Klebsiella pneumoniaeEscherichia coli
Defence Molecules in Microbial Ecosystems: Microcins, a Model of Parasitism
of Iron-Import Pathways
Sylvie REBUFFAT
UMR 5154 CNRS-MNHN
National Museum of Natural HistoryParis, France
Department " Regulations, Developmentand Molecular Diversity "
Thai-French Seminar 2005 « Natural substances and drug delivery », Bangkok, Thailand
Muséum National d’Histoire Naturelle Jardin des Plantes, Paris, France
1635Jardin Royal
des Plantes Médicinales
1793Muséum National
d’Histoire Naturelle
2005
The National Museum of Natural History
Seine
Rue
Buf
fon
Rue
Cuvie
r
Quai St Bernard
Rue Linné
7 Scientific Departments of MNHN
Systematics and EvolutionEcology and Gestion of BiodiversityAquatic Populations and EnvironmentsRegulations, Development and MolecularDiversityHistory of the EarthPrehistoryMan, Nature and Societies
Galeries; Botanical Gardens
UMR 5154
Defence and communication molecules in microbialecosystems (Sylvie Rebuffat)
UMR 5154Chemistry/Biochemistry of Natural Substances
Theme 1: Marine sponges/bacteria associations
- Role of the associated bacteria in defence mechanisms: pathogenicity, opportunism, symbiosis;
* Antimicrobial peptides (Jean Peduzzi)
* Secondary metabolites (Marie-Lise Bourguet-Kondracki)
Lamellodysidea herbacea
Theme 2: Bacterial competitions in the intestinal ecosystem(microcins)
- Structures (primary, 3D)- Mechanisms involved in recognition (receptors) and
antibacterial activity- Biosynthetic pathways (posttranslational modification
enzymes, in vitro reconstitution)
"Molecular Adaptation of Microorganisms to the Environment "
Natural substances as mediation agents : action on protozoan parasites (Bernard Bodo)
Thai-French Seminar, Bangkok 2005 UMR 5154 CNRS-MNHN
Sylvie REBUFFATTheme 2: MicrocinsJean PEDUZZI Delphine DESTOUMIEUX-GARZÓNChristophe GOULARDGérard GASTINETheme 1: Symbiosis in spongesMarie-Lise BOURGUET-KONDRACKIArlette LONGEON
NMR/molecular modellingAlain BLOND Michel CHEMINANT
Doctorants,Post-Doctorants
UMR 5154 CNRS-MNHN"Defence and Communication Molecules in Microbial Ecosystems"
Defence and communication molecules in microbialecosystems (Sylvie Rebuffat)
UMR 5154Chemistry/Biochemistry of Natural Substances
Theme 1: Marine sponges/bacteria associations
Theme 2: Bacterial competitions in the intestinal ecosystem :the microcins
"Molecular Adaptation of Microorganisms to the Environment "
Natural substances as mediation agents : action on protozoan parasites (Bernard Bodo)
Thai-French Seminar, Bangkok 2005
DNA RNA50S30S
50S30S
Ribosomes
ProteinDNA
replication
transcription
translation
Cell wall synthesisinhibition
Cytoplasmic membranealteration
DNA, RNA and proteinsynthesis inhibition
Binding to DNA
Enzymatic activityinhibition
Main targets for antibiotics
Thai-French Seminar, Bangkok 2005 UMR 5154 CNRS-MNHN
DNA RNA50S30S
50S30S
Ribosomes
ProteinDNA
replication
transcription
translation
Cell wall synthesisinhibition
Cytoplasmic membranealteration
Enzymatic activityinhibition
Binding to DNA
DNA, RNA and proteinsynthesis inhibition
Antibioticinactivation
Target modification
Antibiotic efflux Tolerance to the antibiotic
Main bacterial resistances to antibiotics
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
Antimicrobial peptides, a key defence strategy
FUNGIpeptaibolsbleomycins
BACTERIAbacteriocins
colicinsmicrocins
INSECTScecropins
CRUSTACEANSpenaeidins
MAMMALSdefensins
AMPHIBIANSmagainins
dermaseptins
FISHESpardaxin
pleurocidin
thioninsPLANTS
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
DNA RNA50S30S
50S30S
Ribosomes
ProteinDNA
replication
transcription
translation
Cell wall synthesisinhibition
Cytoplasmic membranealteration
Enzymatic activityinhibition
Binding to DNA
DNA, RNA and proteinsynthesis inhibition
Alteration of membrane composition (net surface charge; Lipid A)
Atm. peptide export(efflux pumps)
Atm. peptide degradation(proteolytic enzymes)
UMR 5154 CNRS-MNHN
Main bacterial resistances to antimicrobial peptidesfrom vertebrates
Thai-French Seminar, Bangkok 2005
Enterobacteria secrete, through a conservedgenetic system, antimicrobial peptides/proteins
against which they are immune
STRESSPoor medium
Microcins
Enterobacteria(Escherichia coliKlebsiella pneumoniae)
Mediators of bacterial competitions
Microcins from Enterobacteria
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
O
N
S
N
HN
O
VGIGGGGGGGGG
S
N
O
O
N
HNG
HNGGQGG
S
N
O
HNG
S
N
HNSN
O
N
O O
O
N
HNGGNG
O
GSHI
(G. Jung & coll., 1995)- MccB17: targets DNA gyrase
- MccC7/MccC51: target protein synthesis
(Rebuffat & coll., 2000)(Delepierre & coll., 1995)
N
N N
N
NH2
OOH2C
OH OH
P
O
NH
O
(CH2)3
NH2
M-R-T-G-N-A-DCO
H
- MccJ25: targets RNA polymerase
(Rebuffat & coll., 2000)(Craik & coll., 2003)
-G-G-A-G-H-V-P-E-Y-F-V-G-I-G-T-P-I-S-F-Y-G
1 to 10 kDa peptides
GETDPNTQLLNDLGNNMAWGAALGAPGGLGSAALGAAGGALQTVGQGLIDHGPVNVPIPVLIGPSWNGSGSGYNSATSSSGSGS (Lagos & coll., 1993)
(Pons et coll., 2002)
- MccE492: unmodified ? targets plasmic membrane ?
Microcins, a diverse group of antimicrobial peptides
81
Immunity factor Microcinprecursor
Secretionfactors
Modificationenzymes
A conserved genetic system
Resistance/Tolerance Export
ProcessingPosttranslational modificationsThree-dimensional structure
The microcin biosynthetic pathways
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
?
(Lagos & coll. 1999)
mceG mceHmceBmceA mceJmceDmceC mceImceFmceE
PrecursorImmunity
Export
Microcin E492 genetic system
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
?
(Lagos et al. 1999)
mceG mceHmceBmceA mceJmceDmceC mceImceFmceE
PrecursorImmunity
Export
OH
OH
HN O
O
O
O
OO
O
HN
HN
O
O
OH
OH
HO
OH
Enterobactin
OH
OH
HN O
OHHO
O
DHBS
Posttranslational modification ?
mceDmceC mceI
Enterobactinesterase
AcyltransferaseGlycosyl-
transferase
Siderophores(iron chelators)
Microcin E492 genetic system
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
3.5
(kDa)
14.2
6.5
17.0
26.6
1.0
Silver-stainedTris-Tricine SDS-PAGE
1 1 2
Modified and unmodified microcin E492
SDS-PAGE / Western-blot
UMR 5154 CNRS-MNHN
?
1. Casamino acids 2. Bactotryptone
MccE4922
Thai-French Seminar, Bangkok 2005
3.5
(kDa)
14.2
6.5
17.0
26.6
1.0
Silver-stainedTris-Tricine SDS-PAGE
1
Western Blot (anti-MccE492 antibodies)
1 2MccE492
2
1. Casamino acids 2. Bactotryptone
MccE492m
Modified and unmodified microcin E492
SDS-PAGE / Western-blot
• Identical 37-residue N-terminal sequence
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
3.5
(kDa)
14.2
6.5
17.0
26.6
1.0
Silver-stainedTris-Tricine SDS-PAGE
1
Western Blot (anti-MccE492 antibodies)
1 2MccE492
2
1. Casamino acids 2. Bactotryptone
MccE492m bears an atypical posttranslational modification
MccE492m
Modified and unmodified microcin E492
SDS-PAGE / Western-blot MALDI-TOF-MS
MccE4927887 (MH+)
8718 (MH+) MccE492m
• Identical 37-residue N-terminal sequence
+ 831 Da
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
_________________________________________________
Microorganisms MICs (µM)MccE492m MccE492
_________________________________________________Escherichia coli B 0.30Escherichia coli F 0.30Escherichia coli ML35 0.30Salmonella enteritidis 1.25Enterobacter cloacae NAKlebsiella pneumoniae NA_________________________________________________
NA: non active
Antibacterial activity of MccE492 / MccE492m
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
_________________________________________________
Microorganisms MICs (µM)MccE492m MccE492
_________________________________________________Escherichia coli B 0.04 0.30Escherichia coli F 0.08 0.30Escherichia coli ML35 0.08 0.30Salmonella enteritidis 0.15 1.25Enterobacter cloacae 0.60 NAKlebsiella pneumoniae 2.50 NA_________________________________________________
NA: non active
The antibacterial activity of MccE492 is increased by 4- to 8-times upon modification
Antibacterial activity of MccE492 / MccE492m
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
_________________________________________________
Microorganisms MBCs (µM)MccE492m MccE492
_________________________________________________Escherichia coli B 0.08 0.30Escherichia coli F 0.15 0.60Escherichia coli ML35 0.15 0.60Salmonella enteritidis 0.30 NAEnterobacter cloacae 1.25 NAKlebsiella pneumoniae 10 NA_________________________________________________
NA: non active
MccE492 and MccE492mare bactericidal
Antibacterial activity of MccE492 / MccE492m
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
GETDPNTQLL NDLGNNMAWG AALGAPGGLG SAALGAAGGA LQTVGQGLID HGPVNVPIPV LIGPSWNGSG SGYNSATSSS GSGS
Chymotrypsin digestion of MccE492 and MccE492m
Structure of MccE492 posttranslational modification
• ESI-IT MS studies of MccE492m[74-84]: CID experiments in thepositive and negative modes
• 800 MHz NMR study: TOCSY, NOESY, 1H-13C HMBC experiments
74 84SGYNSATSSS GSGS
HPLC analysis, Purification, Edmansequencing, Mass Spectrometry/NMR
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
692(115) (202) 273 374 461 548 635 779 836
NH2-Asn-Ser-Ala-Thr-Ser-Ser-Ser-Gly-Ser-Gly-Ser-COOH
b-ions
y-ions 937:131213991500(1571)(1658) 108111381225 994 Ser + 831
MccE492m[74-84]: NSATSSSGSGS-
Modification
MccE492m posttranslational modification is located at the peptide C-terminal serine
UMR 5154 CNRS-MNHN
Localization of MccE492 posttranslational modification
Thai-French Seminar, Bangkok 2005
Trimer of DHBS
β-D-glucose
MccE492--
MccE492m posttranslational modification contains a catechol-type siderophore
Structure of MccE492 posttranslational modification
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
Microcin E492m, a siderophore-peptide
positive mode
ESI-IT-MS – positive mode : MccE492m-[74-84] + 2 eq FeCl3
y* = (y - 3H + FeIII)
b4NH2-Asn-Ser-Ala-Thr-Ser-Ser-Ser-Gly-Ser-Gly-Ser
y*3y*4y*5y*6y*7y*8y*9 y*1y*2
+ FeIII
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
Microcin E492m, a siderophore-peptide
GETDPNTQLLNDLGNNMAWGAALGAPGGLGSAALGAAGGALQTVGQGLIDHGPVNVPI
PVLIGPSWNGSGSGYNSATSSSGSGSHOO
H H
OH
OH
HN O
O
O
OH
OO
O
HN
HN
O
O
OH
HO
OH
OH
O
H
HOHO
H
OHH
O
C
ONH
O
O NH
O O
OHN
O
O
O
HOOH
OHHO
OH
OH
O
OHHO
OHOH
HO
OHOHOOH
OH
OH
ONH
O
ONH
O O
OHN
O
O
O
HOOH
OHHO
OH
OH
Salmochelin S4(Bister et al., 2004)
Enterobactin(O’Brien et al., 1970;Pollack & Neilands, 1970)
MccE492m, the first natural siderophore-peptide, is the natural form of MccE492
S. Rebuffat & coll., J Biol Chem 2004
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
C E F G H IBA D J
DHBS, its linear dimer and trimer and its cyclic trimer (enterobactin)are catecholate siderophores involved in Fe3+ import in Enterobacteria
D
• Enterobactin esterase (D) breaks down enterobactin (cyclic trimerof DHBS) into the linear trimer of DHBS, its dimer and monomer
C
• Glycosyltransferase (C) anchors the β-D-glucose moiety to enterobactin
I
• Acyltransferase (I) links β-D-glucose to Ser84
Understanding the microcin E492 genetic system
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
OM
IM
(Cir, Fiu)
H+H+
H+
H+
H+
H+
ATP
ONH
O
ONH
O O
OHN
O
O
O
HOOH
OHHO
OH
OH
Enterobactin
(FepA)
DHBS monomer, dimer, trimer
(FepA, Fiu, Cir)
catechol-typesiderophores
Periplasmicspace
ONH
O
ONH
O O
OHN
O
O
O
HOOH
OHHO
OH
OH
Recognition/import of siderophores
UMR 5154 CNRS-MNHN
X-Pro domain
ExbB/ExbD
complex
ExbB/ExbD
complex
TonB-box
TonB-CTD
Thai-French Seminar, Bangkok 2005
Cytoplasm
TonB
FepA
_________________________________________________
Microorganisms MICs (µM)MccE492m
_________________________________________________E. coli H1443 (wt)E. coli H873 (fepA-)E. coli H1594 (fiu-)E. coli H2222 (cir-)E. coli H1728 (cir-, fiu-)E. coli H1875 (fepA-, cir-)
E. coli H1877 (fepA-, fiu-)E. coli H1876 (fepA-, fiu-, cir-) _________________________________________________
E. coli strains mutated in genes encodingiron-siderophore receptors
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
_________________________________________________
Microorganisms MICs (µM)MccE492m
_________________________________________________E. coli H1443 (wt) 0.04E. coli H873 (fepA-) 0.04E. coli H1594 (fiu-) 0.04E. coli H2222 (cir-) 0.04E. coli H1728 (cir-, fiu-) 0.02E. coli H1875 (fepA-, cir-) 0.04
E. coli H1877 (fepA-, fiu-) 0.32E. coli H1876 (fepA-, fiu-, cir-) 8.50_________________________________________________
E. coli strains mutated in genes encodingiron-siderophore receptors
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
_________________________________________________
Microorganisms MICs (µM)MccE492m MccE492
_________________________________________________E. coli H1443 (wt) 0.04 0.15E. coli H873 (fepA-) 0.04 0.15E. coli H1594 (fiu-) 0.04 0.15E. coli H2222 (cir-) 0.04 0.15E. coli H1728 (cir-, fiu-) 0.02 0.15E. coli H1875 (fepA-, cir-) 0.04 0.15
E. coli H1877 (fepA-, fiu-) 0.32 1.25E. coli H1876 (fepA-, fiu-, cir-) 8.50 >10_________________________________________________
Recognition of MccE492 and MccE492m is mediated by the three catechol-type FeIII-siderophore receptors,
FepA, Fiu and Cir
E. coli strains mutated in genes encodingiron-siderophore receptors
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
_________________________________________________
Microorganisms MICs (µM)MccE492m MccE492
_________________________________________________E. coli H1443 (wt) 0.04 0.15E. coli H873 (fepA-) 0.04 0.15E. coli H1594 (fiu-) 0.04 0.15E. coli H2222 (cir-) 0.04 0.15E. coli H1728 (cir-, fiu-) 0.02 0.15E. coli H1875 (fepA-, cir-) 0.04 0.15
E. coli H1877 (fepA-, fiu-) 0.32 1.25E. coli H1876 (fepA-, fiu-, cir-) 8.50 >10_________________________________________________
Improvement of the MccE492 activity upon modification results from an increase in the affinity of the receptor
for the microcin
E. coli strains mutated in genes encodingiron-siderophore receptors
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
_________________________________________________
Microorganisms MICs (µM)MccE492m MccE492
_________________________________________________E. coli W3110 (wt)E. coli W3110-KP1344 (tonB-)E. coli W3110-KP1344 pMS7*_________________________________________________*plasmid encoding TonB
E. coli strain mutated in TonB gene
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
_________________________________________________
Microorganisms MICs (µM)MccE492m MccE492
_________________________________________________E. coli W3110 (wt) 0.08 0.30E. coli W3110-KP1344 (tonB-) >10 >10E. coli W3110-KP1344 pMS7* 0.04 0.30_________________________________________________*plasmid encoding TonB
Antibacterial activity of MccE492 / MccE492m is TonB-dependent
E. coli strain mutated in TonB gene
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
0
0.4
0.8
1.2
1.6
0 20 40 60 80Time (min)
1µM MccE4921µM MccE492m
A420 nm
E. coli ML35 : (ONPG → ONP: A420 nm )
In vivo / in vitro membrane properties
2 s e c
4 0 p A
MccE492m
4 0 0 m S
0 p A
MccE492
4
Planar lipid bilayers(POPC/DOPE)
Permeabilization of the inner-membrane(S. Rebuffat & coll., Mol. Microbiol. 2003)
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
0
0.4
0.8
1.2
1.6
0 20 40 60 80Time (min)
1µM MccE4921µM MccE492m
A420 nm
E. coli ML35 : (ONPG → ONP: A420 nm )
In vivo / in vitro membrane properties
MccE492 and MccE492m modify the properties of membrane bilayers in vivo and in vitro
(S. Rebuffat & coll., Mol. Microbiol. 2003)
Planar lipid bilayers(POPC/DOPE)
Permeabilization of the inner membrane Voltage-independent single-channels
-100
2
1
0 100
-10
-5
5
10 nA
mV
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
Cir Fiu
C
N
NEx
bB
ExbD
N
NC
C
TonB
MccE492/MccE492m
FepA
Outermembrane
Innermembrane
Periplasmicspace
Mechanism of the antibacterial activity of MccE492
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
?
OM
IM
Ferrichrome
FhuA FhuE Iut
FhuD FepB
FhuBFhuC
FepDGFepC
FecA
FecB
FecCDFecE
FepA Fiu Cir
CoprogenAerobactin
Enterobactin
DHBS Citrate
MccE492
Are other microcins recognized by such receptors?
Parasitism of iron-siderophore receptors by microcinsHydroxamate-type
siderophoresCatechol-typesiderophores
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
Carboxylate-typesiderophores
O
N
S
N
HN
O
VGIGGGGGGGGG
S
N
O
O
N
HNG
HNGGQGG
S
N
O
HNG
S
N
HNSN
O
N
O O
O
N
HNGGNG
O
GSHI
(G. Jung & coll., 1995)- MccB17: targets DNA gyrase
- MccC7/MccC51: target protein synthesis
(Rebuffat & coll., 2000)(Delepierre & coll., 1995)
N
N N
N
NH2
OOH2C
OH OH
P
O
NH
O
(CH2)3
NH2
M-R-T-G-N-A-DCO
H
- MccJ25: targets RNA polymerase
(Rebuffat & coll., 2000)(Craik & coll., 2003)
-G-G-A-G-H-V-P-E-Y-F-V-G-I-G-T-P-I-S-F-Y-G
1 to 10 kDa peptides
GETDPNTQLLNDLGNNMAWGAALGAPGGLGSAALGAAGGALQTVGQGLIDHGPVNVPIPVLIGPSWNGSGSGYNSATSSSGSGS (Lagos & coll., 1993)
(Pons et coll., 2002)
- MccE492: unmodified ? targets plasmic membrane ?
Microcins, a diverse group of antimicrobial peptides
81
MccJ25
S. Rebuffat & coll., Eur. J. Biochem, 2002S. Rebuffat & coll. Biochemistry, 2004
Parasitism of iron-siderophore receptors by microcins
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
Loop Val11-Pro16
"Lasso-type structure": Extraordinary stability
S. Rebuffat & coll., Eur. J. Biochem, 2002S. Rebuffat & coll. Biochemistry, 2004
Parasitism of iron-siderophore receptors by microcins
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
t-MccJ25
A two-peptide fragment
MccJ25
protease
Loop Val11-Pro16
OM
IM
H+
H+
H+H+H+
ATP
Periplasmicspace
UMR 5154 CNRS-MNHN
TonBX-Pro domain
ExbB/ExbD
complex
ExbB/ExbD
complex
TonB-box
TonB-CTD
Recognition mechanism of MccJ25
Thai-French Seminar, Bangkok 2005
Ferrichrome
FhuA is a hydroxamate-typesiderophore receptor(ferrichrome)
FhuA
Phages T1, T5, Φ80? Antibiotics
S. Rebuffat & coll.,Biochem. J. 2005
In vivo/in vitro study of theMccJ25/FhuA interaction
?
OM
IM
H+
H+
H+H+H+
ATP
Periplasmicspace
UMR 5154 CNRS-MNHN
TonBX-Pro domain
ExbB/ExbD
complex
ExbB/ExbD
complex
TonB-box
TonB-CTD
Recognition mechanism of MccJ25
Thai-French Seminar, Bangkok 2005
Ferrichrome
FhuA
MccJ25
In vitro : MccJ25/FhuA molecularinteraction was studied by:
- Gel permeation- Microcalorimetry(determination of the parametersof the interaction)
S. Rebuffat & coll.,Biochem. J. 2005
t-MccJ25
OM
IM
H+
H+
H+H+H+
ATP
Periplasmicspace
UMR 5154 CNRS-MNHN
TonBX-Pro domain
ExbB/ExbD
complex
ExbB/ExbD
complex
TonB-box
TonB-CTD
Recognition mechanism of MccJ25
Thai-French Seminar, Bangkok 2005
Ferrichrome
FhuA
MccJ25t-MccJ25
t-MccJ25 does not interactwith FhuA
In vitro :
MccJ25 interacts with FhuA
OM
IM
H+
H+
H+H+H+
ATP
Periplasmicspace
UMR 5154 CNRS-MNHN
TonBX-Pro domain
ExbB/ExbD
complex
ExbB/ExbD
complex
TonB-box
TonB-CTD
Recognition mechanism of MccJ25
Phage T5Phage T5Phage T5Phage T5Phage T5
Fe3+siderophore
In vivo :
Inhibition of the infection by phage T5: evidence of theMccJ25/FhuA interaction
S. Rebuffat & coll.,Biochem. J. 2005
Thai-French Seminar, Bangkok 2005
FhuAMccJ25 is recognized by thehydroxamate siderophorereceptor FhuA
MccJ25 prevents T5 infection
MccJ25
The Val11-Pro16 β-hairpinregion of MccJ25 is requiredfor recognition by FhuA
t-MccJ25
t-MccJ25 does not preventT5 infection
OM
IM
Ferrichrome
FhuAFhuE Iut
FhuD FepB
FhuBFhuC
FepDGFepC
FecA
FecB
FecCDFecE
FepA Fiu Cir
CoprogenAerobactin
Enterobactin
DHBS Citrate
Hydroxamate-typesiderophores
Catechol-typesiderophores
MccE492
Parasitism of iron-siderophore receptors by microcins
The recognition step of microcins E492 and J25 at the outer membrane of susceptible bacteria involves a parasitism of the receptors
to iron-siderophore complexes
MccJ25
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
Competition
E. coli Mcc-
E. coli Mcc+
mcc
efflux
Microcin Fe3+
import
Parasitism ofiron import pathways
E. coli Mcc-
Bactericidal Activity
Selective advantage
Microcins in microbial competitions
UMR 5154 CNRS-MNHNThai-French Seminar, Bangkok 2005
Defence molecules secreted by microorganisms in confinedecosystems as potent pharmacologically active molecules
Perspectives
Defence molecules from symbiotic bacteria- Marine sponges- Cephalopodes (Nautilus, …)
UMR 5154 CNRS-MNHN
Microcins of the enterobacteria, an attractive model ofmolecules for antibiotherapy- Other natural siderophore-peptides(MccH47, MccM, MIC 1-100 nM)- Siderophore - antimicrobial- conjugates
* Antibiotics, antimalarial agentsDecreased resistance frequency / Increased selectivity
* Adjunctive therapeutic agents- Parasitism of other receptors used by the bacteria for vital functions (MccC51/receptor to nucleosides Tsx, …)
Thai-French Seminar, Bangkok 2005
- UMR 5154 CNRS-MNHN
Jean PEDUZZI Doctorants/Post-DoctorantsDelphine DESTOUMIEUX-GARZÓN Sophie DUQUESNE Christophe GOULARD Fabienne FRANÇOIS-VADROTGérard GASTINE Vanessa PETITAlain BLOND Xavier THOMAS Michel CHEMINANT Gaëlle VASSILIADISSylvie REBUFFAT
- UMR 7613 CNRS Laboratory of Structural Organic and Biological ChemistryUniversity Paris VI; Carlos AFONSO - Jean-Claude TABET
- Laboratory of High-Field NMR, Institute of Chemistry of NaturalSubstances, CNRS, Gif s/Yvette Nicolas BIRLIRAKIS - Eric GUITTET
- Laboratory of Biochemistry of Proteins, DIEP, C.E.A. Saclay Robert THAI
- Institut für Mikrobiologie, University of TübingenKlaus HANTKE - Volkmar BRAUN
- Institute of Biochemistry, Molecular and Cellular Biophysics, University Paris XI, Orsay; Pascale BOULANGER – Lucienne LETELLIER
- Centre of Structural Biology, CNRS, Montpellier; Gérard MOLLE
The siderophore-peptide, and also the unmodifiedmicrocin E492, both use a receptor-mediatedmechanism involving membrane permeabilizationto kill sensitive bacteria. They are potent narrowspectrum antibacterial agents (MIC 1-100 nM).
Microcins parasitize the iron-siderophore receptorsto enter the target bacteria.
They use outstanding strategies to increase theirpotency, such as mimicking the iron-siderophorecomplexes themselves to enter their target cellsmore efficiently.
Microcins constitute an attractive model for thedesign of antibacterial molecules through theircomplex/original mechanisms of action.
Summary, Conclusions
Thai-Franco Seminar, Bangkok 2005 UMR 5154 CNRS-MNHN
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