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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.

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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 Sciences

www.warwick.ac.uk/go/ropergroup


Life death and hydrogen bonds

Bacterial pathogens with multiple antibiotic resistance phenotypes

Staphylococcus aureus

Enterococcus faecalis

Mycobacterium tuberculosis

Klebsiella pneumoniae

Pseudomonas aeruginosa

Acinetobacter spp.

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


Life death and hydrogen bonds

Centre for Disease Control, Al. USA

Antibiotic Resistance: Problem? What Problem?

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).

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)


Life death and hydrogen bonds

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


Life death and hydrogen bonds

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


Life death and hydrogen bonds

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


Life death and hydrogen bonds

Antibiotics: The Targets

Protein Synthesis

Intermediary Metabolism

DNA Replication

Cell Wall (Peptidoglycan) Synthesis


Life death and hydrogen bonds

Choroamphenicol

Vancomycin

Target

Modification

Antibiotic

Modification

b-lactams

Sulphon-

omides

b-lactams

Quinolones

Erythromycin

Reduction of [Antibiotic]

at site of Action (influx/Efflux)

Choroamphenicol

Tetracyclines

Quinolones

Antibiotic Resistance Mechanisms

Resistance

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


Life death and hydrogen bonds

Contents and Aims

To understand the action of cell wall directed antibiotics

and mechanisms of resistance to them, we need:

  • A working knowledge of peptidoglycan biosynthesis:Part 1

  • 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


Life death and hydrogen bonds

Part 1

Peptidoglycan: Structure,

Function, Synthesis and Target


Life death and hydrogen bonds

OM

PG

PG

CM

CM

  • Gram-positive

  • Gram-negative

Peptidoglycan position in Gram-positive and Gram-negative bacteria


Life death and hydrogen bonds

Electron micrograph of a cross section of the

Escherichia coli Cell Wall


Life death and hydrogen bonds

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;

Gram Positive Organisms

Techioic Acids,

mycolylarabinogalactan (Capsule of Mycobacterium tuberculosis)


Life death and hydrogen bonds

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)


Life death and hydrogen bonds

NAM = N-acetyl muramic acid

NAG = N-acetyl glucosamine


Life death and hydrogen bonds

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


Life death and hydrogen bonds

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


Life death and hydrogen bonds

NADP+

NADPH

Pi

PEP

UDP-MurNac

UDP-GlcNac

murB

murA

L-Ala

D-Glu

meso diaminopimelicacid (DAP) or lysine)

D-Ala-D-Ala

ATP

ADP

murC

murD

ATP

ADP

ATP

ADP

murE

ATP

ADP

murF

Gram Negative

(and a few positive)

UDP-MurNAc-pentapeptide

Gram Positive

Schematic Representation of the Cytoplasmic Phase of

Peptidoglycan Synthesis


Life death and hydrogen bonds

MurA; MurB

N-acetyl-

muramyl

Uridine 5’-

diphospho

MurC

L-alanyl

MurD

g-D-glutamyl

MurE

L-Lysyl

D-alanyl

MurF

D-alanine

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

UDP-MurNac Pentapeptide


Life death and hydrogen bonds

UDP GlcNac

Enoyl pyruvoyl

UDP GlcNac

UDP MurNac

UDP MurNacAla

4-[(2-napthyl)methyl]

-D-Glutamate

Fosfomycin

UDP MurNacAla

GluLys/DAP

UDP MurNacAla

GluLys/DAPAlaAla

UDP MurNac

AlaGlu

D-Cycloserine

Cytoplasmic Phase of Peptidoglycan Synthesis


Life death and hydrogen bonds

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


Life death and hydrogen bonds

(Lipid I-Lys)

(Lipid II-Lys)

D-Ala

D-Ala

D-Ala

D-Ala

Undecaprenyl

Phosphate

Undecaprenyl

Phosphate

L-Lys

L-Lys

D-Glu

D-Glu

L-Ala

L-Ala

GlcNac

MurNac

MurNac

Membrane bound intracellular Steps of Peptidoglycan Synthesis

(Streptococcus pneumonaie example)


Life death and hydrogen bonds

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


Life death and hydrogen bonds

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Membrane bound extracellular Steps of Peptidoglycan Synthesis

(Example: Streptococcus pneumoniae: Ser-Ala)


Life death and hydrogen bonds

tunicamycin,

mureidomycin A,

liposidomycin B

ramoplanin

Ser-Ala

Ser-Ala

Ser-Ala

Ser-Ala

amphomycin

bacitracin

moenomycin

penicillins (b-lactams)

vancomycin

(glycopeptides)

The antibiotic targets of the lipid-linked steps of

peptidoglycan synthesis


Life death and hydrogen bonds

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 lipid-sugar 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.


Life death and hydrogen bonds

Part 2

Mechanisms of Action of and

resistance to Cell-Wall Directed

Antibiotics

1) The b-lactams

Penicillin G

Methicillin


Life death and hydrogen bonds

Membrane bound Extracellular Steps of Peptidoglycan Synthesis

Enterococcus faecicum

Staphylococcus aureus

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Life death and hydrogen bonds

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

S. aureusMecA (PbP2a): Monofunctional

Transpeptidase (TP)

TP

Unknown

function

N-terminal

Linker

TP

Linker

TG

S398

E171;E114

S403

N

N

PbPsare Multimodular and Multifunctional enzymes

Class A

Class B

Cytoplasm

Cytoplasm


Life death and hydrogen bonds

Penicillin Binding Proteins (PBPs)

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

1

2

5

3

1

1

4

2

4

5

2

4

3

3

3

2

3

4

4

5

1

2

1

5

1

2

3

4


Life death and hydrogen bonds

Strained, reactive b-lactam ring

b-Lactam Antibiotics

65% world market of antibiotics

>50 marketed drugs of this class

Penicillins

Cephalosporins

Carbapenems

Monobactams

Cephalosporin-penicillin hybrids,

Penems


Life death and hydrogen bonds

Shared spatial structure of the terminal

D-Ala-D-Ala terminus of the peptidoglycan

pentapeptide and b-lactams

b-lactamring


Life death and hydrogen bonds

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….

RESULT:Inhibition of peptidoglycan crosslinking leading to a weakened

cell wall, leading to osmotic rupture of the cell membrane and cell death


Life death and hydrogen bonds

b-lactamases

PbP Remodelling

Principally Gram positive pathogens, e.g. Streptococcus pneumoniae

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

PbP Re-aquisition

Principally Gram positive pathogens, e.g. MRSA, PbP2a

Emergence of penicillin resistance

Antibiotic Inactivation

Target Modification


Life death and hydrogen bonds

Antibiotic Inactivation

Bacillus lichiniformisb-lactamase

Streptomyces D,D, carboxypeptidase

b-Lactamases- Like PbPs but not

b-lactamases evolved from PbPs

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


Life death and hydrogen bonds

1984‘86‘88‘90‘92‘94‘96‘982000

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

Target Modification

Global clonal spread of penicillin resistant pneumococci


Life death and hydrogen bonds

Penicillin

resistant

strains

(mic ≤16 mg/ml)

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

C

D

E

F


Life death and hydrogen bonds

Generation of a penicillin-resistant

pneumococcal PbP2x by homologous recombination


Life death and hydrogen bonds

K-[T/S]-G

K-[T/S]-G

S-X-X-K

S-X-X-K

341

A337

338

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

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

Generalisedactive site scaffold of a PbP with amino acids from penicillin

Resistant PbP2x from S. pneumoniae Sp328 superimposed upon it

Generalised active site of a PbP with amino acids from penicillin

Sensitive PbP2x from S. pneumoniae R6 superimposed upon it

341

HO

Thr337

338


Life death and hydrogen bonds

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 lossof 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


Life death and hydrogen bonds

Vancomycin

Mechanisms of Action of, and

resistance to Cell-Wall Directed

Antibiotics

2) Vancomycin and other Glycopeptides


Life death and hydrogen bonds

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.


Life death and hydrogen bonds

Extracellular surface

Vancomycin (glycopeptides)

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


Life death and hydrogen bonds

Vancomycin: Targets the D-Ala-D-Ala Terminus

Extracellular Peptidoglycan Precursors

Mr=1805


Life death and hydrogen bonds

“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

Emergence of Vancomycin

Resistance

Reduction of [Antibiotic]

at site of Action

Target Modification


Life death and hydrogen bonds

Target Modification mediated Mechanisms

of Vancomycin Resistance

Vancomycin sensitive

Vancomycin Resistant

1000-fold drop in affinity of

vancomycin for its target


Life death and hydrogen bonds

Vancomycin Resistance; Simple and elegant in principle,

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

……..complex in execution

In Gram positive pathogens such as Enterococcusfaecalis and Enterococcusfaecicum

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

Transposonencoded high-level vancomycin resistance operon. Has been transferred from anEnterococcusto S. aureus in a clinical setting !!!!!!!!!


Life death and hydrogen bonds

?

PO4

PO4

VanR

Response

regulator

Sensor

D-lactate

producing

reductase

D-Ala-D-lac

ligase

D-Ala-D-Ala

dipeptidase

Sensing and initiation of gene expression

leading to EnterococcalVancomycin Resistance

Cell membrane

VanS

Cytoplasm

VanR

vanR

vanS

vanH

vanA

vanX

PvanR

PvanH

Regulation

Resistance


Life death and hydrogen bonds

Precursors of Target Modification required for

High Level Vancomycin Resistance

No Vancomycin

+Vancomycin


Life death and hydrogen bonds

Mechanism of Target Modification required for

High Level Vancomycin Resistance

MurAtoMurE

UDPmurNac

AlaGluLys/DAP/Ala-Lac

UDPmurNac

AlaGluLys/DAP/AlaAla

UDPmurNacAlaGluLys/DAP

D-Ala-D-Ala peptidoglycan:

Vancomycin sensitivity

D-Ala-D-Lac peptidoglycan:

Vancomycin Resistance


Life death and hydrogen bonds

Vancomycin sensitive

Vancomycin sensitive

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Summary – Vancomycin Sensitivity

Cell Death


Life death and hydrogen bonds

Vancomycin Resistant

Lac

Lac

Lac

Lac

Lac

Ala

Ala

Ala

Ala

Lac

Ala

Lac

Ala

Ala

Ala

Ala

Lac

Ala

Ala

Ala

Lac

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Lac

Ala

Ala

Lac

Lac

Lac

Lac

Ala

Ala

Ala

Ala

Ala

Ala

Lac

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Lac

Ala

Ala

Ala

Ala

Ala

Ala

Ala

Cell Survival

Summary-Vamcomycin Resistance

Vancomycin Resistant

Operon on


Life death and hydrogen bonds

Overall Summary

  • 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. aureusMecA)

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-alanyl-D-lactate. This is relatively complex to achieve, requiring re-programming of the synthesis of the peptidoglycan.


Life death and hydrogen bonds

References

  • Bacterial antibiotic resistance and discovery:

  • 1.Walsh, C. (2000) Molecular mechanisms that confer antibacterial drug resistance, Nature406, 775-781.

  • 2.Gwynn, M. N., Portnoy, A., Rittenhouse, S. F., and Payne, D. J. (2010) Challenges of antibacterial discovery revisited, Ann N Y AcadSci1213, 5-19.

  • Agarwal, A. K., and Fishwick, C. W. (2010) Structure-based design of anti-infectives, Ann N Y AcadSci1213, 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, ChemBiol7, 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 Reviews32, 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 Biotechnol29, 167-173.

  • 7.Mattei, P. J., Neves, D., and Dessen, A. (2010) Bridging cell wall biosynthesis and bacterial morphogenesis, CurrOpinStructBiol20, 749-755.


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