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Carbon monoxide dehydrogenase: A novel target for the development of new anti-tubercular drugs

From Basic to Application. Carbon monoxide dehydrogenase: A novel target for the development of new anti-tubercular drugs. Young Min Kim Department of Biology, Yonsei University. Molecular Microbiology Laboratory, Yonsei University. Molecular Microbiology Laboratory, Yonsei University. CO.

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Carbon monoxide dehydrogenase: A novel target for the development of new anti-tubercular drugs

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  1. FromBasic to Application Carbon monoxide dehydrogenase: A novel target for the development of new anti-tubercular drugs Young Min Kim Department of Biology, Yonsei University Molecular Microbiology Laboratory, Yonsei University

  2. Molecular Microbiology Laboratory, Yonsei University CO • An air pollutant • A color- and odorless toxic gas • : high affinity to metal-containing enzymes • (e.g. hemoglobin, a-type cytochromes) • Also acts as a neurotransmitter • : the one produced endogenously during heme • degradation

  3. Molecular Microbiology Laboratory, Yonsei University  Production and sources - 12 - 14 x 108 tons per year - Major source : incomplete oxidation of fossil fuels * Constant concentration in atmosphere : 0.03 - 0.9 ppm (50 - 100 ppm in urban district)  Sinks - Major sink (2/3) : Oxidation by hydroxyl radical in the upper atmosphere (troposphere) - Biological oxidation, especially microbial oxidation, plays a major role in the lower atmosphere (soil and water)

  4. Molecular Microbiology Laboratory, Yonsei University Carboxydobacteria • - A group of aerobic bacteria which are able to grow at the expense of carbon monoxide as the sole source of carbon and energy • Facultatively chemolithoautotrophs

  5. Molecular Microbiology Laboratory, Yonsei University Carboxydobacteria • - A group of aerobic bacteria which are able to grow at the expense of carbon monoxide as the sole source of carbon and energy • Facultatively chemolithotrophic bacteria • Gram(-) carboxydobacteria • Mycobacterial carboxydobacteria (Gram-positive) • Gram(+) carboxydobacteria other than • mycobacteria

  6. Molecular Microbiology Laboratory, Yonsei University Carboxydobacteria  Gram negative - Pseudomonas carboxydohydrogena - Hydrogenophaga pseudoflava  Oligotropha carboxidovorans  Gram positive  Mycobacterium sp. strain JC1( Acinetobacter sp. strain JC1) 2002

  7. Molecular Microbiology Laboratory, Yonsei University Bases of CO utilization  CO dehydrogenase  CO-insensitive electron transport system  Calvin cycle * Mycobacterium tuberculosis: reductive TCA cycle (?)

  8. Molecular Microbiology Laboratory, Yonsei University CO dehydrogenases • Aerobic bacteria - carboxydobacteria - metals: Mo, Cu, Fe • Anaerobic bacteria - acetogenic bacteria, methanogenic bacteria, phototrophic bacteria, sulfate reducers - metals: Ni, Fe

  9. Molecular Microbiology Laboratory, Yonsei University CO dehydrogenases in carboxydobacteria  300-380 kD  3 nonidentical subunits (L2M2S2) Molybdopterin cytosine dinucleotide:[2Fe-2S]:FAD (2:4:2)  Inducible by CO * Mycobacterium sp. strain JC1: constitutive & CO-inducible

  10. Molecular Microbiology Laboratory, Yonsei University O. Meyer’s group O. carboxidovorans FAD FeS II FeS I MCD-Mo

  11. CO + H2O CO2 + 2H+ + 2e- Molecular Microbiology Laboratory, Yonsei University Role of CO dehydrogenase during CO oxiadtion • CO + H2OCO-DH(MCD-HCOO- + H+/[Fe-S]/FAD) CO2 • CO-DH(MCD/[Fe-S]/FADH2) • CO-DH(MCD/[Fe-S]/FAD) 2H+ + 2e-

  12. CO + H2O CO2 + 2H+ + 2e- Molecular Microbiology Laboratory, Yonsei University Fate of electrons during CO oxidation • CO + H2OCO-DH(MCD-HCOO- + H+/[Fe-S]/FAD) CO2 • CO-DH(MCD/[Fe-S]/FADH2) • CO-DH(MCD/[Fe-S]/FAD) 2H+ + 2e-

  13. Molecular Microbiology Laboratory, Yonsei University Electron transport system in carboxydobacteria • Organic materials • NADHUQ/Cyt. bCyt. cCyt. aO2 • COUQ/Cyt. bCyt. oO2 • NADH

  14. Molecular Microbiology Laboratory, Yonsei University Electron transport system in carboxydobacteria • Organic materials • NADHUQ/Cyt. bCyt. cCyt. aO2 • COUQ/Cyt. bCyt. oO2 • NADH

  15. CO + H2O CO2 + 2H+ + 2e- Molecular Microbiology Laboratory, Yonsei University CO2 fixation in carboxydobacteria • Calvin cycle • Reductive TCA cycle (Mycoabcterium tuberculosis?) • Hydroxypropionate pathway • Acetyl-CoA pathway

  16. CO + H2OCO-DH(MCD-HCOO- + H+/[Fe-S]/FAD) CO2 • CO-DH(MCD/[Fe-S]/FADH2) • CO-DH(MCD/[Fe-S]/FAD) 2H+ + 2e- CO + H2O CO2 + 2H+ + 2e- Molecular Microbiology Laboratory, Yonsei University Oxidation of CO by CO dehydrogenase Cellular materials Energy

  17. Molecular Microbiology Laboratory, Yonsei University Mycobacteriaand CO

  18. Molecular Microbiology Laboratory, Yonsei University Reclassification of CO-oxidizing isolate Acinetobacter sp. strain JC1 (1985) Mycobacterium sp. strain JC1 (2002)

  19. Growth on CO CO-DH activity RubisCO activity Bacteria Mycobacterium sp. strain JC1 + + + M. tuberculosis H37Ra + + - M. parafortuitum + + + M. vaccae + + + M. gastri + + + M. Smegmatis MC2 + + + M. neoaurum + + + M. peregrinum + + + M. phlei M. flavescens + + + + + + Molecular Microbiology Laboratory, Yonsei University Mycobacterial carboxydobacteria Park et al., J. Bacteriol. 185:142-147 (2003)

  20. 1 2 3 4 5 6 7 8 9 10 A B Molecular Microbiology Laboratory, Yonsei University Activity staining and Immunoblotting of CO-DHs in mycobacteria

  21. Amino acid identity (%) Bacteria Large Medium Small 55.7 38.2 57.7 57.7 36.9 57.4 57.8 40.4 57.0 87.7 71.2 81.7 O. carboxidovorans P. thermocarboxydovorans H. pseudoflava M. tuberculosis Molecular Microbiology Laboratory, Yonsei University Identity of amino acid sequence of Mycobacterium sp. strain JC1 CO-DH to those of CO-DHs in other bacteria * No immunological relationship between CO-DHs in mycobacteria and Gram-negative bacteria

  22. modC moaE cutR cutB1 cutC1 cutA1 orf7 orf8 orf9 orf10 orf11 orf12 orf13 cutB2 cutC2 cutA2 orf17 orf18 orf19 orf20 orf21 rv0377 0376c 0375c 0374c 0373c 0372c 0371c 0370c 0369c 0368c mb0384 0383c 0382c 0381c 0380c 0379c 0378c 0377c 0376c 0375c bcg0415 0414c 0413c 0412c 0411c 0410c 0409c 0408c 0407c 0406c mmar0653 0654 0655 0656 0657 0658 0659 0660 0661 0662 Molecular Microbiology Laboratory, Yonsei University Mycobacterim sp. strain JC1 CO-DH genes and conserved genes up- and downstream of the CO-DH genes in mycobacteria M. tuberculosis H37Rv M. bovis M. bovis BCG M. marinum msmeg0742 0743 0744 0745 0746 0747 0748 0749 0750 M. smegmatis mjls0476 0477 0478 0479 0480 0481 0482 0483 0484 Mycobacterium sp. JLS mkms0498 0499 0500 0501 0502 0503 0504 0505 0506 Mycobacterium sp. KMS mmcs0487 0488 0489 0490 0491 0492 0493 0494 0495 Mycobacterium sp. MCS rha1_ro05229 05230 05231 05232 05233 05234 05235 05236 1 kb R. jostii coxB coxC coxM coxS coxL coxD coxE coxF coxG coxH coxI coxK O. carboxidovorans cutM cutS cutL orf1 H. pseudoflava cutB cutC cutA orf4 P. thermocarboxydovorans trd_A0566 0565 0564 0563 T. roseum coxG coxC coxM coxS coxL coxD coxE coxF R. pomeroyi rcoM bxe_C0029 0030 0031 0032 0033 0034 0035 0036 Park et al., Microbiology 156:999-1008 (2010)

  23. Molecular Microbiology Laboratory, Yonsei University What is the physiological role of CO dehydrogenase in pathogenic mycobacteria?

  24. MW: 28 MW: 30 Molecular Microbiology Laboratory, Yonsei University

  25. Molecular Microbiology Laboratory, Yonsei University NO • An air pollutant • An important signaling molecule in human • body • - Beneficial and detrimental • Kills bacteria in macrophages

  26. Specific activityb Source Bacteria CO-DH NO-DH 1.5 2.0 3.1 0.0 Mycobacterium sp. strain JC1 DSM 3803 M. tuberculosis H37Ra ATCC 35835 M. vaccae ATCC 15483 O. carboxidovorans OM5DSM 1227 6.0 9.9 17.5 18.1 a Activity was determined with cell-free extracts prepared from cells grown at 37oC in SMB-CO. b Nanomoles of INT reduced per milligram of protein per minute. NO source: sodium nitroprusside (SNP) Molecular Microbiology Laboratory, Yonsei University CO-DH and NO-DH activities in mycobacterial and a Gram-negative carboxydobacteriaa Park et al., BBRC 362:449-453 (2007)

  27. Molecular Microbiology Laboratory, Yonsei University CO-DH and NO-DH stained by activity Lane 1: Stained for CO-DH (CO) Lane 2: Stained for NO-DH (SNP)

  28. Molecular Microbiology Laboratory, Yonsei University Purified CO-DH possesses NO-DH activity Purified CO-DH of Mycobacterium sp. strain JC1 - CO-DH activity : 139.3 nmol of INT reduced/mg protein/min - NO-DH activity : 32.6 nmol of INT reduced/mg protein/min

  29. Molecular Microbiology Laboratory, Yonsei University Oxidation of NO by NO-DH activity of CO-DH NO + H2O NO2 + 2H+ + 2e- CO + H2O CO2 + 2H+ + 2e-

  30. Specific activityb Time after inoculation (min) CO-DH NO-DH Bacteria -SNP +SNP –SNP +SNP 5.7 8.5 0.0 17.6 7.6 7.8 0 20 40 5.7 5.7 6.0 17.6 17.0 14.5 1.5 2.1 0.0 0.0 0.0 0.0 1.5 1.4 1.5 0.0 0.0 0.0 Mycobacterium sp. strain JC1 O. carboxidovorans OM5 0 20 40 Molecular Microbiology Laboratory, Yonsei University CO-DH and NO-DH activities in cells incubated in the presence and absence of SNPa a Cells were grown in SMB-CO in the presence (+SNP) and absence (-SNP) of 5 mM SNP. Activity was determined with cell-free extracts prepared from cells harvested at the indicated time intervals after inoculation of the CO-grown cells into the appropriate medium. bNanomoles of INT reduced per milligram of protein per minute.

  31. -SNP +SNP 1 2 3 1 2 3 A B C D Molecular Microbiology Laboratory, Yonsei University Effect of SNP on the expression of CO-DH Immunoblotting of CO-DH (min) 02040 Mycobacterium sp. strain JC1SMB-glucose + SNP Panel A: Mycobacterium sp. JC1, CO, stained with CBB Panel B: Mycobacterium sp. JC1, CO, stained by activity Panel C: Mycobacterium sp. JC1, glucose, stained by activity Panel D: O. carboxidovorans OM5, CO, stained by activity Cells were harvested at the time of inoculation (lane 1) and at 20 min (lane 2) and 40 min (lane 3) after inoculation

  32. Growth on CO CO-DH activity RubisCO activity Bacteria Mycobacterium sp. strain JC1 + + + M. tuberculosis H37Ra + + - M. parafortuitum + + + M. vaccae + + + M. gastri + + + M. Smegmatis MC2 + + + M. neoaurum + + + M. peregrinum + + + M. phlei M. flavescens + + + + + + Molecular Microbiology Laboratory, Yonsei University Mycobacterial carboxydobacteria Park et al., J. Bacteriol. 185:142-147 (2003)

  33. modC moaE cutR cutB1 cutC1 cutA1 orf7 orf8 orf9 orf10 orf11 orf12 orf13 cutB2 cutC2 cutA2 orf17 orf18 orf19 orf20 orf21 rv0377 0376c 0375c 0374c 0373c 0372c 0371c 0370c 0369c 0368c mb0384 0383c 0382c 0381c 0380c 0379c 0378c 0377c 0376c 0375c bcg0415 0414c 0413c 0412c 0411c 0410c 0409c 0408c 0407c 0406c mmar0653 0654 0655 0656 0657 0658 0659 0660 0661 0662 Molecular Microbiology Laboratory, Yonsei University Mycobacterim sp. strain JC1 CO-DH genes and conserved genes up- and downstream of the CO-DH genes in mycobacteria M. tuberculosis H37Rv M. bovis M. bovis BCG M. marinum msmeg0742 0743 0744 0745 0746 0747 0748 0749 0750 M. smegmatis mjls0476 0477 0478 0479 0480 0481 0482 0483 0484 Mycobacterium sp. JLS mkms0498 0499 0500 0501 0502 0503 0504 0505 0506 Mycobacterium sp. KMS mmcs0487 0488 0489 0490 0491 0492 0493 0494 0495 Mycobacterium sp. MCS rha1_ro05229 05230 05231 05232 05233 05234 05235 05236 1 kb R. jostii coxB coxC coxM coxS coxL coxD coxE coxF coxG coxH coxI coxK O. carboxidovorans cutM cutS cutL orf1 H. pseudoflava cutB cutC cutA orf4 P. thermocarboxydovorans trd_A0566 0565 0564 0563 T. roseum coxG coxC coxM coxS coxL coxD coxE coxF R. pomeroyi rcoM bxe_C0029 0030 0031 0032 0033 0034 0035 0036 Park et al., Microbiology 156:999-1008 (2010)

  34. Molecular Microbiology Laboratory, Yonsei University Tuberculosis • A common and often deadly disease caused in human mainly by Mycobacterium tuberculosis • Systemic but usually attacks lung

  35. Molecular Microbiology Laboratory, Yonsei University M. tuberculosis • Infects over a third of the world’s population (~2 billions) - one in 10 asymptomatic latent infections progresses to active TB (8~9 millions per year) • Accounts for 2 million death annually (one every 15-20 seconds) • Infects a fourth of AIDS patients - accounts for a third of AIDS patient’s death

  36. Molecular Microbiology Laboratory, Yonsei University ROS RNS

  37. Molecular Microbiology Laboratory, Yonsei University Strategies of mycobacteria to survive inside host macrophages 1. Prevention of phagosome-lysosome fusion 2. Inactivation of toxic reactive oxygen species by KatG etc. 3. Inactivation of toxic reactive nitrogen species by; - NO dioxygenase activity of Hmp and trHbN - Peroxinitritase activity of KatG and alkylhydroperoxide reductase AhpC ROS RNS

  38. Molecular Microbiology Laboratory, Yonsei University Strategies of mycobacteria to survive inside host macrophages 1. Prevention of phagosome-lysosome fusion 2. Inactivation of toxic reactive oxygen species by KatG etc. 3. Inactivation of toxic reactive nitrogen species by; - NO dioxygenase activity of Hmp and trHbN - Peroxinitritase activity of KatG and alkylhydroperoxide reductase AhpC - NO-DH activity of CO-DH (?) ROS RNS

  39. Molecular Microbiology Laboratory, Yonsei University Effect of CO-DH on the survival of E. coli under NO stress E. coli DH5α, 5 mM SNP, 50 mM Tris-HCl (pH 7.5), RT  50 g of Mycobacterium sp. JC1 purified CO-DH

  40. Molecular Microbiology Laboratory, Yonsei University Effect of NO on the survival of Mycobacterium sp. JC1 cutR- mutant under NO stress Mycobacterium sp. strain JC1wild type andcutR-mutant cells, 5 mM SNP, RT

  41. Molecular Microbiology Laboratory, Yonsei University Effect of NO on the survival of Mycobacterium sp. strain JC1 cutA-mutant under NO stress

  42. Molecular Microbiology Laboratory, Yonsei University Effect of NO on the survival of M. smegmatis cutA-mutant under NO stress

  43. Wild type-1 Wild type-2 CO-DH mutant#1-1 CO-DH mutant#1-2 CO-DH mutant#2-1 CO-DH mutant#2-1 Molecular Microbiology Laboratory, Yonsei University Survival of M. tuberculosis H37Rv in macrophage

  44. Molecular Microbiology Laboratory, Yonsei University Strategies of mycobacteria to survive inside host macrophages 1. Prevention of phagosome-lysosome fusion 2. Inactivation of toxic reactive oxygen species by KatG etc. 3. Inactivation of toxic reactive nitrogen species by; - NO dioxygenase activity of Hmp and trHbN - Peroxinitritase activity of KatG and alkylhydroperoxide reductase AhpC - NO-DH activity of CO-DH

  45. Possible role of CO-DH in pathogenic mycobacteria CO-DH CO NO Molecular Microbiology Laboratory, Yonsei University

  46. Molecular Microbiology Laboratory, Yonsei University Known anti-tuberculosis drugs and genes inducing resistance to the drugs

  47. Molecular Microbiology Laboratory, Yonsei University Known anti-tuberculosis drugs and genes inducing resistance to the drugs Multi-drug resistant TB Extensively drug-resistant TB

  48. No new anti-TB drug has been introduced in the last four decades → needs novel targets with less mutation rate

  49. Molecular Microbiology Laboratory, Yonsei University CO-oxidizing system as a novel target for TB drug screening 1. CO dehydrogenase : screening for natural and synthetic compounds 2. Proteins involved in the expression of CO dehydrogenase

  50. Molecular Microbiology Laboratory, Yonsei University Functional analysis of genes (possibly) involved in the oxidation of CO in mycobactera Mycobacterium tuberculosis 0377 0377 0376c 0376c 0375c 0375c 0374c 0374c 0373c 0373c 0372c 0372c 0371c 0371c 0370c 0370c 0369c 0369c 0368c 0368c Mycobacterium smegmatis Genes Mutant Function Structure orf1 orf6 cutR cutB cutC cutA orf2 orf4 orf5 cutR orf1 cutBCA orf2 orf3 orf4 orf5 orf6 regulation unknown CO-DH unknown Const. exp. (?) unknown unknown regulation O X O O X O X O O O X X O X X X Mycobacterium sp. strain JC1 cutR cutB cutC cutA orf2 orf3 orf4 orf5 Copy I cutB cutC cutA orf2 orf3 orf4 orf1 orf5 orf6 Copy II

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