Defence Molecules in Microbial Ecosystems: Microcins, a ... · Defence Molecules in Microbial...

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




Binding to DNA


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


DNA RNA50S30S

50S30S

Ribosomes

ProteinDNA

replication

transcription

translation




Binding to DNA


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


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


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


?

(Lagos & coll. 1999)

mceG mceHmceBmceA mceJmceDmceC mceImceFmceE

PrecursorImmunity

Export

Microcin E492 genetic system


?

(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


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


3.5

(kDa)

14.2

6.5

17.0

26.6

1.0


1

Western Blot (anti-MccE492 antibodies)

1 2MccE492

2


MccE492m


SDS-PAGE / Western-blot

• Identical 37-residue N-terminal sequence


3.5

(kDa)

14.2

6.5

17.0

26.6

1.0


1

Western Blot (anti-MccE492 antibodies)

1 2MccE492

2


MccE492m bears an atypical posttranslational modification

MccE492m


SDS-PAGE / Western-blot MALDI-TOF-MS

MccE4927887 (MH+)

8718 (MH+) MccE492m

• Identical 37-residue N-terminal sequence

+ 831 Da


_________________________________________________

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


_________________________________________________


_________________________________________________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



_________________________________________________

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



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


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


Trimer of DHBS

β-D-glucose

MccE492--

MccE492m posttranslational modification contains a catechol-type siderophore

Structure of MccE492 posttranslational modification


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


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


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


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


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


_________________________________________________

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 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 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 W3110 (wt)E. coli W3110-KP1344 (tonB-)E. coli W3110-KP1344 pMS7*_________________________________________________*plasmid encoding TonB

E. coli strain mutated in TonB gene


_________________________________________________


_________________________________________________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


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)


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


Cir Fiu

C

N

NEx

bB

ExbD

N

NC

C

TonB

MccE492/MccE492m

FepA

Outermembrane

Innermembrane

Periplasmicspace

Mechanism of the antibacterial activity of MccE492


?

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


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


Loop Val11-Pro16

"Lasso-type structure": Extraordinary stability

S. Rebuffat & coll., Eur. J. Biochem, 2002S. Rebuffat & coll. Biochemistry, 2004



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


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



Ferrichrome

FhuA

MccJ25

In vitro : MccJ25/FhuA molecularinteraction was studied by:

- Gel permeation- Microcalorimetry(determination of the parametersof the interaction)


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



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


Phage T5Phage T5Phage T5Phage T5Phage T5

Fe3+siderophore

In vivo :

Inhibition of the infection by phage T5: evidence of theMccJ25/FhuA interaction



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


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


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


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, …)


- 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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