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
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
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ser-Ala
PP
P
MurG
UDPGlcNAc
MurMSer-tRNA
UDP-MurNAc-pentapeptide
UMP
MraY
P
CYTOPLASM
undecaprenylphosphate
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
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)
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
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ser-Ala
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ser-Ala
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
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
MurNAcL-AlaD-GluL-Lys
GlcNAc-
Ser-Ala
Penicillin binding proteincarboxy-peptidase
D-Ala D-Ala
Membrane bound extracellular Steps of Peptidoglycan Synthesis(Example: Streptococcus pneumoniae: Ser-Ala)
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ser-Ala
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ser-Ala
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
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 proteincarboxy-peptidase
D-Ala
-MurNAcL-AlaD-GluL-LysD-Ala
GlcNAc
Ser-Ala
MurNAcL-AlaD-GluL-Lys
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
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ala-Ser
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ala-Ser
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ala-Ser
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
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
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ser-Ala
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ser-Ala
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ser-Ala
PP
P
trans-glycosylase
EXTRACELLULARSPACE
CELL MEMBRANE, EXTRACELLULAR FACE
-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
MurNAcL-AlaD-GluL-Lys
GlcNAc-
Ser-Ala
Penicillin binding proteincarboxy-peptidase
D-Ala D-Ala
Membrane bound Extracellular Steps of Peptidoglycan Synthesis
Streptococcus pneumoniae
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
(Gly)5
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
PP
P
trans-glycosylase
EXTRACELLULARSPACE
CELL MEMBRANE, EXTRACELLULAR FACE
-MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc MurNAcL-AlaD-GluL-Lys
GlcNAc-
D-Ala
Penicillin binding proteintranspeptidase
D-Ala
-MurNAcL-AlaD-GluL-LysD-Ala
GlcNAc MurNAcL-AlaD-GluL-Lys
GlcNAc-Penicillin binding proteincarboxy-peptidase
D-Ala D-Ala
(Gly)5 (Gly)5
(Gly)5(Gly)5(Gly)5
Staphylococcus aureus
(Gly)5
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
PP
P
trans-glycosylase
EXTRACELLULARSPACE
CELL MEMBRANE, EXTRACELLULAR FACE
-MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc MurNAcL-AlaD-GluL-Lys
GlcNAc-
D-Ala
Penicillin binding proteintranspeptidase
D-Ala
-MurNAcL-AlaD-GluL-LysD-Ala
GlcNAc MurNAcL-AlaD-GluL-Lys
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-carboxy- peptidase
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)
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ala-Ser
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ala-Ser
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
GlcNAc
Ala-Ser
PP
MurNAcL-AlaD-GluL-LysD-AlaD-Ala
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
Ala
AlaAla
Ala
Ala
AlaAla
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AlaAla
AlaAla
AlaAla
Ala Al
aAl
aAl
a
Ala
AlaAla
AlaVancomycin sensitive
Ala
Ala
Ala
AlaAla
Ala
Ala
AlaAla
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AlaAla
AlaAla
AlaAla
Ala Al
aAl
aAl
a
Ala
AlaAla
Ala
Cell Death
Summary – Vancomycin Sensitivity
Vancomycin Resistant
Ala
Ala
Ala
AlaAla
Ala
Ala
AlaAla
Ala
Ala
Ala
Ala
Ala
Ala
Ala
AlaAla
AlaAla
AlaAla
Ala Al
aAl
aAl
a
Ala
AlaAla
Ala
Vancomycin Resistant
Ala
Lac
Ala
LacAla
Lac
Ala
LacAla
Lac
Ala
Lac
Lac
Ala
Lac
Ala
LacAla
LacAla
LacAla
Lac Al
a
Lac
Ala
Lac
AlaLacAla
Lac
Ala
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.