xi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS iii

ABSTRACT (in English) v

ABSTRACT (in Thai) viii

LIST OF TABLES xvii

LIST OF FIGURES xviii

ABBREVIATIONS xxi

CHAPTER I INTRODUCTION 1

CHAPTER II LITERATURE REVIEWS 7

2.1 General information of Penicillium marneffei 7

2.2 Mycology 11

2.3 Antigenic proteins 16

2.3.1 Mannoprotein of Penicillium marneffei 16

2.3.2 Antigenic protein profile and cDNA screening-derived 16

clones

2.4 Putative virulence factors of Penicillium marneffei 18

2.4.1 Adhesins 19

2.4.2 Dimorphic switching 20

2.4.3 Stress response and adaptation proteins 21

2.4.4 Secreted enzymes from Penicillium marneffei 25

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2.4.5 Melanins 26

2.5 Molecular biology of Penicillium marneffei morphogenesis 27

2.5.1 Conidiation of Penicillium marneffei 27

2.5.2 Polar growth and dimorphism of Penicillium marneffei 31

2.6 Genome and mitochondrial genome of Penicillium marneffei 34

2.7 Molecular approaches to study Penicillium marneffei 36

2.7.1 Targeted gene deletion 36

2.7.2 Conditional expression method 40

2.7.3 RNA interference 42

2.7.4 Suppression subtractive hybridization 43

2.7.5 Proteomics 45

2.7.6 Random insertional mutagenesis 47

2.8 Polyamines in fungi 52

CHAPTER III OBJECTIVES 56

CHAPTER IV MATERIALS AND METHODS 57

4.1 Isolation and characterization of ura5 and ura3 genes from 57

Penicillium marneffei

4.1.1 Strains and growth conditions 57

4.1.2 Preparation of genomic DNA 58

4.1.3 Isolation of ura5 cDNA and ura5 genomic DNA of 59

Penicillium marneffei

4.1.4 Cloning the ura3 gene 63

4.1.5 Sequencing and sequence analysis 64

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Page

4.1.6 Total RNA extraction 65

4.1.7 Analysis of genes expression during phase transition 66

and stress conditions

4.2 Isolation and characterization of uracil auxotrophs of 67

Penicillium marneffei

4.2.1 Isolation of uracil auxotrophs of Penicillium marneffei 67

4.2.2 Measurement of enzymatic activity and the protein 68

content

4.3 Agrobacterium-mediated transformation (AMT) system 69

4.3.1 Strains and their maintenance 69

4.3.2 Antifungal susceptibility testing of Penicillium 70

marneffei and Penicillium citrinum

4.3.3 Agrobacterium-mediated transformation 70

4.3.4 PCR for identifying the integrative T-DNA 73

4.3.5 Southern blot analysis for determining the copy number 73

of T-DNA

4.3.6 Mitotic stability of AMT-derived transformants 74

4.4 Screening for mutants defective in morphogenesis, cell wall 74

production and melanin synthesis

4.5 Analysis of integrated genes or promoters 75

4.6 Characterization of selected mutants of interest 76

4.7 Functional analysis of �sadA mutant 77

4.7.1 Complementation of the Penicillium marneffei �sadA

mutation

77

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4.7.2 Phenotypic screening of Penicillium marneffei F4 78

(sadA+), I6 (�sadA), and C3 (�sadAsadA+) strains

CHAPTER V RESULTS 83

5.1 Isolation and sequencing of the ura5 gene 83

5.2 Isolation and sequencing of the ura3 gene 86

5.3 Differential gene expression during phase transition and 89

stress-induced conditions

5.4 Isolation and characterization of uracil auxotrophs of 93

Penicillium marneffei

5.5 Agrobacterium-mediated transformation in Penicillium

marneffei

97

5.6 Confirmation of ble gene integration 99

5.7 Inverse PCR analysis of the T-DNA insertion junctions 102

5.8 Characterization of mutant phenotypes and corresponding 105

mutations

5.9 Isolation of putative mutants defective in cell wall production, 107

melanin synthesis, and dimorphic switch of Penicillium

marneffei

5.10 Agrobacterium-mediated transformation in Penicillium 113

citrinum

5.11 Functional analysis of a mutant defective in conidiation, 115

�sadA mutant

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5.11.1 Isolation of a mutant defective in conidiation from 115

AMT-derived transformants

5.11.2 sadA is required for spermidine synthesis in Penicillium

marneffei

115

5.11.3 Spermidine is required for the conidiation of 118

Penicillium marneffei

5.11.4 Spermidine is essential for growth and conidial 121

germination of Penicillium marneffei

CHAPTER VI DISCUSSION

6.1 Isolation of ura5 and ura3 genes and generation of uracil 125

auxotrophic mutants in Penicillium marneffei

6.2 Agrobacterium mediated transformation in Penicillium 127

marneffei

6.3 Agrobacterium mediated transformation in Penicillium 134

citrinum

6.4 Functional analysis of �sadA mutant 135

CHAPTER VII SUMMARY 140

REFERENCES 144

APPENDICES 176

Appendix A 177

Appendix B 178

Appendix C 179

Appendix D 180

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Page

Appendix E 181

Appendix F 182

Appendix G 183

Appendix H 187

CURRICULUM VITAE 198

xvii

LIST OF TABLES

Table Page

1 Heterologous probe for isolating Penicillium marneffei gene 36

2 Plasmids used for selectable markers in Penicillium marneffei

transformation

38

3 Primers used in gene isolation and sequencing study 80

4 Primers used in gene expression study 81

5 Primers used in Agrobacterium-mediated transformation and gene 82

function studies

6 Relative gene expression levels during phase transition and stress-

induced conditions

7 Colony diameter of uracil auxotrophs and Penicillium marneffei

wild type.

95

8 Comparison of the OPRTase activity of Penicillium marneffei F4 96

and its mutants

9 Average number of bleR transformants generated by 97

Agrobacterium-mediated transformation of Penicillium marneffei

10 Number of positive samples for inverse PCR using different 103

restriction enzymes

11 Genetic defects in AMT-generated mutants of Penicillium marneffei 106

12 Effect of different concentrations of spermidine on the growth and 120

conidiation of Penicillium marneffei

xviii

LIST OF FIGURES

Figure Page

1 Colony appearance and microscopic morphology 13

2 Diagrammatic representation of the Penicillium marneffei life cycle 15

3 Chemical reactions involving reactive oxygen species 22

4 Model describing the pathway proposed to operate in the signal 30

transduction of conidiation induction in Penicillium marneffei

5 Diagrammatic representation of the generation of saprobic mycelial 33

and parasitic yeast forms of Penicillium marneffei (in vitro model)

6 Schematic overview of the A. tumefaciens T-DNA transfer system 50

7 Polyamine biosynthetic pathway in Saccharomyces cerevisiae 53

8 Overview of Agrobacterium-mediated transformation system in 72

Penicillium marneffei

9 The nucleotide sequence and deduced amino acid sequence of the 84

ura5 gene from Penicillium marneffei

10 Alignment of the fungal Ura5 amino acid sequences 85

11 The nucleotide and deduced amino acid sequences of the ura3 gene 87

from Penicillium marneffei

12 Alignment of the fungal Ura3 amino acid sequences 88

13 Differential gene expression during phase transition and 90

stress-induced conditions

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Page

14 Expression of Penicillium marneffei actA and ura5 genes during 92

macrophage infection

15 Uracil requirement of Penicillium marneffei uracil auxotrophs 94

16 Colony appearance of Penicillium marneffei wild type strain and 94

uracil auxotrophs (5-FOAR1 and 5-FOAR8.1)

17 Selection of putative bleomycin-resistant transformants of 98

Penicillium marneffei

18 Determination of T-DNA integration by PCR 100

19 Southern blot analysis of selected AMT-generated transformants of 101

Penicillium marneffei

20 Identification of T-DNA insertion by inverse PCR 103

21 Sequence analysis of the T-DNA insertion junctions from AMT- 104

derived transformants of Penicillium marneffei.

22 A comparison of the colony appearance of the wild type 107

(P. marneffei F4) and the AMT-generated stuA and gasC mutants

23 Putative mutants defective in cell wall production 109

24 Putative mutants defective in melanin synthesis 110

25 Mutants defective in transition from mold to yeast 111

26 Mutants defective in transition from yeast to mold 112

27 Selection of putative bleomycin-resistant transformants of 114

Penicillium citrinum

28 Inverse PCR for identifying sadA gene and Southern blot analysis 116

for determining the copy number of T-DNA in sadA mutant

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Page

29 sadA is required for spermidine synthesis in Penicillium marneffei 117

30 Spermidine is required for the conidiation of Penicillium marneffei 119

31 Spotting assay 122

32 Mutation of sadA affects conidial germination at 27oC and 37o 123C

33 Spermidine is required for conidial germination at both 27o 124C and

37oC

xxi

ABBREVIATIONS

% PercentoC Degree Celsius

µg Microgram

µl Microliter

µm Micrometer

µM Micromolar

AIDS Acquired immunodeficiency syndrome

ATCC American Type Culture Collection

BLAST Basic local alignment search tools

BHI Brain heart infusion

bp Base pair

CBS Centraalbureau voor Schimmelcultures

cDNA Complementary deoxyribonucleic acid

CFU Colony forming unit

cm Centimeter

CMI Cell-mediated immunity

CO2

h hour(s)

Carbon dioxide

DEPC Diethylpyrocarbonate

DMEM Dulbecco’s modified Eagle’s medium

DNA Deoxyribonucleic acid

DNaseI Deoxyribonuclease I

EDTA Ethylenediamine tetraacetic acid

et al. And others

g Gram(s)

x g gravity

gDNA Genomic deoxyribonucleic acid

HIV Human immunodeficiency virus

xxii

H2O2

SDA Sabouraud dextrose agar

Hydrogen peroxide

kb Kilobase

kDa Kilodalton

l liter

lbs Pounds

LB Luria-Bertani media

M Molar

mg Milligram(s)

min Minute(s)

ml Milliliter(s)

mm Millimeter

mM Millimolar

mRNA Messenger ribonucleic acid

MW Molecular weight

NaOH Sodium hydroxide

NCBI National Center for Biological Information

ng Nanogram

nm Nanometer

no. Number

OD Optical density

ORF Open reading frame

PCR Polymerase chain reaction

pfu Plaque forming unit

pI Isoelectric point

RNA Ribonucleic acid

RNase Ribonuclease

ROS Reactive oxygen species

rpm Revolution per minute

rRNA Ribosomal ribonucleic acid

RST Random sequence tag

RT-PCR Reverse transcription-polymerase chain reaction

SD Standard deviation

xxiii

SDB Sabouraud dextrose broth

SDS Sodium dodecyl sulfate

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

s Second

SOD Superoxide dismutase

SSH Suppression subtractive hybridization

Taq Thermus aquaticus

tRNA Transfer ribonucleic acid

U Unit(s)

UV Ultraviolet

V Volt

Wt Weight