DOSR a response regulator essential for hypoxic dormancy in mycobacterium bovis BCG 1

DOSR – A RESPONSE REGULATOR ESSENTIAL FOR HYPOXIC DORMANCY IN Mycobacterium bovis BCG BOON KA KHIU CALVIN (B.Sc Hons.) IMPERIAL COLLEGE OF LONDON A THESIS SUBMITTED FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2003 ii Acknowledgement I would like to express my heartfelt gratitude to my supervisor A/P Thomas Dick for his constant guidance, stimulating discussions and encouragements in the tough times encountered during the course of this project His contributions go beyond scientific thinking He had had a major influence on the further development of my strengths and realization of my weaknesses For this, I am very grateful My sincere thanks to the members of my PhD Supervisory Committee A/P Wang Yue, Dr Anthony Ting and Dr Michael Sprengart for their constructive suggestions and guidance I am very grateful to Dr Robert Qi and Li Rong for performing the mass spectrometric analysis that lead to the identification of DosR, which had been the central feature of this thesis I am also thankful to Dr Alice Tay for providing excellent sequencing services Special thanks to Bernadette Oei-Murugasu for her help and advice during the learning stages, to Indra for his critical comments on the thesis, to Bee Huat, Boon Heng, Michael, Pam, Raymond and other past and present members of the mycobacterium lab for their friendship, advice and stimulating discussions Last but not least, I would like to express my deepest gratitude to my parents for their support and encouragement throughout the years of my education Without them, I would not be writing this thesis Finally, I am very grateful to my beautiful wife Boon Tin for her enduring love and confidence in me that had been my constant source of strength throughout these years iii Table of Contents Acknowledgement ii Table of contents iii List of Figures vii List of Tables x Abbreviations xi Summary xiv Chapter 1 Introduction 1.1 Bacilli Persist in vivo 1.2 Hypoxia could be a Factor in Persistence 1.3 Discovery of the Dormancy Response: Wayne’76 Culture Model 1.4 Temporal Analysis of Dormancy: Wayne’96 Culture Model 1.5 Other ‘Hypoxia’ Culture System 13 1.6 The Hypoxia Induced in vitro Dormancy Response is Poorly Understood 15 Chapter 2 Materials and Methods 2.1 20 Materials 2.1.1 Vectors 20 2.1.1 Bacterial Strains 21 2.1.2 Chemicals and Reagents 21 2.1.3 Growth Media 22 2.2 Mycobacterial Culture 2.2.1 The Wayne Dormancy Culture Model 23 2.2.2 The Aerated Stationary Phase Culture Model 23 2.2.3 Determination of C.F.U 24 iv 2.3 DNA and RNA Methods 24 2.3.1 Restriction Digest of DNA 25 2.3.2 Blunt Ending of DNA Fragments 25 2.3.3 Dephosphorylation of DNA Fragments 26 2.3.4 Ligation of DNA Fragments 26 2.3.5 Agarose Gel Electrophoresis 26 2.3.6 Elution of DNA from Agarose Gels 27 2.3.7 Precipitation of DNA 28 2.3.8 Preparation of E.coli Electrocompetent cells 28 2.3.9 Transformation of E.coli by Electroporation 29 2.3.10 Preparation of BCG Electrocompetent cells 29 2.3.11 Transformation of BCG by Electroporation 29 2.3.12 Mini Preparation of Plasmid DNA 30 2.3.13 Maxi Preparation of Plasmid DNA 31 2.3.14 Mini Preparation of Genomic DNA 32 2.3.15 PCR 33 2.3.16 Sequencing of DNA 34 2.3.17 Two Step RT-PCR 35 2.3.18 Southern Blotting and Hybridization 36 2.3.19 Preparation of BCG Total RNA 38 2.3.20 Northern Blotting and Hybridization 38 2.3.21 Screening of M.bovis BCG Genomic Library 40 2.4 Protein Methods 2.4.1 Preparation of BCG Protein Lysate 42 2.4.2 Determination of Protein Concentration 42 2.4.3 Two-dimensional Gel Electrophoresis 43 2.5 Computational Analysis 45 Chapter 3 Identification of Dormancy Induced Proteins 47 v 3.1 Analysis of the Dormancy Response in BCG using 2-D Gel 48 Electrophoresis 3.2 Protein Identification via Mass Spectrometry 53 3.3 Transcript Levels of Dormancy Induced Proteins 57 3.4 Analysis of Dormancy Induced Proteins in Aerated Stationary Phase Cultures 60 3.5 Computational Analysis of Dormancy Induced Proteins 64 3.5.1 HspX 64 3.5.2 23kD Putative Response Regulator 65 3.5.3 32kD Conserved Hypothetical Protein 67 3.5.4 16kD Conserved Hypothetical Protein 67 3.6 Conclusion 70 Chapter 4 Functional Chracterization of the Dormancy Specific Response Regulator Rv3133c 4.1 71 Molecular Characterization of the dosR Locus; Generation of Gene Replacement and Rescue Constructs 72 4.1.1 Genomic Organization of the BCG Rv3133c locus 72 4.1.2 Generation of BCG ∆dosR::km Replacement Construct 75 4.1.3 Isolation of Single Recombinants Clones for the Generation of ∆dosR::km 78 4.1.4 Isolation of ∆dosR::km from Single Recombinants 83 4.1.5 Generation of BCG ∆Rv3132c::km Replacement Construct 87 4.1.6 Isolation of Single Recombinants Clones for the Generation of ∆Rv3132c::km 89 4.1.7 Isolation of ∆Rv3132c::km from Single Recombinants 92 4.1.8 Construction of Rescue Construct pCB4 for ∆dosR::km and ∆Rv3132c:: km 98 vi 4.2 Phenotypic Analysis of ∆dosR::km and ∆Rv3132c::km 99 4.2.1 Reduction in Viability of BCG ∆dosR::km 99 4.2.2 Moderate Survival Phenotype of BCG ∆Rv3132c:: km 103 4.2.3 Regulation of Dormancy Induced Proteins by DosR 106 4.2.4 Minor Role of Rv3132c in the Regulation of the Dormancy Induced Proteins 4.2.5 108 Wild type-like Survival of BCG ∆dosR::km and ∆Rv3132c:: km in Aerated Stationary Phase Cultures 4.3 Conclusion 108 112 Chapter 5 Discussion 113 5.1 Proteins Induced in the Wayne’96 Dormancy Culture System 114 5.1.1 HspX 115 5.1.2 Rv2626c 116 5.1.3 Rv2623 117 5.1.4 DosR 118 5.2 Transcript Levels of the Four Dormancy Induced Proteins are Elevated in Dormant Bacilli 119 5.3 DosR is the Master Regulator of Dormancy 121 5.4 Rv3132c is Required but not Essential for Dormancy 122 5.5 DosR but not Rv3132c is Essential for the Regulation of the Dormancy Induced Proteins 124 5.6 DosR Function is Conserved in M smegmatis 127 5.7 Hypoxic Dormant Bacilli and Persistence in vivo 128 References 134 vii List of Figures Chapter Figure 1.3.1 The Wayne’76 standing culture model Figure 1.4.1 The Wayne’96 in vitro dormancy culture model 12 Figure 1.6.1 Hallmarks of bacilli grown in the Wayne’76 and’96 in vitro dormancy culture model 18 Chapter Figure 3.1.1 Growth of BCG in the Wayne dormancy culture system 50 Figure 3.1.2 Temporal proteome of BCG grown in the Wayne dormancy culture system using pH to 10 isoelectric focusing strips 51 Figure 3.1.3 Temporal proteome of BCG grown in the Wayne dormancy culture system using pH to isoelectric focusing strips 52 Figure 3.1.4 Under Loading Experiments using pH to isoelectric focusing strips 54 Figure 3.2.1 Protein identification by mass peptide fingerprinting and sequence tag analysis 55 Figure 3.3.1 Steady state levels of mRNAs of dormancy-induced proteins in growing and hypoxic stationary phase cultures Figure 3.4.1 Growth of BCG cultures in aerated roller bottles 59 62 Figure 3.4.2 Temporal proteome of BCG grown in aerated roller bottle cultures using pH to isoelectric focusing strips 63 Figure 3.5.2.1 Multiple alignment of Rv3133c with other response regulators with known phosphorylation sites Figure 3.5.1 Domain structure of the four dormancy induced proteins 66 69 Figure 4.1.1.1 The genomic organization of the Rv3133c locus in Mycobacterium tuberculosis and Mycobacterium bovis BCG 73 viii Figure 4.1.2.1 Summary of the cloning strategy for the construction of the dosR gene replacement construct Figure 4.1.2.2 dosR locus and gene replacement constructs 76 77 Figure 4.1.3.1 Streak plates of wild type strains, dosR single and double recombinants mutants 79 Figure 4.1.3.2 PCR strategy for screening of ∆dosR::km legitimate single recombination events 81 Figure 4.1.3.3 PCR analysis using genomic DNA extracted from single recombinants and double recombinants isolated during the generation of ∆dosR::km 82 Figure 4.1.4.1 PCR strategy for screening of ∆dosR::km 84 Figure 4.1.4.2 Southern blot analysis of dosR gene replacement mutants 86 Figure 4.1.5.1 Summary of the cloning strategy for the construction of the Rv3132c gene replacement construct Figure 4.1.5.2 dosR locus, Rv3132c gene replacement construct 88 89 Figure 4.1.6.1 Streak plates of wild type strains, Rv3132c single and double recombinants mutants 90 Figure 4.1.6.2 PCR strategy for screening of ∆Rv3132c::km legitimate single recombination events Figure 4.1.7.1 PCR strategy for screening of ∆Rv3132c::km 91 93 Figure 4.1.7.2 PCR analysis using genomic DNA extracted from single recombinants and double recombinants isolated during the generation of ∆Rv3132c::km Figure 4.1.7.3 Southern blot analysis of Rv3132c gene replacement mutants 94 96 Figure 4.1.7.4 Summary of dosR locus, gene replacement constructs and rescue plasmid 97 Figure 4.2.1.1 Growth of wild type BCG, ∆dosR::km1 and ∆dosR::km1 (pCB4) strains in the Wayne dormancy culture system 101 Figure 4.2.1.2 Survival of wild type BCG and ∆dosR::km1 and ∆dosR::km1 (pCB4) strains in the Wayne dormancy culture system 102 ix Figure 4.2.2.1Growth of wild type BCG, ∆Rv3132c::km1 ,∆Rv3132c::km1 (pCB4) and ∆dosR::km1 strains in the Wayne dormancy culture system 104 Figure 4.2.2.2Survival of wild type BCG, ∆Rv3132c::km1 and ∆Rv3132c::km1 (pCB4) strains in the Wayne dormancy culture system 105 Figure 4.2.3.1Two-dimensional gel electrophoresis analyses of protein extracts from wild type BCG and ∆dosR::km1, ∆dosR::km1 (pCB4) and ∆Rv3132c::km1 strains grown in the Wayne dormancy culture system 107 Figure 4.2.5.1Growth of wild type BCG, ∆dosR::km1 and ∆Rv3132c1::km strains in the aerated stationary phase culture system 110 Figure 4.2.5.2Survival of wild type BCG, ∆dosR::km1 and ∆Rv3132c1::km strains in the aerated stationary phase culture system 111 Chapter Figure 5.5.1 A working model for the molecular mechanisms of the dormancy response 126 x List of Tables Chapter Table 3.2.1 Dormancy induced proteins identified by nanoelectrospray tandem mass spectrometry Table 3.3.1 56 Primer sequences used for RT-PCR and isolation of probes for Northern hybridization 58 Chapter Table 4.1.1.1 Sequences of primers employed in the sequencing of the genomic fragment containing the BCG Rv3133c locus 74 Table 4.1.3.1 Primer sequences used for PCR screening of clones isolated during the generation of ∆dosR::km and ∆Rv3132c:: km 80 xi Abbreviations A600 Absorbance at λ600nm Amp ampicilin AmpR ampicillin resistant ATP adenosine 5’-triphosphate BCG Mycobacterium bovis BCG BSA bovine serum albumin o degrees Celsius C cDNA complementary deoxyribonucleic acid c.f.u colony forming units CHAPS 3-3-cholamidopropyl-dimethylammonio-1-1-propane sulfonate Ci Curie µCi microCurie DEPC diethylenepyrocarbonate DNA deoxyribonucleic acid DNase deoxyribonuclease DTT 1,4-dithiothreitol E.coli Escherichia coli EDTA ethylenediaminetetraacetic acid g gram µg microgram Get gentamycin GetR gentamycin resistant GetS gentamycin sensitive Hyg hygromycin HygR hygromycin resistant xii Kan kanamycin KanR kanamycin resistant KanS kanamycin sensitive kb kilobases kD kilodalton Klenow large fragment of E.coli DNA polymerase I kV kilo volts L litre µl microlitre M moles per litre mA miliamperes µA microamperes µl microlitre µM micromolar mg miligrams ml millilitre MOPs 3-N-morpholinopropmesulfonic acid mRNA messenger RNA nm nanometer OD optical density PBS phosphate-buffered saline PDA piperazine diacrylamide p.f.u plaque forming unit RNA ribonucleic acid RNase ribonuclease rpm revolutions per minute xiii SDS sodium dodecyl sulfate Suc sucrose SucR sucrose resistant S sucrose sensitive Suc TCEP Tris-carboxyethyl-phosphine TEMED NNN’N’-tetra-methylethylenediamide Tris tris (hydroxymethyl) aminomethane UV Ultra-violet V Volts X-gal 5-bromo-4-chloro-3-indolyl-β-galactopyranoside xiv Summary The persistence of Mycobacterium tuberculosis (MTB) despite long chemotherapy is a major obstacle in the effective treatment and eradication of the disease As such, understanding persistence is vital to the global control of tuberculosis Several lines of evidence indicate that hypoxia could be a factor in persistence The discovery that MTB has the ability to adapt and survive hypoxia in vitro by shifting down to nonreplicative drug resistant dormant form raises the question whether the bacilli in vivo are in a similar physiological state and whether they play a role in the observed persistence of the disease However, the molecular mechanisms of the hypoxiainduced dormancy response are poorly understood The lack of molecular dormancy markers and dormancy mutants hamper investigators from providing direct evidence that hypoxic dormant bacilli exist in vivo and contribute to the persistence of the disease The first part of this study aims to further define the hypoxic dormancy response by identifying dormancy dependent proteins via two-dimensional electrophoresis Using the Wayne’96 in vitro dormancy culture system and the attenuated BCG strain of the tubercle bacilli as a model organism, the temporal proteome profile during the dormancy response was defined Four proteins were found to be induced upon entry into dormancy They are the alpha-crystallin homologue HspX, a response regulator Rv3133c, and the conserved hypothetical proteins Rv2623 and Rv2626c Induction of Rv3133c and Rv2623 appears to be dormancy specific Hence these proteins are useful markers for the demonstration of hypoxic dormant bacilli in vivo Response regulators are phosphorylation dependent transcription factors known to be involved in adaptation of bacteria to diverse conditions Therefore, the hypoxic dormancy specific up regulation of Rv3133c response regulator indicated that this protein could play a role in the adaptation to dormancy survival and the induction of the other dormancy-induced proteins xv In the second part of this work, a functional characterization of Rv3133c was carried out Inspection of the Rv3133c locus revealed that the Rv3132c gene, which encodes a histidine protein kinase, overlaps the Rv3133c gene by base pair thereby indicating that the two proteins could form a ‘dormancy’ two-component signalling system To define the function of Rv3133c and its candidate cognate sensor kinase Rv3132c, gene replacement mutants were constructed and analysed in the Wayne’96 in vitro culture system The Rv3133c mutant showed a drastic loss of viability during hypoxic dormancy Thus, the loss of this dormancy specific response regulator resulted in the loss of the ability of the bacilli to adapt and to survive hypoxic dormancy In addition, the loss of induction of the other three dormancy-induced proteins was observed in the Rv3133c mutant background Hence, the induction of these dormancy proteins is dependent on Rv3133c Based on these two functions, dormancy survival and regulation, the Rv3133c gene was named dosR for dormancy survival regulator In contrast, the Rv3132c mutant displayed a moderate dormancy phenotype This suggests that other ‘dormancy’ kinases may be involved in the regulation of DosR Taken together, this work provides conclusive evidence that dosR is the master regulator of the dormancy response The dosR mutant is the first dormancy specific mutant It represents a useful tool to investigate the relevance of hypoxic dormant bacilli in persistence in vivo  ... Rv2626c 11 6 5 .1. 3 Rv2623 11 7 5 .1. 4 DosR 11 8 5.2 Transcript Levels of the Four Dormancy Induced Proteins are Elevated in Dormant Bacilli 11 9 5.3 DosR is the Master Regulator of Dormancy 12 1 5.4 Rv 313 2c... BCG, ? ?dosR: :km1 and ∆Rv 313 2c1::km strains in the aerated stationary phase culture system 11 0 Figure 4.2.5.2Survival of wild type BCG, ? ?dosR: :km1 and ∆Rv 313 2c1::km strains in the aerated stationary... ? ?dosR: :km and ∆Rv 313 2c:: km in Aerated Stationary Phase Cultures 4.3 Conclusion 10 8 11 2 Chapter 5 Discussion 11 3 5 .1 Proteins Induced in the Wayne’96 Dormancy Culture System 11 4 5 .1. 1 HspX 11 5 5 .1. 2