life death and hydrogen bonds:

Life Death and hydrogen bonds: Bacterial peptidoglycan biosynthesis

and its relationship to antibiotic resistance and the development of new

antibacterials David I Roper

School of Life Scienceswww.warwick.ac.uk/go/ropergroup

Bacterial pathogens with multiple antibiotic resistance phenotypes

Mycobacterium tuberculosis

Pseudomonas aeruginosa

Enterococcus faecalisStaphylococcus aureus

http://www.denniskunkel.com (2007)

Klebsiella pneumoniae Acinetobacter spp.

Antibiotic Resistance: Problem? What Problem?

Centre for Disease Control, Al. USA

But, two years earlier the first cases of penicillin-resistance in clinical isolates of Streptococcus pneumoniae were reported. In the US, penicillin resistance is currently encountered in >30% of pneumococcal infections (Doern et al 1999, Emerg. Infect. Dis. 5, 757)

Virtually all prescribed antibiotics were identified between 1940-1960. Their success led to the following testimony to Congress:

“The United States is ready to close the book on infectious disease and shift its resources to new dimensions of health, such as chronic diseases”

The US Surgeon General, Washington, 1969

(Institute of Medicine (1992) Emerging infections – Microbial threats to health in the United States. National Academy Press, Washington, DC).

Antibiotic Resistance Mechanisms: Nature or nuture?

The problem of resistance is promoted by a number of factors:

Most current antimicrobials are derived from natural sources wherein a resistance mechanism is necessary to protect the producing organism:

Natural Antibiotic Resistance amongst 480 Streptomyces Strains Isolated from Soil

D’Costa et al. (2006) Science 311, 374-377

Antibiotic Resistance Mechanisms:Nature or nuture?

The problem of resistance is promoted by a number of factors:Most current antimicrobials are derived from natural sources wherein a resistance mechanism is necessary to protect the producing organism, but can be spread by gene transfer particularly under conditions where there is positive selection

Thus, with natural product antibiotics (or derivatives thereof)The question of resistance is not if, but when……..Thanks to the medical profession and agricultural industry, this is Sooner rather than later• over prescription/use of antibiotics• Clinical environments that have allowed the spread of e.g. vancomycin resistance

from Enterococcus sp. to clinicalStaphylococcus aureus strains to create VRSA• Agricultural use of antibiotics as growth promoters

24 TONS of a vancomycin derivative used for animal health – 1000X more than was used to treat human infections that year

Pigs analysed for vancomycin resistant EnterococciContained the same resistance genes as those

isolated from human patients with VRE infections

Dainish Government banned use of vancomycin derivatives in animal feed

Denmark, 1994

Antibiotics: The TargetsProtein Synthesis Intermediary MetabolismDNA Replication

Cell Wall (Peptidoglycan) Synthesis

Antibiotic Resistance Mechanisms

Resistance

Target Modification

Antibiotic Modification

Reduction of [Antibiotic]at site of Action (influx/Efflux)

Choroamphenicol

b-lactams

Vancomycin

b-lactams

Sulphon-omides

QuinolonesErythromycin

Tetracyclines ChoroamphenicolQuinolones

Antibiotic Resistance, if not man made, has been greatly accelerated by man

Contents and AimsTo understand the action of cell wall directed antibiotics and mechanisms of resistance to them, we need:

1) A working knowledge of peptidoglycan biosynthesis: Part 1

2) An appreciation of how this process is targeted by antibiotics such as the b-lactams and vancomycin and a knowledge of mechanisms of resistance that have allowed pathogenic bacteria to evade the bacteriacidal effects of these cell-wall directed antibiotics: Part 2

Part 1Peptidoglycan: Structure,

Function, Synthesis and Target

Gram-negative

CM

OM

PG

Gram-positive

PG

CM

Peptidoglycan position in Gram-positive and Gram-negative bacteria

Electron micrograph of a cross section of theEscherichia coli Cell Wall

The Essential Role of the Peptidoglycan

A Scaffold providing:

An anchoring point for those components of the bacteriumthat interact with its environment (which could be you….):

extracellular proteins;

Techioic Acids,

mycolylarabinogalactan (Capsule of Mycobacterium tuberculosis)

Supporting and protective mesh surrounding and protecting thecytoplasmic membrane from physical forces such as osmotic pressure

Gram Positive Organisms

Peptidoglycan synthesis is Unique to and essential for bacterial cell

viability

The peptidoglycan synthesising enzymes are therefore good

targets for antibiotics (both natural and man-made)

NAM = N-acetyl muramic acidNAG = N-acetyl glucosamine

MurNAcL-Ala

D-Gln

L-Lys

D-Ala

D-Ala

MurNAcL-Ala

D-Gln

L-Lys

D-Ala

MurNAcL-Ala

D-Gln

L-Lys

D-Ala

D-Ala

MurNAcL-Ala

D-Gln

L-Lys

D-AlaL-Ala L-Ala

MurNAcL-Ala

D-Gln

L-Lys-

D-Ala

D-Ala

Gly-Gly-Gly-Gly-Gly-

MurNAcL-Ala

D-Glu

DAP

D-Ala

D-Ala

MurNAcL-Ala

D-Glu

DAP

D-Ala

MurNAcL-Ala

D-Gln

L-Lys

D-Ala

Normal S.pneumoniaedirect cross-link:

Indirect cross-links found in penicillin-resistant Streptococcus pneumoniae:

Common examples of bacterial peptidoglycan structure

GlcNAc -GlcNAc

1

2

3

4

5

1

2

3

4

5

1

2

3

4

5

GlcNAc

1

2

3

4

5

Normal Escherichia coli and Bacillus subtilis direct cross-link

-GlcNAc-

Indirect cross-links found in penicillin-resistant Staphylococcus aureus________________________________

__________________________________

GlcNac = N-acetyl-glucosamine; MurNac = N-acetyl muramic acid

Cytoplasmic Synthesis of a UDP-Sugar Pentapeptide

Membrane-bound (intracellular) Attachment to a lipid carrier, addition of crosslinking amino

acids and an extra carbohydrate

Extracellular Crosslinking of pentapeptide and carbohydrate to yield final polymer

Peptidoglycan Synthesis - The Essentials

UDP-MurNac UDP-GlcNacmurB murA

PEPPiNADPHNADP+

Schematic Representation of the Cytoplasmic Phase ofPeptidoglycan Synthesis

L-Ala

D-Glu

meso diaminopimelic acid (DAP) or lysine)

D-Ala-D-Ala murF

UDP-MurNAc-pentapeptide

murC

murD

murE

ATP

ADP

ATP

ADP

ATP

ADP

ATP

ADP OH

NH2

NH2

OOH

O NH2

NH2

OOH

Gram Negative(and a few positive) Gram Positive

Uridine 5’-diphospho

N-acetyl-muramyl

L-alanyl

g-D-glutamyl

L-Lysyl

D-alanyl

D-alanine

NH2

NH

NH

NH

NH

OOH

O

O

O

O

OH

NH

O

PO

POH

O O OOOH

O

OH OH

N

NH

O

OO

O

O

OH

NO

OH MurA; MurB

MurC

MurD

MurE

MurF

Structure of the end product of the Cytoplasmic Phase of Peptidoglycan Synthesis

UDP-MurNac Pentapeptide

OH

NH

O

PO

POH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NO

OHPO

POH

O O OOOH

O

OH OH

N

N

O

O

OH

O

OH

NO

OH PO

POH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NO

OH

OH

PO

POH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NO

OH

OH

NH2

NH

NH

O

O

O

OH

NH

O

PO

POH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NOOH

OH

OH O

NH

O

O

OH

NH

O

P

OPOH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NO

OH

OH

OH O

NH2

NH

NH

NH

NH

OOH

O

O

O

O

OH

NH

O

PO

POH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NOOH

m urI

NADPH

L-Ala, ATPADP + Pi

m urCm urB

NADP+

D-GluATP

ADP + Pi

m urD

ADP + Pi

m urA

PEP

L-lys, ATP

m urEm urF

ddl

D-Ala-D-ALa,ATPADP + Pi

D-Ala + D-AlaATP

ADP + Pi

or mesoDAP

L-Glu

L-AlaAlanine Racemase

L,L-Diaminopimelate (DAP)

DAPF

Cytoplasmic Phase of Peptidoglycan Synthesis

Fosfomycin

D-Cycloserine

4-[(2-napthyl)methyl]-D-Glutamate

UDP GlcNac Enoyl pyruvoylUDP GlcNac

UDP MurNac UDP MurNacAla

UDP MurNacAlaGlu

UDP MurNacAlaGluLys/DAP

UDP MurNacAlaGluLys/DAPAlaAla

MurNAcL-AlaD-GluL-LysD-AlaD-Ala

PP


GlcNAc

PP


GlcNAc

Ser-Ala

PP

P

MurG

UDPGlcNAc

MurMSer-tRNA

UDP-MurNAc-pentapeptide

UMP

MraY

P

CYTOPLASM

undecaprenylphosphate

PP


GlcNAc

Ser

MurNAla-tRNA

Lipid 1 Lipid 2

CYTOPLASMIC FACE OF THECELL MEMBRANE

O

OH

HN

NH

HN

NH

OO

O

COOH

O

HN

NH

COOH

H2N

O

O

PO

OHO

PHO

O

OOO

OHO

HN

HO

HO

O

O

(Lipid II-Lys)

D-Ala

D-Ala

L-Lys

D-Glu

L-Ala

GlcNac

MurNac

UndecaprenylPhosphate

O

OH

HN

NH

HN

NH

OO

O

COOH

O

HN

NH

COOH

H2N

O

O

PO

OHO

PHO

O

OOHO

O

(Lipid I-Lys)

D-Ala

D-Ala

L-Lys

D-Glu

L-Ala

MurNac

UndecaprenylPhosphate

Membrane bound intracellular Steps of Peptidoglycan Synthesis(Streptococcus pneumonaie example)

PP


GlcNAc

Ser-Ala


GlcNAc

Ser-Ala

PP


GlcNAc

Ser-Ala

PP

P

trans-glycosylase

EXTRACELLULARSPACE

CELL MEMBRANE,EXTRACELLULARFACE

-MurNAcL-AlaD-GluL-LysD-AlaD-Ala

GlcNAc

Ser-Ala

MurNAcL-AlaD-GluL-Lys

GlcNAc-

Ser-Ala

D-Ala

Penicillin binding proteintranspeptidase

D-Ala

-MurNAcL-AlaD-GluL-LysD-Ala

GlcNAc

Ser-Ala


GlcNAc-

Ser-Ala

Penicillin binding proteincarboxy-peptidase

D-Ala D-Ala

Membrane bound extracellular Steps of Peptidoglycan Synthesis(Example: Streptococcus pneumoniae: Ser-Ala)

PP


GlcNAc

Ser-Ala


GlcNAc

Ser-Ala

PP


GlcNAc

Ser-Ala

PP

P

trans-glycosylase

EXTRACELLULARSPACE

CELL MEMBRANE,EXTRACELLULARFACE


GlcNAc

Ser-Ala


GlcNAc-

Ser-Ala

D-Ala


D-Ala


GlcNAc

Ser-Ala


GlcNAc-

Ser-Ala

Penicillin binding proteinTrans-peptidase

D-Ala D-Ala

The antibiotic targets of the lipid-linked steps of peptidoglycan synthesis

tunicamycin,mureidomycin A,liposidomycin B ramoplanin

vancomycin(glycopeptides)

moenomycin

penicillins (b-lactams)

amphomycin bacitracin


PP


GlcNAc

PP


GlcNAc

Ala-Ser

PP


GlcNAc

Ala-Ser


GlcNAc

Ala-Ser

PP


GlcNAc

Ala-Ser

PP

P

PP

P

Pundecaprenyl phosphate

Lipid-linked steps of peptidoglycan assembly

MraY

MurG

UDPGlcNAc

MurM/N

Ala-tRNA Ser-tRNA

trans- glycosylase

UDP-MurNAc- pentapeptide

UMP

peptidoglycan cross-linking

CYTOPLASM

CELL SURFACE

Ser-Ala

Ser-AlaSer-AlaSer-Ala

Summary

4) The third phase on the extracellular face of the cell membrane polymerises the lipid sugar to form the peptidoglycan

1) Peptidoglycan synthesis is a three phase process

2) The first cytoplasmic phase forms a UDP-sugar linked pentapeptide precursor

3) The second phase on the cytoplasmic face of the cell membrane forms a lipid-sugar linked pentapeptide precursor

5) All phases are subject to the action of one or more antibiotics, however, clinically, the most exploited antibiotics target the third phase of

peptidoglycan synthesis.

1) The b-lactams

N

SHN

H H

CO2HO

O

Ph

N

SHN

H H

CO2HO

O

OCH3OCH3

MethicillinPenicillin G

Part 2Mechanisms of Action of and

resistance to Cell-Wall Directed Antibiotics

PP


GlcNAc

Ser-Ala


GlcNAc

Ser-Ala

PP


GlcNAc

Ser-Ala

PP

P

trans-glycosylase

EXTRACELLULARSPACE

CELL MEMBRANE, EXTRACELLULAR FACE


GlcNAc

Ser-Ala


GlcNAc-

Ser-Ala

D-Ala


D-Ala


GlcNAc

Ser-Ala


GlcNAc-

Ser-Ala


D-Ala D-Ala

Membrane bound Extracellular Steps of Peptidoglycan Synthesis

Streptococcus pneumoniae

PP


GlcNAc

(Gly)5


GlcNAc

PP


GlcNAc

PP

P

trans-glycosylase

EXTRACELLULARSPACE



GlcNAc MurNAcL-AlaD-GluL-Lys

GlcNAc-

D-Ala


D-Ala



GlcNAc-Penicillin binding proteincarboxy-peptidase

D-Ala D-Ala

(Gly)5 (Gly)5

(Gly)5(Gly)5(Gly)5

Staphylococcus aureus

(Gly)5

PP


GlcNAc MurNAcL-AlaD-GluL-LysD-AlaD-Ala

GlcNAc

PP


GlcNAc

PP

P

trans-glycosylase

EXTRACELLULARSPACE




GlcNAc-

D-Ala


D-Ala



GlcNAc-Penicillin binding proteincarboxy-peptidase

D-Ala D-Ala

Enterococcus faecicum

D-Asn D-Asn

D-Asn

D-Asn

D-Asn

PbPs are Multimodular and Multifunctional enzymes

N N

S403

S398 E171;E114

TP TPUnknownfunction

N-terminalLinker

Linker TG

S. aureus MecA (PbP2a): MonofunctionalTranspeptidase (TP)

S. aureus PbP2 bifunctional Trans-peptidase (TP)/Transglycosylase (TG)

Cytoplasm Cytoplasm

Class B Class A

Penicillin Binding Proteins (PBPs)

A group of transpeptidases (class A & B) and d,d carboxypeptidases (class C) that utilise a serine active site nucleophile:

MurNAc

L-Ala

D-Glu

L-Lys

D-Ala

ONH

GlcNAc

Ser

O-O2C

H

NH3+

Lys

MurNAc

L-Ala

D-Glu

L-Lys

D-Ala

O

GlcNAc

Ser

O

NH3+

Lys

MurNAc

L-Ala

D-Glu

L-Lys

D-Ala

O

GlcNAc

MurNAc

L-Ala

D-Glu

L-Lys

D-Ala

D-Ala

GlcNAc

NH

MurNAc

L-Ala

D-Glu

L-Lys

D-Ala

CO2-

cross-linkedpeptidoglycan

tetrapeptide sidechain

Transpeptidase

GlcNAc

D-Ala

D,D-carboxypeptidase

MurNAc

L-Ala

D-Glu

L-Lys

D-Ala

ONH

GlcNAc

-O2C

H2N

HO

H

Ser

OH

NH3+

Lys

1234

5

12345 1

234

51234

12345

1234

b-Lactam Antibiotics

N

SHN

H H

CO2HO

O

Ph

Strained, reactive b-lactam ring

65% world market of antibiotics

>50 marketed drugs of this classPenicillinsCephalosporinsCarbapenemsMonobactamsCephalosporin-penicillin hybrids,Penems

Shared spatial structure of the terminal D-Ala-D-Ala terminus of the peptidoglycan

pentapeptide and b-lactams

b-lactam ring

b-Lactams Antimicrobial Suicide SubstratesAntimicrobial Potency arises because the drug simultaneously targets mutiple enzymes in peptidoglycan synthesis (7 PbPs in E. coli, 5 in S. pneumoniae)

Antimicrobial Potency arises because the drug exploits its strained b-lactam ring structure and the catalytic apparatus of the PbP to spring a trap on the unsuspecting enzyme….

Ser

OH

NH3+

Lys

N

S

O

-O2C

H3CH3C

H H HN COR

Ser

ONH2

Lys

NH

SO

-O2C

H3CH3C

H H HN COR

PBP's

irreversible acylation(or slow hydrolysis)

RESULT: Inhibition of peptidoglycan crosslinking leading to a weakened cell wall, leading to osmotic rupture of the cell membrane and cell death

Emergence of penicillin resistance

Target ModificationAntibiotic Inactivation

b-lactamases

Principally Gram negative enteric and Pseudomonad pathogens, exception: Staphylococcus aureus

PbP RemodellingPrincipally Gram positive pathogens, e.g. Streptococcus pneumoniae

PbP Re-aquisitionPrincipally Gram positive pathogens, e.g. MRSA, PbP2a

b-Lactamases- Like PbPs but not

Ser

OH

NH3+

Lys

NS

O

-O2C

H3CH3C

H H HN COR

Ser

ONH2

Lys

NH

SO

-O2C

H3CH3C

H H HN COR

Ser

OHNH3

+

Lys

NH CO2-

S

-O2C

H3CH3C

H H HN COR

PBP's

irreversible acylation(or slow hydrolysis)

b-lactamases

H2O

Developed catalytic apparatus to hydrolyse the b-lactam ring in a manner analogous to the mechanism of PbP carboxypeptidase hydrolysis

b-lactamases evolved from PbPs

Antibiotic InactivationStreptomyces D,D, carboxypeptidaseBacillus lichiniformis b-lactamase

Target Modification

Global clonal spread of penicillin resistant pneumococci

1984‘86 ‘88 ‘90 ‘92 ‘94 ‘96 ‘98 2000Serotype 23F

Spain UK France South Korea USA South Africa Hungary

Iceland Bulgaria Portugal Germany

Thailand Colombia The Netherlands

Argentina Denmark Japan Malaysia Singapore Taiwan

Mosaic Gene Structure In Pneumococcal pbp2x generated from homologous recombination with homologues from

closely related Streptococci

Ser

A

B

C

D

E

F

pbp2x

Transpeptidase DomainPenicillin sensitive

strains(mic 0.02 mg/ml)

Penicillin resistant

strains(mic ≤16 mg/ml)

Generation of a penicillin-resistant pneumococcal PbP2x by homologous recombination

Generalised active site of a PbP with amino acids from penicillin Sensitive PbP2x from S. pneumoniae R6 superimposed upon it

S-X-X-K

K-[T/S]-G

[S/Y]-X-[N/C]

338

341

HO Thr337

Generalised active site scaffold of a PbP with amino acids from penicillin Resistant PbP2x from S. pneumoniae Sp328 superimposed upon it

S-X-X-K

K-[T/S]-G

[S/Y]-X-[N/C]

338A337

341

PbP2x crystal structure reveals penicillinresistance by target modification has a cost

• PbP2x sequences with up to 20% divergence between resistant and sensitive strains, aquired through homologous recombination

• Key mutations distort the transpeptidase active site

• Optimal distances between conserved active site residues changed, causing simultaneous loss of catalytic activity (to 1 thirtieth of rate shown by sensitive PbP2x) and aquisition penicillin resistance.

• Implied consequence is that penicillin resistance exacts a price on cell wall synthesis, whose rate of cross linking will be impaired

Mechanisms of Action of, and resistance to Cell-Wall Directed

Antibiotics2) Vancomycin and other Glycopeptides

+

H

-

O

N

O

O

ON

HO Cl

N

O H

O2C

HO

HOOH

H

H

NH O

O

NH

O

Cl

OH

H

ONH2

HN

OH NH2

Me

HH

OHOCH2

HO

HO

O

OOH

Me

NH2

Me

H

Vancomycin

Vancomycin - A Vital Antibiotic• Vancomycin is the last line of defence against Gram-positive bacteria where other treatments fail,

Staphylococci. Streptococci, Corynebacteria, Clostridia and particularly MRSA.

• ContraindicationsDeafness, Severe hypertension (red man syndrome), nausea, diarrhoea, vomiting,

may lead to other fungal and gram-negatives.

• Glycopeptide resistant Enterococci (GRE) known since the late 1980s

Some GREs are untreatable due to multiple antibiotic resistance mechanisms.

Vancomycin : Mode of Action• Vancomycin is not an enzyme inhibitor.

• Vancomycin binds to the D-alanyl-D-alanine termini of peptidoglycan units prior their incorporation in the cell wall:

• By doing so, it prevents transpeptidation reactions from crosslinking adjacent peptidoglycan chains, weakening the cell wall leading to osmotic stress and lysis

Vancomycin (glycopeptides)


PP


GlcNAc

PP


GlcNAc

Ala-Ser

PP


GlcNAc

Ala-Ser


GlcNAc

Ala-Ser

PP


GlcNAc

Ala-Ser

PP

P

PP

P

Pundecaprenyl phosphate

Lipid-linked steps of peptidoglycan assembly

MraY

MurG

UDPGlcNAc

MurM/N

Ala-tRNA Ser-tRNA

trans- glycosylase

UDP-MurNAc- pentapeptide

UMP

peptidoglycan cross-linking

CYTOPLASM

CELL SURFACE

Extracellular surface

+

H

-

VANCOMYCIN

N-Ac-D-Ala-D-Ala

O

N

O

O

ON

HO Cl

N

O H

O2C

HO

HOOH

H

H

NH O

O

N

O

H

O

Cl

OH

H

ONH2

HN

OH NH2

Me

H

H

OHOCH2

HO

HO

O

OOH

Me

NH2

Me

O-N

O

Me

NR

H

H

Me

OH

H

H

Vancomycin: Targets the D-Ala-D-Ala TerminusExtracellular Peptidoglycan Precursors

Mr=1805

Emergence of Vancomycin Resistance

Target ModificationReduction of [Antibiotic]at site of Action

“Visa”: Vancomycin-intermediate resistant

Staphylococcus aureus – resistance by decreased permeability using a

thicker peptidoglycan layer

mic: ≥16 mg/ml (Sensitive: 0.02 mg/ml)

Peptidoglycan Remodelling

Principally Gram positive pathogens,e.g. Enterococci and more recently (2002) Staphylococcus aureus (“VRSA”)

mic: ≥500 mg/ml

+

H

-

VANCOMYCIN

N-Ac-D-Ala-D-Lactate

O

N

O

O

ON

HO Cl

N

O H

O2C

HO

HOOH

H

H

NH O

O

N

O

H

O

Cl

OH

H

ONH2

HN

OH NH2

Me

H

H

OHOCH2

HO

HO

O

OOH

Me

NH2

Me

O-

O

O

Me

NR

H

H

Me

OH

H

+

H

-

VANCOMYCIN

N-Ac-D-Ala-D-Ala

O

N

O

O

ON

HO Cl

N

O H

O2C

HO

HOOH

H

H

NH O

O

N

O

H

O

Cl

OH

H

ONH2

HN

OH NH2

Me

H

H

OHOCH2

HO

HO

O

OOH

Me

NH2

Me

O-

N

O

Me

NR

H

H

Me

OH

H

H

Vancomycin sensitive Vancomycin Resistant1000-fold drop in affinity of vancomycin for its target

Target Modification mediated Mechanismsof Vancomycin Resistance

In Gram positive pathogens such as Enterococcus faecalis and Enterococcus faecicumVancomycin resistance is more complex than target modification mediated penicillin resistance, because modification of a single (PbP) gene can be sufficient for b-lactam resistance. Vancomycin resistance, however, requires modification of complex metabolites such as those at the end of peptidoglycan synthesis and so requires expression of many different genes involved in the synthesis of the new target.

Loss of a single hydrogen bonding interaction by interconverting D-Alanine to D-lactate at the end of the peptidoglycan peptide eliminates the interaction of vancomycin with its target

Vancomycin Resistance; Simple and elegant in principle,

……..complex in execution

……mechanism to spread between pathogensTransposon encoded high-level vancomycin resistance operon. Has been transferred from an Enterococcus to S. aureus in a clinical setting !!!!!!!!!

PvanR PvanH

PO4

VanS

VanRPO4

?

VanR

Cytoplasm

D-lactateproducingreductase

D-Ala-D-lacligase

D-Ala-D-Aladipeptidase

Resistance

vanS vanH vanA vanX

Responseregulator

Sensor

Regulation

Cell membrane

vanR

Sensing and initiation of gene expression leading to Enterococcal Vancomycin Resistance

-

-

NADPH

+ -

D-Ala + ATP

H3N O CO2

O

O CO2

HO CO2

NADP+

ADP + Pi

Pyruvate

D-Lactate

D-Alanyl-D-lactate

VanH

VanAVanX

+ -

D-Ala + ATP

H3N NH

CO2

O

ADP + Pi

D-Alanine

D-Alanyl-alanine

DDL

+H3N

OH

O

No Vancomycin +Vancomycin

Precursors of Target Modification required forHigh Level Vancomycin Resistance

D-Ala-D-Ala peptidoglycan: Vancomycin sensitivity

NH2

NH

NH

O

O

O

OH

NH

O

PO

POH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NOOH

OH

OH OOH O

O

NH2

NH

NH

NH

OOH

O

O

O

O

OH

NH

O

PO

POH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NOOH

OH O

NH2

NH

NH

NH

NH

OOH

O

O

O

O

OH

NH

O

PO

POH

O O OOOH

O

OH OH

N

N

O

OO

O

O

OH

NOOH

ADP + Pi

D-Ala-D-Ala Ligase

D-Ala-D-ALa

D-Ala + D-Ala

ATPVanAD-Ala-D-Lac Ligase

D-Ala-D-Lac,ATP

D-Ala + D-Lac

ADP + Pi

ADP + Pi

m urF

Py ruv ate

NADH

NAD+ Van H:D-Lactate dehydrogenase

m urF

ATP

VanX:D-Ala-D-AlaDipeptidase

UDPmurNac AlaGluLys/DAP/Ala-Lac UDPmurNac AlaGluLys/DAP UDPmurNac

AlaGluLys/DAP/AlaAla

MurA to MurE

D-Ala-D-Lac peptidoglycan:Vancomycin Resistance

Mechanism of Target Modification required forHigh Level Vancomycin Resistance

Vancomycin sensitive

Ala

Ala

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Ala

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Ala

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AlaAla

AlaAla

Ala Al

aAl

aAl

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AlaVancomycin sensitive

Ala

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Cell Death

Summary – Vancomycin Sensitivity

Vancomycin Resistant

Ala

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Vancomycin Resistant

Ala

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LacAla

LacAla

Lac Al

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Lac

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Operon on

Cell Survival

Summary-Vamcomycin Resistance

Overall Summary

4) Resistance to vancomycin is caused by modification of its target causing loss of a single hydrogen bond interaction, by replacment of D-alanyl-D-alanine with D-alanyl-D-lactate. This is relatively complex to achieve, requiring re-programming of the synthesis of the peptidoglycan.

1) Penicillin acts as a suicide substrate that chemically modifies a group of mechanistically related cell wall polymerizing/modifying enzymes: the Penicillin binding proteins

2) Penicillin resistance involves antibiotic inactivation (b-lactamases); target modification (Streptococcal PbPs) or wholesale target replacement (S. aureus MecA)

3) Vancomycin acts not on a protein but on late peptidoglycan intermediates to which it binds via their terminal D-alanyl-D-alanine residues. This sterically prevents transpeptidation of cell wall precursors leading to cell lysis.

ReferencesBacterial antibiotic resistance and discovery:1. Walsh, C. (2000) Molecular mechanisms that confer antibacterial drug resistance, Nature 406, 775-781.2. Gwynn, M. N., Portnoy, A., Rittenhouse, S. F., and Payne, D. J. (2010) Challenges of antibacterial discovery revisited, Ann N Y Acad Sci 1213, 5-19.3. Agarwal, A. K., and Fishwick, C. W. (2010) Structure-based design of anti-infectives, Ann N

Y Acad Sci 1213, 20-45.

Vancomycin resistance4. Healy, V. L., Lessard, I. A., Roper, D. I., Knox, J. R., and Walsh, C. T. (2000) Vancomycin

resistance in enterococci: reprogramming of the D-ala-D-Ala ligases in bacterial peptidoglycan biosynthesis, Chem Biol 7, R109-119.

Peptidoglycan specificA comprehensive and detailed set of reviews of many aspects of bacterial cell wall biogeneisis

and inhibition.5. Ende, J. C. a. A. V. D. (2008) Peptidoglycan: the bacterial Achilles heel, FEMS Microbiol

Reviews 32, 147-408.6. Bugg, T. D., Braddick, D., Dowson, C. G., and Roper, D. I. (2011) Bacterial cell wall

assembly: still an attractive antibacterial target, Trends Biotechnol 29, 167-173.7. Mattei, P. J., Neves, D., and Dessen, A. (2010) Bridging cell wall biosynthesis and bacterial

morphogenesis, Curr Opin Struct Biol 20, 749-755.

life death and hydrogen bonds:

Documents

vancomycin resistance

problem of resistance

penicillin resistance

natural antibiotic resistance

resistance genes

lactamssulphono resistance

natural product antibiotics

natural sources