Download Document

Document related concepts

Vectors in gene therapy wikipedia , lookup

Proteolysis wikipedia , lookup

Oxidative phosphorylation wikipedia , lookup

Amino acid synthesis wikipedia , lookup

Lipid signaling wikipedia , lookup

Evolution of metal ions in biological systems wikipedia , lookup

Artificial gene synthesis wikipedia , lookup

Signal transduction wikipedia , lookup

Penicillin wikipedia , lookup

Transcript
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 Sciences
www.warwick.ac.uk/go/ropergroup
Bacterial pathogens with multiple antibiotic
resistance phenotypes
Staphylococcus aureus
Enterococcus faecalis
Klebsiella pneumoniae
Pseudomonas aeruginosa
Mycobacterium tuberculosis
Acinetobacter spp.
http://www.denniskunkel.com (2007)
Antibiotic Resistance: Problem? What Problem?
Virtually all prescribed antibiotics were identified between
1940-1960. Their success led to the following testimony
toBut,
Congress:
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
“The United States is ready to close the book on
>30% of pneumococcal infections
infectious
disease
andDis.
shift
its resources to new
(Doern et al 1999,
Emerg. Infect.
5, 757)
dimensions of health, such as chronic diseases”
The US Surgeon General,
Washington, 1969
Centre for Disease Control, Al. USA
(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:
Natural Antibiotic Resistance amongst 480
Streptomyces Strains Isolated from Soil
Most current antimicrobials
are derived from natural
sources wherein a resistance
mechanism is necessary to
protect the producing
organism:
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
Denmark, 1994
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 Enterococci
Contained the same resistance genes as those
isolated from human patients with VRE infections
Dainish Government banned use of vancomycin derivatives
in animal feed
Antibiotics: The Targets
DNA Replication
Protein Synthesis
Intermediary Metabolism
Cell Wall (Peptidoglycan) Synthesis
Antibiotic Resistance Mechanisms
Choroamphenicol
Vancomycin
b-lactams
Target
Modification
Antibiotic
Modification
Sulphonomides
b-lactams
Quinolones
Erythromycin
Resistance
Reduction of [Antibiotic]
at site of Action (influx/Efflux)
Tetracyclines
Choroamphenicol
Quinolones
Antibiotic Resistance, if not man made, has been greatly accelerated by
man
Contents and Aims
To 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 blactams 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 1
Peptidoglycan: Structure,
Function, Synthesis and Target
Peptidoglycan position in Gram-positive
and Gram-negative bacteria
OM
PG
PG
CM
CM
Gram-positive
Gram-negative
Electron micrograph of a cross section of the
Escherichia coli Cell Wall
The Essential Role of the Peptidoglycan
A Scaffold providing:
Supporting and protective mesh surrounding and protecting the
cytoplasmic membrane from physical forces such as osmotic pressure
An anchoring point for those components of the bacterium
that interact with its environment (which could be you….):
extracellular proteins;
Techioic Acids,
Gram Positive Organisms
mycolylarabinogalactan (Capsule of Mycobacterium tuberculosis)
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 acid
NAG = N-acetyl glucosamine
Common examples of bacterial peptidoglycan structure
Normal S.pneumoniae
direct cross-link:
Indirect cross-links found in
penicillin-resistant Streptococcus pneumoniae:
__________________________________
MurNAc GlcNAc
MurNAc
L-Ala
1 L-Ala
D-Gln
2 D-Gln
3 L-Lys
MurNAc -GlcNAc
MurNAc
L-Ala
1
L-Ala
D-Gln
L-Lys
2
D-Gln
L-Lys
D-Ala
3
L-Lys L-Ala L-Ala D-Ala
4 D-Ala
4
D-Ala
5 D-Ala
5
D-Ala
Normal Escherichia coli and
Bacillus subtilis direct cross-link
Indirect cross-links found in
penicillin-resistant Staphylococcus aureus
________________________________
MurNAc -GlcNAcMurNAc GlcNAc
MurNAc
L-Ala
MurNAc
L-Ala
1
L-Ala
D-Gln
1
L-Ala
D-Glu
2
D-Gln
L-Lys
2
D-Glu
DAP
3 L-Lys- Gly-Gly-Gly-Gly-Gly-D-Ala
3
DAP
D-Ala
4
D-Ala
4
D-Ala
5
D-Ala
5
D-Ala
GlcNac = N-acetyl-glucosamine; MurNac = N-acetyl muramic acid
Peptidoglycan Synthesis - The Essentials
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
Schematic Representation of the Cytoplasmic Phase of
Peptidoglycan Synthesis
NADP+
UDP-MurNac
ATP
murC
ADP
ATP
murD
ADP
ATP
murE
NADPH
murB
ADP
PEP
murA
D-Glu
meso diaminopimelic acid (DAP) or lysine)
OH
OH
O
O
murF
UDP-GlcNac
L-Ala
ADP
ATP
Pi
D-Ala-D-Ala
UDP-MurNAc-pentapeptide
NH 2
NH 2
OH
H 2N
O
Gram Negative
(and a few positive)
H 2N
Gram Positive
Structure of the end product of the Cytoplasmic Phase of
Peptidoglycan Synthesis
OH
HO
N
O
O
NH
O
OH
O
HO
O
O
O
O
P
P
O
OH
OH
N
O
O
Uridine 5’diphospho
N-acetylmuramyl
MurA; MurB
O
HN
L-alanyl
O
MurC
NH
O
MurD
HO
g-D-glutamyl
O
HN
L-Lysyl
NH2
O
MurE
NH
D-alanyl
O
HN
D-alanine
HO
O
MurF
UDP-MurNac
Pentapeptide
Cytoplasmic Phase of Peptidoglycan Synthesis
UDP GlcNac
H
O
N
Enoyl pyruvoyl
UDP GlcNac
O
H
O
O
O
H
H
O O
O
O O
O
P
P
O N
O O
H
H
O
P
E
P
O
H
O
H
N
m
urA
O
H
N
UDP MurNac
N
AD
P
H
O
O
+
N
AD
P
H
O
O
H
H
O O
O
O O
O
O
H
P
P
N
O
O O
H
O
N
N
O
H
O
m
urB
O
O
H
H
O O
O
O O
O
P
P
O N
O O
H
O
AD
P+P
i
H
O
O
H
O
H
N
O
m
urC
N
O
O
H
AD
P+P
i
m
urD
D
-G
lu
ATP
L-G
lu
N
O
H
O
O
O
H
UDP MurNacAla
GluLys/DAP
H
O
H
O O
O
O O
O
O
H
P
PO N
O O
H
O
N
O
H
O
O
N
O
H
O
AD
P+P
i
D-A
la-D-A
La,
A
T
P
O
H
N
O
O
H
N
N
H
O
O H
O
N
H2
N
H2
O
AD
P+P
i
O
D-Cycloserine
H
O
O
H
A
T
P
H
O
D
A
PF
UDP MurNac
AlaGlu
ddl
H
N
D-A
la+D-A
la
L-A
la
A
lanineRacem
ase
N
H
O
H
O
O
H
N
O
O
L-lys, ATP
AD
P+P
o
rm
esoD
AP
i
H
O
H
N
N
H
O
O
O
O
O
O
N
H
O H
O
O
O
H
H
O
O
O
O
PO P
O
O
O
H
N
m
urE
H
N
O
m
urF
O
UDP MurNacAla
GluLys/DAPAlaAla
N
O
H
N
O
O
H
O
O
H
H
O O
O
O O
O
O
H
P
P
O N
O O
H
O
H
N
O
O
H
N
O
m
urI
H
O
O
H
O
H
O
4-[(2-napthyl)methyl]
-D-Glutamate
O
O
H
H
O O
O
O O
O
P
P
N
O
O O
H
N
O
O
H
O
Fosfomycin
L-Ala, ATP
O
O
O
UDP MurNacAla
L,L-D
iam
inopim
elate
(D
AP
)
O
Peptidoglycan Synthesis - The Essentials
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
Membrane bound intracellular Steps of Peptidoglycan Synthesis
(Streptococcus pneumonaie example)
(Lipid I-Lys)
(Lipid
II-Lys)
Lipid 2
Lipid 1
P
undecaprenyl
phosphate
COOH
COOH
D-Ala
NH
O
D-Ala
HN
CYTOPLASM
O
UMP
UDP-MurNAcpentapeptide
L-Lys
MraY
O
D-Glu
P
COOH
MurG
UDPGlcNAc
L-Lys
Undecaprenyl
D-Glu
Phosphate
L-Ala
MurNAc
NH
H2 N
D-Ala
D-Ala
P
P
H2 N
D-Ala
O
D-Ala
NH
D-Ala
MurM
D-Ala
D-Ala
COOH
HN
CYTOPLASMIC FACE OFO THE
L-Ala
CELL MEMBRANE
O
L-Ala
NH
NH
O
O
O
HN
HO
O
O
P
P
O
O
OH O
O HO
OH
MurNac
D-Ala
D-Ala
Ser-tRNA
Ala-tRNA
D-Ala
L-Lys Ser-Ala
L-Lys Ser
Undecaprenyl
D-Glu
D-Glu
O
Phosphate
L-Lys
L-Ala
L-Ala
NH
MurNAc GlcNAc
MurNAc GlcNAc
MurNAc
GlcNAc
O
P
P
P
D-GluP
P
P
L-LysHN
D-Glu
L-Ala
HN
O
MurN
GlcNac
O
O
HN
O
HO
HN
O
HO
HO
O
O
O
P
P
O
O
OH O
O HO
OH
MurNac
Peptidoglycan Synthesis - The Essentials
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
Membrane bound extracellular Steps of Peptidoglycan Synthesis
(Example: Streptococcus pneumoniae: Ser-Ala)
CELL MEMBRANE,
CELL MEMBRANE,
EXTRACELLULAR
EXTRACELLULAR
FACE
FACE
P
P
P
P
P
P
Penicillinbinding
binding
Penicillin
protein
protein
carboxy-peptidase
transpeptidase
P
P
P
P
P
P
P
P
MurNAc GlcNAc MurNAc GlcNAc
MurNAc GlcNAc
MurNAc GlcNAc MurNAc GlcNAc
MurNAc GlcNAc
transL-Ala
L-Ala
L-Ala
transL-Ala
L-Ala
L-Ala
glycosylase
D-Glu
D-Glu
D-Glu
glycosylase
D-Glu
D-Glu
D-Glu
L-Lys Ser-Ala
L-Lys Ser-Ala
L-Lys Ser-Ala
L-Lys Ser-Ala
L-Lys Ser-Ala
L-Lys Ser-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
EXTRACELLULAR
EXTRACELLULAR
SPACE
SPACE
D-Ala
D-Ala
-MurNAcGlcNAc
GlcNAcMurNAc
MurNAc GlcNAcGlcNAc-MurNAc
L-Ala
L-Ala
L-Ala
L-Ala
D-Glu
D-Glu
D-Glu
D-Glu
L-Lys Ser-Ala
L-LysSer-Ala
Ser-Ala
L-Lys
L-Lys
D-Ala Ser-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
Penicillinbinding
binding
Penicillin
protein
protein
Trans-peptidase
carboxy-peptidase
D-Ala
D-Ala
-MurNAcGlcNAc
GlcNAcMurNAc
MurNAc GlcNAcGlcNAc-MurNAc
L-Ala
L-Ala
L-Ala
L-Ala
D-Glu
D-Glu
D-Glu
D-Glu
L-Lys
L-Lys Ser-Ala
L-Lys
Ser-Ala L-Lys Ser-Ala
Ser-Ala
D-Ala
D-Ala
D-Ala
D-Ala
The antibiotic targets of the lipid-linked steps of
peptidoglycan synthesis
tunicamycin,
mureidomycin A,
liposidomycin B
Lipid-linked steps
of peptid oglycan
assembly
UDP-Mur NAcpentapept ide
D-Ala
D-Ala
L-Lys
D-Glu
L-Ala
UMP
MraY
CYT OPLA SM
ramoplanin
MurNAc
P
P
CELL SURFACE
amphomycin
P
undecapre nyl
phosphate
UDPGlcNAc
P
P
P
P
MurG
bacitracin
peptidogly can
cross-linking
D-Ala
D-Ala
L-Lys
D-Glu
L-Ala
MurNAc GlcNAc
P
P
P
P
MurNAc GlcNAc MurNAc GlcNAc
L-Ala
L-Ala
D-Glu
D-Glu
L-Lys Ala-Ser
L-Lys Ala-Ser
Ser-Ala
Ser-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
L-Lys Ala-Ser
Ser-Ala
D-Glu
L-Ala
MurNAc GlcNAc
P
P
MurM/N
Ala-tRNA
Ser-tRNA
P
P
MurNAc GlcNAc
transglycosylase
L-Ala
D-Glu
L-Lys Ala-Ser
Ser-Ala
D-Ala
D-Ala
moenomycin
penicillins (b-lactams)
vancomycin
(glycopeptides)
Summary
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 lipidsugar linked pentapeptide precursor
4) The third phase on the extracellular face of the cell membrane polymerises
the lipid sugar to form the peptidoglycan
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.
Part 2
Mechanisms of Action of and
resistance to Cell-Wall Directed
Antibiotics
1) The b-lactams
H H
N
Ph
O
OCH3 OCH3
H
S
N
H H
N
O
O
CO2H
Penicillin G
H
S
N
O
CO2H
Methicillin
Membrane bound Extracellular Steps of
Peptidoglycan Synthesis
Staphylococcus
aureus
Enterococcus
faecicum
Streptococcus
pneumoniae
EXTRACELLULAR FACE
CELL MEMBRANE,
MEMBRANE, EXTRACELLULAR
FACE
CELL
P
P
P
P
P
P
Penicillin binding
binding
Penicillin
protein
protein
transpeptidase
transpeptidase
P
P
P
P
MurNAc GlcNAc
GlcNAc MurNAc
MurNAc GlcNAc
GlcNAc
MurNAc
transL-Ala
L-Ala
transL-Ala
L-Ala
glycosylase
D-Glu
D-Glu
glycosylase
D-Glu
D-Glu
D-Asn
D-Asn
(Gly)
(Gly)
L-Lys Ser-Ala
L-Lys Ser-Ala
5
5
L-Lys
L-Lys
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
EXTRACELLULAR
EXTRACELLULAR
SPACE
SPACE
D-Ala
D-Ala
-MurNAc GlcNAc
GlcNAc
-MurNAc
L-Ala
L-Ala
D-Glu
D-Glu
L-Lys
L-Lys
D-Asn
(Gly)5
D-Ala Ser-Ala
D-Ala
D-Ala
D-Ala
MurNAc GlcNAcGlcNAcMurNAc
L-Ala
L-Ala
D-Glu
D-Glu
L-Lys (Gly)
5
L-Lys
Ser-Ala
D-Ala
D-Ala
P
P
P
P
MurNAc GlcNAc
GlcNAc
MurNAc
L-Ala
L-Ala
D-Glu
D-Glu
(Gly)
D-Asn
L-Lys Ser-Ala
5
L-Lys
D-Ala
D-Ala
D-Ala
D-Ala
Penicillin binding
binding
Penicillin
protein
protein
carboxy-peptidase
carboxy-peptidase
D-Ala
D-Ala
-MurNAc GlcNAc
GlcNAc
-MurNAc
L-Ala
L-Ala
D-Glu
D-Glu
L-Lys
L-Lys
(Gly)5
D-Asn
Ser-Ala
D-Ala
D-Ala
MurNAc GlcNAcGlcNAcMurNAc
L-Ala
L-Ala
D-Glu
D-Glu
L-Lys Ser-Ala
(Gly)5
L-Lys
D-Ala
D-Ala
PbPs are Multimodular and Multifunctional enzymes
Class A
Class B
S. aureus MecA (PbP2a): Monofunctional
Transpeptidase (TP)
TP
Unknown N-terminal
function
Linker
S. aureus PbP2 bifunctional Transpeptidase (TP)/Transglycosylase (TG)
TP
Linker
S398
TG
E171;E114
S403
Cytoplasm
N
Cytoplasm
N
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 GlcNAc
-
MurNAc GlcNAc
MurNAc GlcNAc
1
2
3
4
-
O 2C
L-Ala
D-Glu
L-Lys
D-Ala
NH
5 D-Ala
O
1
2
3
4
O
H
NH3+
5
NH
L-Ala
Transpeptidase
D-Ala
L-Lys
D-Ala
H2N
O
MurNAc GlcNAc
NH3+
Ser
Lys
O
4
L-Lys 3
D-Glu 2
L-Ala 1
D-Glu
O
5
O2 C
1 L-Ala
2 D-Glu
3 L-Lys
4 D-Ala
O
H
MurNAc GlcNAc
1 L-Ala
2 D-Glu
3 L-Lys
4 D-Ala
CO2-
H
tetrapeptide
sidechain
D-Ala
N
H
D-Ala
L-Lys
D-Glu
L-Ala
NH3+
Lys
D,D-carboxypeptidase
Lys
O
Ser
H
Ser
O
cross-linked
peptidoglycan
5
4
3
2
1
MurNAc GlcNAc
b-Lactam Antibiotics
65% world market of antibiotics
>50 marketed drugs of this class
H H
Penicillins
N
Cephalosporins
Ph
Carbapenems
O
Monobactams
Cephalosporin-penicillin hybrids, O
Penems
H
S
N
CO2H
Strained, reactive b-lactam ring
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 Substrates
Antimicrobial 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….
H
H H N
COR
H
S
H3C
H3C
N
O
O
-
O2C
H
H3C
H3C
-
NH3+
H
S
H
N COR
O
NH
O2C
O
NH2
Ser
Ser
Lys
Lys
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
Antibiotic Inactivation
b-lactamases
Principally Gram negative enteric and
Pseudomonad pathogens, exception:
Staphylococcus aureus
Target Modification
PbP Remodelling
Principally Gram positive pathogens,
e.g. Streptococcus pneumoniae
PbP Re-aquisition
Principally Gram positive pathogens,
e.g. MRSA, PbP2a
Bacillus lichiniformis
b-lactamaseInactivation
Streptomyces D,D, carboxypeptidase
Antibiotic
b-Lactamases- Like PbPs but not
H
H H N COR
S
H3C
H3C
-
N
O2C
O
O
H
H3C
H3C
S
H
H HN
COR
O
NH
O
-
O2C
NH3+
H2O
NH2
Ser
H3C
b-lactamases H3C
Ser
Lys
Lys
S
H
H HN
COR
CO2-
NH
OH
-
O2C
NH3
+
Ser
Lys
PBP's
irreversible acylation
(or slow hydrolysis)
b-lactamases evolved from PbPs
Developed catalytic apparatus to hydrolyse the b-lactam ring in a
manner analogous to the mechanism of PbP carboxypeptidase
hydrolysis
Target Modification
Global clonal spread of penicillin resistant pneumococci
1984‘86 ‘88 ‘90 ‘92 ‘94 ‘96 ‘98 2000
Serotype 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
Transpeptidase Domain
Penicillin
sensitive
strains
(mic 0.02 mg/ml)
pbp2x
Ser
A
B
Penicillin
resistant
strains
C
(mic ≤16 mg/ml)
D
E
F
Generation of a penicillin-resistant
pneumococcal PbP2x by homologous recombination
K-[T/S]-G
K-[T/S]-G
S-X-X-K
S-X-X-K
341
341
A337
Thr337
338338
[S/Y]-X-[N/C]
[S/Y]-X-[N/C]
Generalised
active of
site
a PbP
with
aminoacids
acids from
from penicillin
Generalised active
site scaffold
a of
PbP
with
amino
penicillin
Sensitive PbP2x from S. pneumoniae R6 superimposed upon it
Resistant PbP2x
from S. pneumoniae Sp328 superimposed upon it
PbP2x crystal structure reveals penicillin
resistance 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
Antibiotics
2) Vancomycin and other Glycopeptides
O
Me
OH
NH2
HO
HOCH2
Me
O
O
O
HO
O
H
N H
OH
O
H
N
H
-O C
2
Cl
Cl
HO
O
O
H
H
H
N
O
O
H
N
O
H
N
H
H
O
NH2
HO
OH
HO
Vancomycin
N
H
O
Me
NH+
2
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.
• Contraindications
Deafness, 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
s
UDP-Mur NAcpentapept ide
UMP
D-Ala
D-Ala
L-Lys
D-Glu
L-Ala
D-Ala
D-Ala
L-Lys
D-Glu
L-Ala
MurNAc GlcNAc
P
P
MurG
•Vancomycin is not an enzyme
inhibitor.
MraY
PLA SM
MurNAc
UDPGlcNAc
MurM/N
Ala-tRNA
Ser-tRNA
D-Ala
D-Ala
L-Lys Ala-Ser
D-Glu
L-Ala
MurNAc GlcNAc
P
P
•Vancomycin
bindsPP to the D-alanyl-D-alanine termini of peptidoglycan
P
units prior their incorporation in the cell wall:
P
P
P
URFACE
Extracellular surface
yl
peptidogly can
cross-linking
P
P
MurNAc GlcNAc MurNAc GlcNAc
L-Ala
L-Ala
D-Glu
D-Glu
L-Lys Ala-Ser
L-Lys Ala-Ser
D-Ala
D-Ala
D-Ala
D-Ala
P
P
MurNAc GlcNAc
transglycosylase
L-Ala
D-Glu
L-Lys Ala-Ser
D-Ala
D-Ala
Vancomycin (glycopeptides)
•By doing so, it prevents transpeptidation reactions from crosslinking
adjacent peptidoglycan chains, weakening the cell wall leading to
osmotic stress and lysis
Vancomycin: Targets the D-Ala-D-Ala Terminus
Extracellular Peptidoglycan Precursors
VANCOMYCIN
Me
O
OH
NH2
HO
HOCH2
Me
O
O
O
HO
O
Cl
HO
H
-O C
2
H
N H
OH
O
H
N
O
H
H
N
H
O
O
H
N
H
O
N
H
H
O
NH2
HO
OH
HO
R
Mr=1805
Cl
O
H Me
O
N
H
O-
H
N
O
O
H Me
N-Ac-D-Ala-D-Ala
N
H
O
Me
NH+
2
Emergence of Vancomycin
Resistance
Reduction of [Antibiotic]
at site of Action
Target Modification
“Visa”:
Peptidoglycan
Remodelling
Vancomycin-intermediate resistant
Staphylococcus aureus – resistance by
decreased permeability using a
thicker peptidoglycan layer
mic: ≥16 mg/ml (Sensitive: 0.02 mg/ml)
Principally Gram positive pathogens,e.g.
Enterococci and more recently (2002)
Staphylococcus aureus (“VRSA”)
mic: ≥500 mg/ml
Target Modification mediated Mechanisms
of Vancomycin Resistance
VANCOMYCIN
O
VANCOMYCIN
OH
Me
O
OH
Me
NH2
NH2
HO
HOCH2
H
O
O
H
N H
H
H
N
H
H
N
H
O
N
H
H
N
H
O
NH2
OH
HO
R
H Me
O
N
H
H
N
NH+
-O C
2
H
H
N H
H
H
N
H
H
O
H Me
N-Ac-D-Ala-D-Ala
Vancomycin sensitive
H
N
H
N
H
H
Me
NH2+
O
O
NH2
OH
HO
O-
O
O
N
HO
O
O
Me
2
O
HO
O
OH
O
H
N
O
O
Cl
O
Cl
HO
OH
O
H
N
-O C
2
Cl
Cl
HO
O
HO
O
Me
O
O
O
HO
O
HO
HOCH2
Me
O
O
R
O-
H Me
O
O
N
H
O
O
H Me
N-Ac-D-Ala-D-Lactate
Vancomycin Resistant
1000-fold drop in affinity of
vancomycin for its target
Vancomycin Resistance; Simple and elegant in principle,
Loss of a single hydrogen bonding interaction by interconverting DAlanine to D-lactate at the end of the peptidoglycan peptide eliminates the
interaction of vancomycin with its target
……..complex in execution
In Gram positive pathogens such as Enterococcus faecalis and Enterococcus
faecicum
Vancomycin 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.
……mechanism to spread between pathogens
Transposon encoded high-level vancomycin resistance operon. Has
been transferred from an Enterococcus to S. aureus in a clinical setting
!!!!!!!!!
Sensing and initiation of gene expression
leading to Enterococcal Vancomycin Resistance
?
Cell membrane
VanS
PO4
Cytoplasm
PO4
VanR
VanR
PvanR
vanR
vanS
Response
regulator
Sensor
Regulation
PvanH
vanH
vanA vanX
D-lactate
producing
reductase
D-Ala-D-lac D-Ala-D-Ala
ligase
dipeptidase
Resistance
Precursors of Target Modification required for
High Level Vancomycin Resistance
No Vancomycin
+Vancomycin
CO2
O
Pyruvate
NADPH
VanH
NADP+
+
H3N
OH
D-Alanine
O
VanX
CO2
HO
D-Ala + ATP
DDL
D-Lactate
D-Ala + ATP
VanA
ADP + Pi
+
H3N
O
N
H
CO2
D-Alanyl-alanine
ADP + Pi
+
H3N
O
CO2
O
D-Alanyl-D-lactate
Mechanism of Target Modification required for
High Level Vancomycin Resistance
MurA to MurE
UDPmurNac
AlaGluLys/DAP/Ala-Lac
HO
N
OH
O
O
OH
HO
O
O
O
P O P
O
O
OH
HO
OH
O
OH
N
N
O
N
O
O
OH
HO
O
O
O
P O P
O
O
OH
O
OH
OH
N
O
N
O
O
O
OH
HO
O
O
O
P O P
O
O
OH
O
OH
N
O
N
HN
O
O
HN
O
O
O
m urF
NH
O
HO
O
N
HN
O
UDPmurNac
AlaGluLys/DAP/AlaAla
UDPmurNac AlaGluLys/DAP
NH
O
O
m urF
NH
O
HO
HO
O
HO
O
HN
HO
D-Ala-D-Lac,
ADP + Pi ATP
O
O
OH
VanA
ADP + Pi
D-Ala-D-Lac
Ligas e
HO
O
NH2
O
NH
O
O
ATP
D-Ala + D-Lac
O
D-Ala-D-ALa
NH2
O
HO
HN
HO
VanX:
D-Ala-D-Ala
Dipe ptidas e
ATP
NH2
O
NH
D-Ala + D-Ala
ADP + Pi
D-Ala-D-Ala
Ligas e
O
HN
HO
O
Van H:
D-Lactate
de hydroge nas e
NAD+
NADH
Py ruv ate
D-Ala-D-Lac peptidoglycan:
Vancomycin Resistance
O
HN
D-Ala-D-Ala peptidoglycan:
Vancomycin sensitivity
Summary – Vancomycin Sensitivity
Ala
Ala
Vancomycin sensitive
Ala
Ala
Ala
Ala
Vancomycin sensitive
Ala
Ala
Cell Death
Summary-Vamcomycin Resistance
Ala
Ala
Vancomycin Resistant
Ala
Lac
Lac
Ala
Vancomycin Resistant
Operon on
Ala
Ala
Ala
Lac
Cell Survival
Overall Summary
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.
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-alanylD-lactate. This is relatively complex to achieve, requiring re-programming of the
synthesis of the peptidoglycan.
References
Bacterial 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 resistance
4.
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 specific
A 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.