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

Table of Contents Chapter 1 1.2 Hypoxia could be a Factor in Persistence 3 1.3 Discovery of the Dormancy Response: Wayne’76 Culture Model 5 1.4 Temporal Analysis of Dormancy: Wayne’96 C

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

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

Table of Contents

Chapter 1

1.2 Hypoxia could be a Factor in Persistence 3

1.3 Discovery of the Dormancy Response: Wayne’76 Culture Model 5

1.4 Temporal Analysis of Dormancy: Wayne’96 Culture Model 9

1.5 Other ‘Hypoxia’ Culture System 13 1.6 The Hypoxia Induced in vitro Dormancy Response is Poorly Understood 15

Chapter 2

2.1 Materials

2.2 Mycobacterial Culture

2.2.1 The Wayne Dormancy Culture Model 23 2.2.2 The Aerated Stationary Phase Culture Model 23

2.3 DNA and RNA Methods 24

2.3.2 Blunt Ending of DNA Fragments 25 2.3.3 Dephosphorylation of DNA Fragments 26

2.3.6 Elution of DNA from Agarose Gels 27

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

2.3.18 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

Chapter 3

3 Identification of Dormancy Induced Proteins 47

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

3.5 Computational Analysis of Dormancy Induced Proteins 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

Chapter 4

4 Functional Chracterization of the Dormancy Specific Response

4.1 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

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

4.1.7 Isolation of ∆Rv3132c::km from Single Recombinants 92 4.1.8 Construction of Rescue Construct pCB4 for ∆dosR::km and

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

4.2.5 Wild type-like Survival of BCG ∆dosR::km and ∆Rv3132c:: km

in Aerated Stationary Phase Cultures 108

Chapter 5

5.1 Proteins Induced in the Wayne’96 Dormancy Culture System 114

5.1.3 Rv2623 117

5.2 Transcript Levels of the Four Dormancy Induced Proteins are

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

5.6 DosR Function is Conserved in M smegmatis 127

5.7 Hypoxic Dormant Bacilli and Persistence in vivo 128

List of Figures

Chapter 1

Figure 1.3.1 The Wayne’76 standing culture model 9

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 3 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 3 to 10 isoelectric focusing strips 51

Figure 3.1.3 Temporal proteome of BCG grown in the Wayne dormancy

culture system using pH 4 to 7 isoelectric focusing strips 52

Figure 3.1.4 Under Loading Experiments using pH 4 to 7 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 59

Figure 3.4.1 Growth of BCG cultures in aerated roller bottles 62

Figure 3.4.2 Temporal proteome of BCG grown in aerated roller bottle

cultures using pH 4 to 7 isoelectric focusing strips 63

Figure 3.5.2.1 Multiple alignment of Rv3133c with other response regulators

with known phosphorylation sites 66

Figure 3.5.1 Domain structure of the four dormancy induced proteins 69 Figure 4.1.1.1 The genomic organization of the Rv3133c locus in

Mycobacterium tuberculosis and Mycobacterium bovis BCG 73

Figure 4.1.2.1 Summary of the cloning strategy for the construction of the

dosR gene replacement construct 76

Figure 4.1.2.2 dosR locus and gene replacement constructs 77

Figure 4.1.3.1 Streak plates of wild type strains, dosR single and double

Figure 4.1.3.2 PCR strategy for screening of ∆dosR::km legitimate single

Figure 4.1.3.3 PCR analysis using genomic DNA extracted from single

recombinants and double recombinants isolated during the

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 88

Figure 4.1.5.2 dosR locus, Rv3132c gene replacement construct 89

Figure 4.1.6.1 Streak plates of wild type strains, Rv3132c single and double

Figure 4.1.6.2 PCR strategy for screening of ∆Rv3132c::km legitimate single

Figure 4.1.7.1 PCR strategy for screening of ∆Rv3132c::km 93

Figure 4.1.7.2 PCR analysis using genomic DNA extracted from single

recombinants and double recombinants isolated during the

Figure 4.1.7.3 Southern blot analysis of Rv3132c gene replacement mutants 96

Figure 4.1.7.4 Summary of dosR locus, gene replacement constructs and

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

Figure 4.2.2.1Growth of wild type BCG, ∆Rv3132c::km1 ,∆Rv3132c::km1

(pCB4) and ∆dosR::km1 strains in the Wayne dormancy culture

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

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 5

Figure 5.5.1 A working model for the molecular mechanisms of the

List of Tables

Chapter 3

Table 3.2.1 Dormancy induced proteins identified by nanoelectrospray

Table 3.3.1 Primer sequences used for RT-PCR and isolation of probes

Chapter 4

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

Abbreviations

A 600 Absorbance at λ600nm

Amp ampicilin

Amp R ampicillin resistant

ATP adenosine 5’-triphosphate

BCG Mycobacterium bovis BCG

BSA bovine serum albumin

o C degrees Celsius

cDNA complementary deoxyribonucleic acid

c.f.u colony forming units

CHAPS 3-3-cholamidopropyl-dimethylammonio-1-1-propane sulfonate

µCi microCurie

DEPC diethylenepyrocarbonate

DNA deoxyribonucleic acid

DNase deoxyribonuclease

DTT 1,4-dithiothreitol

EDTA ethylenediaminetetraacetic acid

g gram

µg microgram

Get gentamycin

Get R gentamycin resistant

Get S gentamycin sensitive

Hyg hygromycin

Hyg R hygromycin resistant

Kan kanamycin

Kan R kanamycin resistant

Kan S kanamycin sensitive

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

SDS sodium dodecyl sulfate

Suc sucrose

Suc R sucrose resistant

Suc S sucrose sensitive

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

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 non-replicative 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 hypoxia-induced 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 3 dormancy-induced proteins

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 1 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