The antibiotics
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1. A brief history of the fight against pathogenic microorganisms
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Germ theory of disease Microorganisms are the cause of many infectious diseases Tuberculosis Anthrax Cholera Plague
Robert KOCH 1843-1910
Mycobacterium tuberculosis Bacillus anthracis Vibrio cholerae Yersinia pestis
Louis PASTEUR 1822-1895
1877 1882 1883 1894
Joseph LISTER 1827- 1912
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How to kill the germs (without killing the patient) ?
Kills 99,99% of germs
Kills 99,99999% of germs in 30 seconds 4
How to kill the germs (without killing the patient) ? "If we picture an organism as infected by a certain species of bacterium, it will . . . be easy to effect a cure if substances have been discovered which have a specific affinity for these bacteria and act…on these alone. . . while they possess no affinity for the normal constituents of the body. . . such substances would then be . . . magic bullets" Molecular interaction "corpora non agunt nisi fixata" Things do not interact unless they make contact The beginning of chemotherapy
Paul ERLICH 1854-1915 5
Synthetic antimicrobial agents (1) P. Erlich
Selective toxicity
Selective dyes?
Trypan blue active against trypanosomes that cause sleeping sickness
Arsphenamine (Salvarsan) active against Treponema pallidum that causes syphyllis
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Synthetic antimicrobial agents (2) G. Domagk 1895 - 1964
Prontosil
Sulfanilamide
4-[(2,4-diaminophenyl)azo]benzenesulfonamide
Active against Gram+ cocci, e.g. Streptococci 7
Natural products with antimicrobial activity (1) Antibiosis: an association between two populations of organisms that is detrimental to one of them (by opposition to symbiosis). Observed between microorganisms (Joubert, Villemin Pasteur 1889) Ernest DUCHESNE Contribution to the study of vital competition in micro-organisms: antagonism between moulds and microbes Ph. D. thesis 1897 Antagonism between Penicillium glaucum and various - bacteria
Ernest DUCHESNE 1874-1912
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Natural products with antimicrobial activity (2) Inhibition of Staphylococci by Penicillium notatum (Fleming, 1928)
Purification and characterization of penicillin (H. Florey, E. Chain, 1940)
penicillin
Alexander FLEMING 1881 - 1955
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Natural products with antimicrobial activity (3) Dubos based his early experiments on the principle of “antibiosis” and “the supremely simple working hypothesis that soil as a self-purifying environment could supply an agent to destroy disease-causing bacteria”. In 1939, Dubos isolated gramicidin the first natural antibiotic discovered through a deliberate, systematic search for antibacterial compounds and the first clinically tested antibiotic agent.
Gramicidin produced by Bacillus brevis Active against Gram+ bacteria
René DUBOS 1901-1982
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Natural products with antimicrobial activity (4) Systematic search for antibacterial compounds produced by soil bacteria
actinomycin D 1940
streptomycin 1943
Selman WAKSMAN 1888 - 1973 11
Definition(s) of "antibiotic" An antibiotic is a chemical substance, produced by microorganisms, which has the capacity to inhibit the growth and even to destroy bacteria and other microorganisms, in dilute solutions. (Waksman, 1951) An antibiotic is a substance produced by microorganisms or a synthetic or semisynthetic compound, which kill or prevent the growth of other microorganisms.
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The "golden age" of antibiotic
(Hopwood D., 2007, in: Streptomyces in nature and 13 medicine. The antibiotic makers. Oxford Ed.)
The "golden age" of antibiotic
It’s time to close the book on infectious diseases, declare the war against pestilence won, and shift national resources to such chronic problems as cancer and heart disease. William H. STEWART, US Surgeon General, 1969
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The end of "golden age"
ASM Press 2005
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Use (and misuse) of antibiotics: selection of resistant strains
Recognizing bacteria as evolving entities and understanding their evolution should help us to control that evolution, allowing us to prolong the useful lifespan of antibiotics.
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2. The antibiotics Classification Mode of action Resistance
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Classification(s) of antibiotics Several thousands of antibiotics About 100 used in human medicine Different classifications: - whether they are bacteriostatic or bactericidal - according to their mode of synthesis natural products, products obtained by hemi-synthesis, products obtained by total chemical synthesis
- according to their chemical structure - according to their target
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Classification by chemical structure (1) Aminoglycosides (streptomycine, gentamicin…) Beta-lactams (penicillins, cephalosporins…) Peptides (Gramicidin…) Glycopeptides (Vancomycin…) Lipopeptides (Daptomycin…)
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Classification by chemical structure (2) Macrolides (Erythromycin, spiramycin…) Lincosamides (Lincomycin, clindamycin…) Streptogramins (Pristinamycin…) Cyclines (Tetracyclin…) Phenicols (chloramphenicol…) Rifamycins (Rifampicin…) ….
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Classification by chemical structure (3) Sulfonamides (Sulfanilamide, Sulfadiazine…) Quinolones (Nalidixic acid…) Fluoroquinolones (Ciprofloxacin…) Oxazolidinones (Linezolid…) ….
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Classification by target (1) Peptidoglycan biosynthesis Beta-lactams, glycopeptides Membrane function Peptides, lipopeptides Protein synthesis Aminogycosides, macrolides, lincosamides, streptogramines, phenicols, cyclines, oxazolidinones
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Classification by target (2) RNA synthesis Rifamycins DNA Quinilones, fluoroquinolones Biosynthesis of folic acid Sulfonamides
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Antibiotic resistance
Intrinsic resistance Acquired resistance (mutation, gene acquisition)
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Mechanisms of antibiotic resistance 1 impermeability, efflux 2 inactivation of the antibiotic 3 target modification or substitution
Antibiotic
1 2
Target
3
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Dissemination of antibiotic resistance
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3. The biosynthesis of antibiotics
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Streptomyces: the antibiotic makers A single group of bacteria, the Streptomyces, synthesizes about half of all antibiotics and is at the origin of about two thirds of those used in medicine.
Streptomyces also synthesize other bioactive natural products which are used in medicine (as immuno-suppressant, anti-tumoral drug…) or in agriculture (as herbicide, insecticide..)
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Streptomyces Bacteria Actinobacteria (Gram+, high GC) Actinobacteridae Actinomycetales Streptomycineae Streptomycetaceae Streptomyces
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Streptomyces Actinomycetales Corynebacterineae Corynebacterium, Mycobacterium
Frankineae Frankia
Streptomycineae Streptomycetaceae Streptomyces > 400 species
Kitasatospora 30
Streptomyces morphological differenciation Streptomyces are Gram+ filamentous and sporulating soil bacteria.
Colonies of Streptomyces coelicolor
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Streptomyces morphological differenciation
18 h
30 h
10 h 2 days spore
3 days 5 to 10 days
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Streptomyces morphological differenciation
18 h
30 h
10 h 2 days spore
3 days 5 to 10 days
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Streptomyces morphological differenciation
18 h
30 h
10 h 2 days spore
3 days 5 to 10 days
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Streptomyces morphological differenciation
18 h
30 h
10 h 2 days spore
3 days 5 to 10 days
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Streptomyces morphological differenciation
18 h
30 h
10 h 2 days spore
3 days 5 to 10 days
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Streptomyces morphological differenciation
18 h
30 h
10 h 2 days spore
3 days 5 to 10 days
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Streptomyces morphological differenciation
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Primary and secondary metabolism
Primary metabolism: the synthesis of all the components required for growth, development and reproduction Secondary metabolism: the synthesis of compounds that are dispensable for growth and have no apparent function for the producing cell Antibiotics are secondary metabolites
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Metabolism
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Metabolism DNA Nucleoid
PO4 3-
ATP
SO4 2-
RNA
Cytosol
Amino acids
Precursors metabolites
Carbon source
Nucleotides
Proteins Sugars
Reductive Power
Ribosomes
Peptidoglycan
NH4 + Fatty acids
Lipids
Central Metabolism
Biosyntheses
Polymerizations
Envelope
Assembly
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Metabolism Secondary metabolites
DNA Nucleoid
PO4 3-
ATP
SO4 2-
RNA
Cytosol
Amino acids
Precursors metabolites
Carbon source
Nucleotides
Proteins Sugars
Reductive Power
Ribosomes
Peptidoglycan
NH4 + Fatty acids
Lipids
Central Metabolism
Biosyntheses
Polymerizations
Envelope
Assembly
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Two types of biosynthetic systems are involved in the synthesis of various secondary metabolites •Two larges families of secondary metabolites, the polyketides and the non-ribosomal peptides Polyketides
Non-ribosomal peptides H2NSNONHOHNOHOONHOHNOHNONHOHNOHNONHONHHNOOOHOH2NNH2NNH
Spiramycin Streptomyces ambofaciens
PolyKetide Synthases (PKS)
Bacitracin Bacillus subtilis
Non-Ribosomal Peptide Synthetases (NRPS) 43
Polyketides
From Hopwood Chem. Rev. 1997, 97: 2465 44
Polyketide/Fatty acid Biosynthesis
From Hopwood Chem. Rev. 1997, 97: 2465
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Polyketide/Fatty acid Biosynthesis
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PKS of type I and type II Type I (modular) PKS
Type II PKS : individual proteins for AT, KS, etc… 47
Synthesis of a polyketide by a type I PKS
from Hopwood Chem. Rev. 1997, 97: 24654 8
Synthesis of a polyketide by a type I PKS
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Synthesis of a polyketide by a type I PKS
From Hopwood Chem. Rev. 1997, 97: 2465 50
PKS as assembly lines
and NRPS too 51
Non-Ribosomal Peptide Synthetases (NRPS)
Cy
A
T
Initiation module
C
Me
A
E
T
Elongation Module
C
T
C
A
T
Te
Termination module
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Non-Ribosomal Peptide Synthetases (NRPS)
Bruner et al., Structure (2002) 10, 301-310
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PKS and NRPS
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PKS and NRPS
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4. Where will new antibiotics come from?
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The need for new antibiotics 1960
1940
1980
2000
Sulfamides 1936 β-lactams 1940 Chloramphenicol Tetracyclines 1949
Oxazolidinones 2000
Aminoglycosides 1950 Macrolides 1952 Glycopeptides 1958 Quinolones Streptogramines 1962
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New antibiotics ? - Natural Products/Chemical synthesis - Hemi-synthesis - New targets, new screens - New bacterial producing strains - Explore and exploit the unsuspected diversity of already know producers - Combinatorial biology, hybrid antibiotics - Metagenomics
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Streptomyces coelicolor – the model for a genus of antibiotic-producing bacteria
• Linear Chromosome 8.7 Mb • >7,800 predicted genes • ~250 secondary metabolism (mostly in chromosome arms) 59
Putative gene clusters for secondary metabolite biosynthesis Biosynthetic System
Metabolite
Size (kb)
Location
Type II PKS Type II PKS PKS NRPS; Type I Mod PKS NRPS NRPS NRPS NRPS Type I Mod PKS Type I Mod PKS Type I It PKS Chalcone synthase Chalcone synthase Chalcone synthase Sesquiterpene cyclase Sesquiterpene cyclase Squalene-Hopene cyclase Phytoene synthase Siderophore synthetase Siderophore synthetase Type II FAS Butyrolactone synthase Deoxysugar
Actinorhodin Grey spore pigment Methylenomycin Prodiginines CDA Coelichelin? Coelibactin? Unknown Unknown Unknown Polyunsaturated fatty acid? Tetrahydroxynaphthalene? Unknown Unknown Geosmin Unknown Hopanoids? Isorenieratine? Desferrioxamines Unknown Unknown SCB1? Unknown
22 8 20 33 80 20 26 14 70 10 19 1 3.5 1 2 2.5 15
5071-5092 5314-5320 SCP1 5877-5898 3210-3249 0489-0499 7681-7691 6429-6438 6273-6288 6826-6827 0124-0129 1206-1208 7669-7671 7222 6073 5222-5223 6759-6771 0185-0191 2782-2785 5799-5801 1265-1273 6266 0381-0401
5 4 10 1 20
Other Streptomyces genome projects Streptomyces avermitilis • Complete genome sequence published in 2003 • Up to 30 gene clusters may direct production of secondary metabolites
Streptomyces ambofaciens • Sequencing of chromosome arms in progress • Multiple gene clusters for secondary metabolite biosynthesis already identified 61
Putative secondary metabolites in the genome of Streptomyces avermitilis
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Putative secondary metabolites in the genome of Streptomyces ambofaciens TIR
TIR
Alpomycin 2-methylisoborneol
Spiramycin
Desferrioxamine
Isorenieratene ?
PKS, type II
NRPS-PKS
NRPS
PKS, type I
terpenes
Others
Coelichelin
Alpomycin
Congocidine Lantibiotic + in the central part: • SGP (Type II PKS) • Syringolide-like cluster • Hopanoids • Geosmine • Albaflavenone • DesD-like • DesD-like
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Hybrid antibiotics
CarE: acyl-transferase
Expression of carE in S. ambofaciens
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Mutasynthesis
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Combinatorial biology
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Combinatorial biology
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Metagenomics
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