Life Death and hydrogen bonds:

1. 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

2. Bacterial pathogens with multiple antibiotic resistance phenotypes Staphylococcus aureus Enterococcus faecalis Mycobacterium tuberculosis Klebsiella pneumoniae Pseudomonas aeruginosa Acinetobacter spp. http://www.denniskunkel.com (2007)

3. 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)

4. 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

5. 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

6. 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

7. Antibiotics: The Targets Protein Synthesis Intermediary Metabolism DNA Replication Cell Wall (Peptidoglycan) Synthesis

8. 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

9. 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

10. Part 1 Peptidoglycan: Structure, Function, Synthesis and Target

11. OM PG PG CM CM • Gram-positive • Gram-negative Peptidoglycan position in Gram-positive and Gram-negative bacteria

12. Electron micrograph of a cross section of the Escherichia coli Cell Wall

13. 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)

14. 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)

15. NAM = N-acetyl muramic acid NAG = N-acetyl glucosamine

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

17. 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

18. 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

19. 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

20. 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

21. 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

22. (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)

23. 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

24. C E L L M E M B R A N E , E X T R A C E L L U L A R F A C E P P P P P P P M u r N A c G l c N A c M u r N A c G l c N A c M u r N A c G l c N A c t r a n s - L - A l a L - A l a L - A l a g l y c o s y l a s e D - G l u D - G l u D - G l u L - L y s S e r - A l a L - L y s S e r - A l a L - L y s S e r - A l a D - A l a D - A l a D - A l a P e n i c i l l i n b i n d i n g D - A l a D - A l a D - A l a p r o t e i n t r a n s p e p t i d a s e E X T R A C E L L U L A R S P A C E D - A l a P e n i c i l l i n b i n d i n g - M u r N A c G l c N A c M u r N A c G l c N A c - p r o t e i n - M u r N A c G l c N A c M u r N A c G l c N A c - L - A l a L - A l a c a r b o x y - p e p t i d a s e L - A l a L - A l a D - G l u D - G l u D - G l u D - G l u L - L y s L - L y s S e r - A l a L - L y s L - L y s S e r - A l a S e r - A l a S e r - A l a D - A l a D - A l a D - A l a D - A l a D - A l a D - A l a Membrane bound extracellular Steps of Peptidoglycan Synthesis (Example: Streptococcus pneumoniae: Ser-Ala)

25. 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

26. 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.

27. Part 2 Mechanisms of Action of and resistance to Cell-Wall Directed Antibiotics 1) The b-lactams Penicillin G Methicillin

28. Membrane bound Extracellular Steps of Peptidoglycan Synthesis Enterococcus faecicum Staphylococcus aureus C E L L M E M B R A N E , E X T R A C E L L U L A R F A C E C C E E L L L L M M E E M M B B R R A A N N E E , , E E X X T T R R A A C C E E L L L L U U L L A A R R F F A A C C E E P P P P P P P P P P P P P P M M u u r r N N A A c c G G l l c c N N A A c c M M u u r r N N A A c c G G l l c c N N A A c c M M u u r r N N A A c c G G l l c c N N A A c c t t r r a a n n s s - - L L - - A A l l a a L L - - A A l l a a L L - - A A l l a a g g l l y y c c o o s s y y l l a a s s e e D D - - G G l l u u D D - - G G l l u u D D - - G G l l u u D-Asn D-Asn D-Asn (Gly)5 (Gly)5 (Gly)5 L L - - L L y y s s L L - - L L y y s s L L - - L L y y s s D D - - A A l l a a D D - - A A l l a a D D - - A A l l a a P P e e n n i i c c i i l l l l i i n n b b i i n n d d i i n n g g D D - - A A l l a a D D - - A A l l a a D D - - A A l l a a p p r r o o t t e e i i n n t t r r a a n n s s p p e e p p t t i i d d a a s s e e E E X X T T R R A A C C E E L L L L U U L L A A R R S S P P A A C C E E D D - - A A l l a a P P e e n n i i c c i i l l l l i i n n b b i i n n d d i i n n g g - - M M u u r r N N A A c c G G l l c c N N A A c c M M u u r r N N A A c c G G l l c c N N A A c c - - p p r r o o t t e e i i n n - - M M u u r r N N A A c c G G l l c c N N A A c c M M u u r r N N A A c c G G l l c c N N A A c c - - L L - - A A l l a a L L - - A A l l a a c c a a r r b b o o x x y y - - p p e e p p t t i i d d a a s s e e L L - - A A l l a a L L - - A A l l a a D D - - G G l l u u D D - - G G l l u u D D - - G G l l u u D D - - G G l l u u L L - - L L y y s s (Gly)5 L L - - L L y y s s L L - - L L y y s s L L - - L L y y s s D-Asn (Gly)5 (Gly)5 D-Asn (Gly)5 D D - - A A l l a a D D - - A A l l a a D D - - A A l l a a D D - - A A l l a a D D - - A A l l a a D D - - A A l l a a Streptococcus pneumoniae P P P P P P P M u r N A c G l c N A c M u r N A c G l c N A c M u r N A c G l c N A c t r a n s - L - A l a L - A l a L - A l a g l y c o s y l a s e D - G l u D - G l u D - G l u L - L y s S e r - A l a L - L y s S e r - A l a L - L y s S e r - A l a D - A l a D - A l a D - A l a P e n i c i l l i n b i n d i n g D - A l a D - A l a D - A l a p r o t e i n t r a n s p e p t i d a s e E X T R A C E L L U L A R S P A C E D - A l a P e n i c i l l i n b i n d i n g - M u r N A c G l c N A c M u r N A c G l c N A c - p r o t e i n - M u r N A c G l c N A c M u r N A c G l c N A c - L - A l a L - A l a c a r b o x y - p e p t i d a s e L - A l a L - A l a D - G l u D - G l u D - G l u D - G l u L - L y s L - L y s S e r - A l a L - L y s L - L y s S e r - A l a S e r - A l a S e r - A l a D - A l a D - A l a D - A l a D - A l a D - A l a D - A l a

29. 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

31. 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

32. 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

33. Shared spatial structure of the terminal D-Ala-D-Ala terminus of the peptidoglycan pentapeptide and b-lactams b-lactamring

34. 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

35. 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

36. 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

37. 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 Target Modification Global clonal spread of penicillin resistant pneumococci

38. 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

39. Generation of a penicillin-resistant pneumococcal PbP2x by homologous recombination

40. 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

41. 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

42. Vancomycin Mechanisms of Action of, and resistance to Cell-Wall Directed Antibiotics 2) Vancomycin and other Glycopeptides

43. 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.

44. 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

45. Vancomycin: Targets the D-Ala-D-Ala Terminus Extracellular Peptidoglycan Precursors Mr=1805

46. “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

47. Target Modification mediated Mechanisms of Vancomycin Resistance Vancomycin sensitive Vancomycin Resistant 1000-fold drop in affinity of vancomycin for its target

48. 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 !!!!!!!!!

49. ? 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

50. Precursors of Target Modification required for High Level Vancomycin Resistance No Vancomycin +Vancomycin