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Bacterial Diseases

Bacterial Diseases. Bubonic Plague. Tuberculosis. Cholera. Sepsis. Lyme Disease. Antibiotics. 1865 – Pasteur - Decay due to living organisms. 1867 – Lister – phenol is disinfectant. 1929 – Penicillin discovered. 1933 – Sulfa drugs synthesized.

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Bacterial Diseases

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  1. Bacterial Diseases Bubonic Plague Tuberculosis Cholera Sepsis Lyme Disease Antibiotics 1865 – Pasteur - Decay due to living organisms 1867 – Lister – phenol is disinfectant 1929 – Penicillin discovered 1933 – Sulfa drugs synthesized 1969 – US surgeon Gen “end infectious diseases??? Today – bacteria with multi-drug resistance. Concern over resistance to ‘last resort’ antibiotics.

  2. Enterococcus faecalis A leading cause of hospital infections vanomycin = antibiotic of last resort E faecalis resistant strains for years can transfer resistance genes to Staphylococcus aureus in lab - MRSA virulent cause of pneumonia, endocarditis, sepsis etc.

  3. Examples of Antibiotic Targets Cell Wall Formation - penicillin, cephalosporins, vancomycin Folate biosynthesis – sulfa drugs & DHPS Replication – novobiocin & DNA Gyrase Transcription – rifampicin & RNA Pol Translation – puromycin & ribosome ‘A’ Fatty Acid synthesis – triclosan & enoyl reductase

  4. Killing Bacteria without Resistance Drastically alter Bacterial environment so that multiple systems become inoperative. Therefore, many genes would have to mutate to cause resistance. Bleach (NaOCl) – Oxidize multiple targets in bacteria Detergents/soap/alcohol – disrupt membrane Heat/pH extremes - denature proteins UV irradiation – grossly damage DNA Antimicrobial Peptides (AMPs) – lyse membranes

  5. chromosome Plasmids in bacteria often contain genes critical for ….. antibiotic resistance, toxins, natural product metabolism F factor plasmid (for sexual transmission of plasmids) plasmids Bacteria Bacteria can transfer antibiotic resistance plasmids between species

  6. Practices that Foster Resistance 1. taking antibiotics for non-bacterial illness 2. not taking all of antibiotic 3. non-human use of antibiotics antibiotics as growth promoters in animals Resistant Bacteria ― strategies 1. mutated target enzyme – evasion strategy 2. enzyme to destroy antibiotic – attack strategy 3. efflux channel – bailout strategy

  7. Fighting Back at Resistant Bacteria 1. Develop new drugs for same targets 2. Develop ‘co’-drugs 3. Find new targets for Drugs 4. Find new classes of drugs

  8. Sulfthiazoleresistance ― case study 1985 – 5 isolates of resistant StreptoccoccusPyogenessaved from patients in Sweden Hospital 1990’s – Genomes from normal and resistant isolates compared – highly mutated genes cloned & expressed in E. coli. Pathway genes: folC- folE - folP - folQ - folK folE = GTP cyclohydrolase folQ = dihydroneopterinaldolase folK = hydroxymethydihydropterinpyrophosphatase converts GTP intodihydropteridin unit folP = DHPS (dihydropteroatesynthetase) adds PABA unit folC = dihydrofolatesynthetaseadds glutamate unit DHPS gene found to be mutated. (evasion strategy)

  9. H2N- -COOH O HN- -C- NH-CH-COO- CH2 CH2 COO- O N HN H2N- N N Dihydrofolate Biosynthesis Pathway genes: folC- folE - folP - folQ - folK folE = GTP cyclohydrolase folQ = dihydroneopterinaldolase folK = hydroxymethydihydropterinpyrophosphatase converts GTP intodihydropteridin unit folP = DHPS (dihydropteroatesynthetase) adds PABA unit ― 16% divergence folC = dihydrofolatesynthetaseadds glutamate unit DHPS DHFS PABA → → dihydrofolate

  10. NH2 NH2 O=S=O | NH2 O=S=O N-H N S sulfanilamide sulfathiazole

  11. sulfonamide E. Coli - DHPS

  12. E. Coli - DHPS

  13. KM(inhib) = KM (1 + [I]/Ki) KMKi G1 (suscep)0.7mM 0.2mM G56 (res) 2.5mM 27.4mM Difference 3.6x 137x DHPS Kinetics

  14. penicllin and b-lactams inhibit the cell wall synthesis in bacteria They are analogs of the peptide component of the bacterial cell wall Lactams contain a 4-membered ring with an amide nitrogen and a keto group. penicillins and cephalosporins are antibiotic classes that possess lactam ring Penicillin inhibits last connection in making bacterial cell wall … GlycopeptideTranspeptidase b-lactamases of varying specificities are often found in ‘R’ plasmids of resistant bacteria. b-lactamasesdestroy b-lactams by cleaving (O=C ― N) in lactam structure. Attack strategy destroys antibiotic before it can kill bacteria .

  15. b-lactam antibiotic Glycopeptide transpeptidase

  16. b-lactam antibiotic Glycopeptide transpeptidase

  17. Polysaccharide X-X-X-A-A X-X-X-A-A X-X-X-A-A X-X-X-A-A G-G-G-G-G G-G-G-G-G G-G-G-G-G G-G-G-G-G Peptidoglycan

  18. Bacterial Cell Wall Completion X-X-X-A X-X-X-A X-X-X-A-A X-X-X-A-A G-G-G-G-G G-G-G-G-G G-G-G-G-G G-G-G-G-G

  19. R C = O H - N S CH3 C - C C - CH3 C - N C O COO- b-lactamase CH3 CH3 - N - C - C - N - C O COO- penicillin mimics AA seq of peptide linker -D-Ala-D-Ala

  20. penicillin R C = O H - N S CH3 C - C C - CH3 C - N C O COO- clavulanate CH3 C - C C - CH3 C - N C O COO- O O given along with penicillin it will inhibit penicillinase

  21. C O OH Cl O O OH O Cl O HO N-CH3 N N N O N O NH2 N O HOOC Vancomycin binds to D-Ala – D-Ala peptide unit OH OH OH Resistance due to target mutation in peptidoglycan – D Ala to D – lactate giving 3x less drug affinity due to missing H-bond. replacing C=O with CH2 produces 100x activity to mutant retains only 3% activity to sensitive bacteria. C&E News Feb 13, 2006

  22. D-Ala – D-Ala Vancomycin (blue)

  23. Efflux Pumps ― bailout strategy Many efflux pumps expel a broad range of compounds – may have normal anti-toxin function. efflux pump inhibitors, like b-lactamase inhibitors, could well be analogs of the original antibiotic and have mild antibiotic activity as well.

  24. E. Coli ACRB Multi-drug efflux transporter

  25. Efflux Pumps antibiotic EP inhibitor efflux pump bacteria cell membrane antibiotic target

  26. Pdb – 2f2m EmrE tetraphenylphosphonium

  27. Cl Cl O Cl O Triclosan inhibits enoyl reductase

  28. Fatty Acid Synthesis acetylCoA + HCO3- + ATP  malonyl CoA +ADP acetylCoA + ACP  acetyl-ACP + CoA malonylCoA + ACP  malonyl-ACP + CoA acetyl-ACP +malonyl-ACP  acetoacetyl-ACP + CO2 + ACP acetoacetyl-ACP + NADPH  hydroxybutyryl-ACP + NADP+ hydroxybutyryl-ACP  Crotonyl-ACP + H2O Crotonyl-ACP + NADPH  butyryl-ACP + NADP+ Enoyl-ACP reductase

  29. triclosan Enoyl reductase (step in fatty acid synthesis)

  30. triclosan Enoyl reductase (step in fatty acid synthesis)

  31. Parikh et. al. (2000) Biochemistry 39, 7645-7650 Triclosan inhibits enoyl-ACP reductase from Mycobacterium Tuberculosis Ki ~ 0.22 mM for crotonyl-ACP & NADH Y158 F Ki ~ 47 & 36 mM M161  V triclosan resistant Ki ~ 4.3 mM also less sensitive to isoniazid triclosan could stimulate TB resistant strains of mycobacterium

  32. New Antibiotics oxazolidimones (linezolid) – binds 30S subunit of ribosome and prevents mRNA & fMet-tRNA binding. gemifloxacin – DNA gyrase inhibitor used on respiratory tract infections daptomycin – blocks peptidoglycan and lipoteichoic acid synthesis (cell wall formation) works on vanomycin resistant enterococci BPI Protein - (bacterial permeability increasing) naturally found in bactria killing wbc’s – good in combo Antimicrobial Peptides – defensins & protegrins may function as voltage-gated pores specific for acidicphospholipids found only in bacteria

  33. New Targets for Antibiotics sortase – cleaves loosely bound surface proteins in gram (+) bacteria to activate infectivity proteins. (doesn’t kill bacteria) deformylase – removes formyl group from amino end of bacterial polypeptides – includes actinonen (natural cpd) Efflux Pump Inhibitors

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