Molecular basis of glycopeptide resistance - ESCMID

Molecular basis of

glycopeptide resistance

Patrice CourvalinInstitut Pasteur

Mechanism of action of glycopeptides

glycopeptide

transglycosylasetranspeptidase

Cytoplasm Membrane Wall

UDPD-Ala-D-Ala-Tri

UDPTri

D-AlaDdl ligase

D-Ala-D-Ala

D-Ala-D-Ala-Tri

L-Ala

Tri-D-Ala-D-Ala

racemase

addingenzyme

Glycopeptide resistance in enterococci

Resistance Acquired Intrinsic

Type VanA VanB VanD VanC

MIC (mg/L)Vancomycin 64 - 1000Teicoplanin 16 - 512 0.5 - 1 4 - 64

VanE

160.5 0.5 - 1

Expression Inducible Constitutive Inducible Constitutive

Location Plasmid Chromosome Chromosome ChromosomeChromosome

Modified target D-Ala-D-Lac D-Ala-D-Ser

4 - 1000 64 - 128 2 - 32

Inducible

VanG

8-160.5

Combinatorial biochemistry of glycopeptide resistance

racemaseD-Ala

DdlD-Ala-D-Ala Penta PentaL-Ala

addingenzymeTri


racemaseD-Ala

DdlD-Ala-D-Ala Penta PentaL-Ala

pyruvateVanH VanA

D-Lac D-Ala-D-Lac

addingenzyme

Pentadepsi Pentadepsi

Tri

dehydrogenase ligase

required for glycopeptide resistance

D,D-dipeptidase

vanAvanH vanX

N

H ON

H O-

O

O

H3C

H

H

O O

OH

N

O

O

NH2

ON

O

NH2+

Cl

N

O

O

HN

Cl

OH

OHN

OHHO

NH

O

HO

HOOC

OCH2OH

OHOH

O

OH3C

OHNH2

CH3

HH

H

CH3

D-Ala D-AlaAcetyl

O

O-

O

H

O O

OH

N

O

O

NH2

ON

O

NH2+

Cl

N

O

O

HN

Cl

OH

OHN

OHHO

NH

O

HO

HOOC

OCH2OH

OHOH

O

OH3C

OHNH2

CH3

HH

H

CH3

H O

N

O

H3C

H

D-Ala D-LacAcetyl

Binding of vancomycin to its target


Ddl

racemase D-Ala

VanX

futile cycle D-Ala-D-Ala Penta PentaL-Ala

pyruvateVanH VanA

D-Lac D-Ala-D-Lac

VanYTetra

addingenzyme


Tri


required for glycopeptide resistance

vanAvanH vanX

accessory proteins

vanY vanZ

unknownTeRD,D-dipeptidase

D,D-carboxy-peptidase


Pentadepsi

Preg

mRNA

racemase D-Ala

Ddl

VanX


pyruvateVanH VanA

D-Lac D-Ala-D-Lac

VanYTetra

addingenzyme

Pentadepsi

Tri

response regulator

histidine protein kinase dehydrogenase ligase

regulation required for glycopeptide resistance

vanR vanS vanAvanH vanX

accessory proteins

vanY vanZ

unknownTeRD,D-dipeptidase D,D-carboxy-

peptidase

mRNA

Pres

Two-component regulatory system

SensorNH2

HisCOOH

Regulator COOHAsp

P

Kinase

Signal

ATPP

(DNA binding domain)

membrane

cytoplasm

Receptor EffectorNH2

Induction of VanA-type resistance

Activation

vanAvanH vanXvanR

P-VanR VanR

P-VanS

vanS

ATP ADP

VanR

VanS Membrane

Cytoplasm

PP resregvanY

Induction of VanX D,D-dipeptidase in VanB- and VanA-type enterococci

Glycopeptide (mg/l)

1000

800

600

400

200

0

Spec

ific

activ

ity (U

)

501610420.05 0.2 0.5 1

VanB, Te

VanA, Vm

VanB, Vm

VanA, Te

Transfer of physically unlinked van genes

vanAvanH

vanXPX

PA

PH


Pentadepsi

Preg

mRNA

racemase D-Ala

Ddl

VanX


pyruvateVanH VanA

D-Lac D-Ala-D-Lac

VanYTetra

addingenzyme

Pentadepsi

Tri

response regulator

histidine protein kinase dehydrogenase ligase

regulation required for glycopeptide resistance

vanR vanS vanAvanH vanX

accessory proteins

vanY vanZ

unknownTeRD,D-dipeptidase D,D-carboxy-

peptidase

mRNA

Pres

VanA-type MRSA

S. aureus

NY-VRSA

PA-VRSA

MI-VRSA

Vancomycin

32 (LLR)

32 (LLR)

1024 (HLR)

Teicoplanin

8 (S)

8 (S)

128 (R)

VRSA-5 256 (HLR) 128 (R)

MIC (mg/L) of:

MI-VSSA 2 0.5

E. faecalis VanA 1024 128

Structure of Tn1546

transposition regulation accessory proteinsrequired forglycopeptide resistance

vanR vanS vanAvanH vanXORF2ORF1

IRL

vanY vanZ

IRRtransposase resolvase dehydrogenase ligase dipeptidase

carboxy-unknownpeptidaseregulator sensor

CATGCTAGC AAGTTGGGGTAGCGTC...TACAACGCTAAG...GTACGATCG TTCAACCCCATCGCAG...ATGTTGCGATTC...

pLW1043

...AAATTAGCATCATGCTAGC AAGTT AAGCGAACACTGACATGAT...

...TTTAATCGTAGTACGATCG TTCAA TTCGCTTGTGACTGTACTA ...

...CTTAACGTTGTA.... GACGGTACCCC AAGTTAAGCGAACA

...GAATTGCAACAT... CTGCCATGGGG TTCAA TTCGCTTGT

Target

5 bp 5 bp38 bp 38 bp

IR-left IR-right

Tn1546

pLW1043

pAM829

Integration of the Tn1546 element

Organisation of Tn1546 in VanA-type MRSA

IS1251

IS1216V

IRR

vanZvanYvanXvanAvanHvanR vanSORF2

PA-VRSA

MI-VRSA-VRSA-5

vanZvanYvanXvanAvanHvanR vanSORF1 ORF2

IRR

NY-VRSA

IS1251

IS1216V vanZvanYvanXvanAvanHvanR vanSORF2

Spontaneous overnight loss of glycopeptide resistance in VanA-type MRSA

% loss (range)Strain

NY-VRSA

PA-VRSA

MI-VRSA

Average of three independent experiments

47 (45-48)

49 (47-50)

0 (0-0)

0.25 (0-1)VRSA-5

Growth of VanA-type MRSA

Time (h)

O.D

. (60

0 nm

)

Vm0 / Vm0

Vm0 / Vm6

0

1

2

3

4

5 MI-VRSA

1 2 3 4 5 6 7 8 24

0

1

2

3

4

5 NY-VRSAVm0 / Vm0

Vm0 / Vm6

1 2 3 4 5 6 7 8 249

VRSA-5

1

2

3

4

5Vm0 / Vm0

Vm0 / Vm6

1 2 3 4 5 6 7 248

1 2 3 4 5 6 7 8 240

1

2

3

4

5 PA-VRSA Vm0 / Vm0

Vm0 / Vm6

S. aureusVancomycin Oxacillin

BHI BHI + Oxa BHI BHI + Vmb

MI-VRSA 1024 2 > 256 2

VRSA-5 128-256 1 128 1

PA-VRSA 24-32 0.03 4-8 0.03

NY-VRSA 32-64 0.25 32-64 0.25

VanA-type MRSA: MICs (mg/L) by agar dilution

a oxacillin, 2-8 µg/ml.b vancomycin, 8 µg/ml.

Ox (8 mg/L) Vm (8 mg/L)

VmTe

Ox

Vm

Ox Vm Ox

VanA-type MRSA : Combination of glycopeptides and oxacillin

Em

Pe

SpVmTe

Ox

Em

Pe

Sp

VmTe

Ox

Em

Pe

Sp

Time (h)

Log

10cf

u/m

lControlVm 2Ox 2Vm 2 + Ox 2

0

2

4

6

8

10

12

14

16

0 3 246

NY-VRSA

VanA-type MRSA: time-kill curves

0

2

4

6

8

10

12

14

16

0 3 246

VRSA-5ControlVm 8Ox 8Vm 8 + Ox 8

VanA-type MRSA: Synthesis of peptidoglycan

The end

GCG CGA AGC AGA AAA AGT GGG CGA AGC GGT TTA GGA CTT GCC ATC GTA

GCG --- --- --- --- --- --- CGA AGC GGT TTA GGA CTT GCC ATC GTA

A R S R K S G R S G L G L A I V

inducibleconstitutive

VmR TeR constitutive

∗

NH2

Cytoplasm

Η Ν G1 G2F

COOH

Membrane

Mutations in the VanSB sensor

VmR TeR

Inducible

VmR TeR

Inducible

∗

∗

Constitutive glycopeptide resistance

transcription

SBP

kinase

Induced

phosphatase

SB

RBP

SB

Non induced

transcription

P

vanRB vanYB

PresvanSB

Preg

+

Constitutive

vanXBVanHB vanB

RBRB

RB

RBP

RBP

vanW

SB SB

Glycopeptide dependence

racemaseD-Ala

DdlL-Ala

pyruvate VanHB VanBD-Lac D-Ala-D-Lac

addingenzyme


Tri


resistance

D,D-dipeptidase

vanRB vanSB vanBvanHB vanXBvanYB vanW

regulator sensor

regulation

unknownD,D-carboxy-peptidase

Glycopeptide independence by constitutivity

racemaseD-Ala

DdlL-Ala


addingenzyme


Tri


resistance

D,D-dipeptidase


regulator sensor

regulation


Glycopeptide independence by reversion

racemaseD-Ala

DdlL-Ala


addingenzyme


Tri


resistance

D,D-dipeptidase


regulator sensor

regulation


Penta

van genetic tinkering

VmR TeS

(Vmi )

VmD TeD

(Vmi Tei )

(Vmi )VmDddl

vanS Te 10-7

Vm 10-8

VmR TeR

(Vmi Tei )ddl+

ddl+

vanS Te 10-7

Vm Te10-7

vanS

Molecular basis of glycopeptide resistance - ESCMID

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