglycomics.org/fg/
“Glycan array”
Covalent link with glass slide functionalized with N-
hydroxylsuccimidine
“Printed array”
� Lectin labelled with fluorophore (or provided with Ab)
�Concentraton from 0,01 to 200 ug/mL
Glycochips : Glycan arrays and printed arrays
Specificity of P. aeruginosa LecB by glycan array
0
2000
4000
6000
8000
10000
1 20 39 58 77 96 115 134 153 172 191 210 229 248 267 286 305 324 343 362 381 400 419 438 457
RF
U
Chart No
PA-IIL (1ug/mL) Slide#12863 CFG#2058 ALexa488 5/26/10 Hong
ααααFuc1-2Gal
βGal1-3[ααααFuc1-4]GlcNAc
βGal1-4α[α[α[α[Fuc1-3]GlcNAc
Ϛغ
•Lavoisier and Laplace (1784)
• Measure heat associated with any process
Titration microcalorimetry
ɐϛ
22
Titration microcalorimetry
aKlnRTG −=°∆
°∆−°∆=°∆ STHG
Free Energy Enthalpy Entropy
ϛ
n = 0.77
Ka=2.3 106 M-1
Kd=4.1 10-7 M
∆∆∆∆G= -36.3 kJ.mol-1
∆∆∆∆H= -41.1 kJ.mol-1
-T∆∆∆∆S= 4.1 kJ.mol-1
∆∆∆∆G ∆∆∆∆H -T∆∆∆∆S
MeFuc: 0.44 mM
PA-IIL: 55.8 µM
Thermodynamics of LecB/ααααMeFuc interaction
ϛ
Kd= 0.21 10-6 M
∆∆∆∆G ∆∆∆∆H -T∆∆∆∆S
Lewis a
Kd= 1.02 10-6 M
∆∆∆∆G ∆∆∆∆H -T∆∆∆∆S
Lewis a Lewis x
LecB/Lewis epitope interactions
ITC
OCH2OH
HO
OHOH
O
O
CH2OH
AcHNOH
O
OHHO
CH3
O
OH
Gal
GlcNAc
Fuc
βGal1-3[ααααFuc1-4]GlcNAc
Lewis x
OCH2OH
HO
OHOH
O O
NHAcOH
CH2OH
O
OHHO
CH3
O
OH
Gal
GlcNAc
Fuc
βGal1-4α[α[α[α[Fuc1-3]GlcNAc
ϛ
Surface Plasmon Resonance
ϛ
Sugar on the surface
Lectin on the surface
Chip CM5 + Streptavidin + Biot-lectin
Chip NTA + HisTag-lectin
Chip CM5 + Lectin (amine coupling)
CM5 Fc
1Fc2
Fc1Fc1
CM5
NTA
CM5
+
Ni2+
NT
A
CM5
Chip CM5 + Streptavidin + Biot-PAA-Fuc
DetectionRegeneration
Glycan
Glycan
Glycan
???
???
???
EDTA
)
Lewis a Lewis x
LecB/Lewis epitope interactions
-50
0
50
100
150
-1000 0 1000 2000 3000 4000 5000
Lewis a (RU)
Lewis x (RU)
Kon (Lex): 486 Ms-1
Kon (Lea): 1446 Ms-1
SPR
OCH2OH
HO
OHOH
O
O
CH2OH
AcHNOH
O
OHHO
CH3
O
OH
Gal
GlcNAc
Fuc
OCH2OH
HO
OHOH
O O
NHAcOH
CH2OH
O
OHHO
CH3
O
OH
Gal
GlcNAc
Fuc
βGal1-3[ααααFuc1-4]GlcNAcβGal1-4α[α[α[α[Fuc1-3]GlcNAc
ϛ
X-ray crystallography
㦀Ϝ
Mitchell et al (2002)
Nature Struct. Biol.
@ϛ
O2
O3
O4
C6
Specificity of LecB for fucose
Mitchell et al. & Imberty (2003) Nature Struct. Biol.
㦀Ϝ
Crystal structure of the LecB/Lewis a complex
KD Lewis a
0.21 µµµµM
.Perret et al. (2005) Biochem. J
KD ααααMeFuc
0.5 µµµµM
ITC
OCH2OH
HO
OHOH
O
O
CH2OH
AcHNOH
O
OHHO
CH3
O
OH
Gal
GlcNAc
Fuc
묰Ϝ
Lewis X
Crystal structure
NHAc
OH
O
OH
OH
O
OHCH
3
O
O
CH2OH
OH
CH2OHO
OH
OH
3
4
Fuc
Gal
GlcNAc
Lewis a
CH2OH
OH
OH
CH2OHOO
OH
OH
O
OH
OH
O
OHCH
3
O
NHAc3
4
Fuc
Gal
GlcNAc
Model
KD Lewis a
0.21 µµµµM
KD Lewis x
1.02 µµµµM
쁠Ϝ
LecB / P. aeruginosaLecA / P. aeruginosa
Bc2L-A / B. cenocepacia BambL / B. ambifaria
See Poster P171 / Aymeric Audfray
Relation between topology and function ?
뮰ϝ
P. aeruginosa
B . cenocepacia
B . ambifaria
Non Sec Le- Non Sec Le+ ALe- A Le+ B Le+ O Le- O Le+
Collab Jacques Le Pendu
Specificity of bacterial lectins towards histo-blood group epitopes
no selectivityspecificity
for Le+
specificity for secretor
뮰ϝ
- Lectins : distribution and role in infection
- Structure, specificity, multivalency of lectins
- Lectins as drug targets against infection
Learning objectives
-A wide range of tools is available for studying protein/carbohydrate
- Some microbial lectins have high affinity to glycans
- Multivalency and topology play a role in lectin function
-Lectins are ubiquitous and interprete the glycocode
- Many lectins are involved in host-pathogen interactions
- LecA and LecB from Pseudomonas aeruginosa are virulence factors
뮰ϝ
Antibacterial compounds - strategies
Monovalent MultivalentOligovalent
Imberty et al, Chemistry 2009
Glycomimetics
Aglycone
“polar gate”
Cluster effect
Potent
aggregation
Cluster effect
Chelate effect
Specificity
솠Ϝ
0
5
10
15
20
25
23
20
22
19
18
21
13
16
14
15
12
Lewis a
fucose
Met-a-L-Fuc
48
49
50
51
52 5 4 1 9
10
55
57
56
59
43
41
30
27
28
26
24
inhibiteurs
potentiel d'inhibition
Série 1 (l-Fuc(α1-4)GlcNac)
Série 2 (Dendrimers of Fuc14GN))
Natural ligands
Série 3 (Isoxazole-(R))
Série 4 (Triazole-(R))
Série 5 (addition at C5)
Série 6 (other additions at C1)
ELLA tests on series of glycomimetics
(coll. René Roy, UQAM)
뮰ϝ
9.8 8.28.3
KD
3.2 10-7 M
Marotte et al. Imberty & Roy (2007) ChemMedChem
뮰ϝ
0
5
10
15
20
25
23
20
22
19
18
21
13
16
14
15
12
Lewis a
fucose
Met-a-L-Fuc
48
49
50
51
52 5 4 1 9
10
55
57
56
59
43
41
30
27
28
26
24
inhibiteurs
potentiel d'inhibition
Série 1 (l-Fuc(α1-4)GlcNac)
Série 2 (Dendrimers of Fuc14GN))
Natural ligands
Série 3 (Isoxazole-(R))
Série 4 (Triazole-(R))
Série 5 (addition at C5)
Série 6 (other additions at C1)
슐Ϝ
OO
N
HO
OO
N
O
OO
NO
ON
O
OHHO
OHO
OHHO
OHO
OHHO
OHO
OHHO
OH
9.8 7.1 7.1 6.8
OO
O
OH
HO
OH
O
OOH
OHHO
N
N
N
O
N
N
N
NO2
N
N
N
O
OH
HO
N
AcHN
OH
N
N
OOH
OHHO
O
OH
HO
OH
9.89.69.8 4.2
KD
7.1 10-7 M
뮰ϝ
0
5
10
15
20
25
23
20
22
19
18
21
13
16
14
15
12
Lewis a
fucose
Met-a-L-Fuc
48
49
50
51
52 5 4 1 9
10
55
57
56
59
43
41
30
27
28
26
24
inhibiteurs
potentiel d'inhibition
Série 1 (l-Fuc(α1-4)GlcNac)
Série 2 (Dendrimers of Fuc14GN))
Natural ligands
Série 3 (Isoxazole-(R))
Série 4 (Triazole-(R))
Série 5 (addition at C5)
Série 6 (other additions at C1)
뮰ϝ
O O OO
HOAcNH
OH
O
O
OH
OHOH
NNN
O
OHAcNH
OH
O
O
HO
OHOH
NN N
O O
C H N O
CP43B
Best multivalent inhibhitors
Kd = 90 10-9 M
∆∆∆∆G = -40.2 kJ.mol-1
∆∆∆∆H = -69.5 kJ.mol-1
-T∆∆∆∆S = 29.3 kJ.mol-1
Kd = 100 10-9 M
∆∆∆∆G = -39.9 kJ.mol-1
∆∆∆∆H = -37.2 kJ.mol-1
-T∆∆∆∆S = -2.7 kJ.mol-1
∆∆∆∆G ∆∆∆∆H -T∆∆∆∆S
∆∆∆∆G ∆∆∆∆H -T∆∆∆∆S
Marotte et al. Imberty & Roy (2007) Org. Biol. Chem.
쎀ϜLecA LecB
P. aeruginosa lectins inhibitors
Natural 100 µµµµM 210 nM
Monosacch
derivatives 6 µµµµM 290 nM
Oligomers 176 nM 100 nM
뮰ϝ
- Lectins : distribution and role in infection
- Structure, specificity, multivalency of lectins
- Lectins as drug targets against infection
Learning objectives
-A wide range of tools is available for studying protein/carbohydrate
- Some microbial lectins have high affinity towards glycans
- Multivalency and topology play a role in lectin function
-Lectins are ubiquitous and interprete the glycocode
- Many lectins are involved in host-pathogen interactions
- LecA and LecB from Pseudomonas aeruginosa are virulence factors
- Glycomimetics are efficient inhibitors of lectin and bactiera adhesion
- Thermodynamic should be considered for designing ligands
뮰ϝ
Conclusions and perspectives
- Microbes use our sugars during infection process
- The redundancy of glycan-binding proteins make them difficult to target
- High affinity glycocompounds can be used as anti-infectious agents
- Engineering of bacterial lectins to “neolectins” provide tools with controlled valency and specificity
- Bacterial lectins are excellent tools for research