ISOLATION AND THE ELUCIDATION OF CYTOTOXICITY...

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UNIVERSITI PUTRA MALAYSIA

CYTOTOXICITY MECHANISM OF A FUNGAL INHIBITOR FROM A SOIL-DERIVED STREPTOMYCES SP.

JEE JAP MENG

FPSK(p) 2010 8

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CYTOTOXICITY MECHANISM OF A FUNGAL INHIBITOR FROM A

SOIL-DERIVED STREPTOMYCES SP.

By

JEE JAP MENG

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

JULY 2010

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Doctor of Philosophy

CYTOTOXICITY MECHANISM OF A FUNGAL INHIBITOR FROM A

SOIL-DERIVED STREPTOMYCES SP.

By

JEE JAP MENG

JULY 2010

Chairman : Professor Seow Heng Fong, PhD

Faculty : Medicine and Health Sciences

Effective fungal growth inhibitors are important to drive the development of

antifungal compound. In the search for fungal inhibitors, actinomycete H7372 was

isolated from a mangrove soil sample from Sabah. Subsequently, in a yeast cell-

based screening system, the crude acetone extract prepared from the fermentative

culture of H7372 was found to inhibit the growth of the yeasts. The purposes of this

study were to establish the phylogenetic position of H7372 and to isolate,

characterize and examine the toxicity mechanism of the active fungal growth

inhibitor produced by H7372. The partial sequence of 16S rRNA gene was amplified

from H7372 for phylogenetic analysis. An active compound was isolated from crude

acetone extract of mannitol-soybean fermentation culture and its structure was

elucidated. Antifungal properties of the isolated active compound against Candida

spp and Aspergillus spp were characterised by minimum inhibitory concentration and

time-kill kinetic studies. The consequences of C. glabrata treatment with the active

compound were examined by electron microscopy and cDNA microarray.

Phylogenetic analysis placed H7372 to its closest relative, S. kasugaensis M338-M1.

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The isolated active compound, designated as J5, was determined to be the natural

(11S, 13S, 9S, 8R)-cycloheximide. All Candida species tested (except C. albicans)

and only A. niger were sensitive to J5, with MIC at 24H ranging from 0.313 to 40

µg/ml. The degree of susceptibility shown by some species of Candida, from the

most to the least susceptible, were C. krusei, C. glabrata, C. rugosa and C.

parapsilosis. J5 is a fungistatic compound which showed total fungicidal effect at 12

times of its MIC when applied to C. glabrata. Treatment with J5 demonstrated

profound intracellular and cell surface modifications, such as by marked cell wall

thickening, confused cytoplasm, mitochondria loss, and irregular plasma membrane

invaginations with detachment of the protoplast from the cell wall. cDNA

microarray revealed a total of 60 genes affected by J5 treatment, corresponding to

genes involved in protein synthesis, plasma membrane and H+

pumps, mitochondria

maintenance and nutrient metabolism. In conclusion, H7372 is a Streptomyces sp.

which is closely related to S. kasugaensis M338-M1. The active compound, J5, is a

cis-cycloheximide. Comprehensive susceptibility profiles of Candida and

Aspergillus species toward J5 were established for the first time and generated new

MIC readings for C. krusei (0.313 µg/ml), C. rugosa (0.625 µg/ml), C. glabrata (2.5

µg/ml), C. parapsilosis (2.5 µg/ml), C. tropicalis (5 µg/ml) and A. niger (40 µg/ml).

Ultrastructures of J5-treated C. glabrata revealed new evidence on the toxicity

mechanisms of cycloheximide on plasma membranes and mitochondria. The gene

expression profiles for J5, the cycloheximide treatment, were revealed for the first

time in yeast.

Abstrak tesis yang dikemukakan kepada senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Doktor Falsafah

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MEKANISMA SITOTOSIKSITI PADA SATU PERENCAT KULAT

DARIPADA STREPTOMYCES SPESIS TANAH

Oleh

JEE JAP MENG

JULAI 2010

Pengerusi : Profesor Seow Heng Fong, PhD

Fakulti : Perubatan dan Sains Kesihatan

Bahan perencat pertumbuhan kulat yang berkesan adalah penting untuk menerajui

perkembangan kompaun anti-kulat. Dalam usaha pencarian bahan perencat

pertumbuhan kulat, aktinomysit H7372 telah dipencil daripada tanah di negeri Sabah.

Proses penyaringan yang menggunakan yis dua hybrid mendapati ekstrak mentah

fermentasi H7372 boleh merencat pertumbuhan yis. Justeru itu, kajian ini bertujuan

untuk megetahui filogeni H7372 dan untuk memencil, memeriksa mekanisma

tosiksiti bahan aktif yang dihasilkan daripada H7372. Gen 16S RNA telah

diamplifikasi daripada H7372 sebagai bahan genetik untuk kajian filogeni. Satu

bahan aktif telah dipencil daripada ekstrak mentah aseton kultur fermentasi mannitol-

kacang soya dan struktur kimia bahan aktif ini juga ditentukan. Kesan perencatan

antikulat bahan aktif kepada spesis Candida dan Aspergillus ditentukan sebagai

kepekatan perencatan mimima (MIC) dan kinetik perencatan semasa. Kesan rawatan

bahan aktif pada C. glabrata telah diperiksa melalui mikroskop elektron. Analisis

filogenetik mnedapati spesis saudara terdekat kepada H7372 adalah S. kasugaensis

M338-M1. Analisis struktur kimia mendapati bahan aktif yang telah dipencil, yang

dinamakan sebagai J5 adalah cis-cycloheximide. Semua spesis Candida (kecuali C.

albicans) dan hanya Aspergillus niger adalah sensitif kepada J5, dengan MIC 24 jam

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berjulat daripada 0.313 ke 40 µg/ml. Darjah sensitiviti yang disusun daripada spesis

paling sensitif adalah seperti C. krusei, C. glabrata, C. rugosa dan C. parapsilosis. J5

bersifat fungistatik, hanya menjangkal pembunuhan sepenuhnya pada kepekatan 12

kali MICnya. Rawatan J5 pada C. glabrata menunjukkan kesan-kesan modifikasi

yang drastik pada luar permukaan sel and intrasellular seperti penebalan dinding sel,

kerosakan sitoplasma, kehilangan mitokondria dan juga invaginasi pada plasma

membran yang abnormal dengan susutan protoplas daripada dinding cell. Mikroarrai

cDNA memaparkan 60 gen terkait dengan rawatan J5, gen-gen ini diklasifikasi

kepada sintesis protein, plasma membran dan pam proton, penyelengaraan

mitokondria dan metabolisme nutrien. Kesimpulannya, H7372 merupakan spesis

Streptomyces yang berkait rapat dengan S. kasugaensis M338-M1. Kompaun aktif,

J5 adalah cis-cycloheximide. Profail kepekaan komprehensif spesis-spesis Candida

dan Aspergillus kepada cycloheximide semulaji telah ditentukan buat kali pertama

dan menghasilkan nilai MIC baharu kepada C. krusei (0.313 µg/ml), C. rugosa

(0.625 µg/ml), C. glabrata (2.5 µg/ml), C. parapsilosis (2.5 µg/ml), C. tropicalis (5

µg/ml) dan A. niger (40 µg/ml). Ultrastruktur C. glabrata selepas rawatan J5

menunjukkan bukti-bukti baharu mekanisma tosiksiti cycloheximide pada plasma

membran dan mitokondria. Profil ekpresi gen J5, iaitu rawatan cycloheximide telah

diwujudkan buat kali yang pertama.

ACKNOLEDGEMENTS

I wish to acknowledge generous individuals whose valuable supports made this study

a success. I convey my most sincere thank to my supervisors, Professor Seow Heng

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Fong, Assoc. Prof. Chong Pei Pei and Professor Tan Wen Siang for their invaluable

advices, guidance and criticisms during my study.

Special thank goes to Professor Ho Coy Choke for providing the H7372, sincere

guidance, critical comments, useful discussion and proof reading of this thesis. I

sincerely thank Dr. Chang Leng Chee in University of Hawaii Hilo, USA for her

kind assistance in the elucidation of J5 structure. I thank Mr. Ho Oi Kuan from

electron microscopic unit of IBS, UPM for his patient guidance and excellent

technical support in electron microscopy studies of my yeast samples. I thank Dr.

Takuji Kudo from RIKEN, Japan for his invaluable review, advice and taught on

taxonomy of H7372. I thank Professor Ng Kee Peng from UMMC for providing the

clinical Candida isolates. Many thanks go to Dr. Phelim Yong, Madam Juita and all

members in immunology laboratory (2005-2009), UPM for their help in routine

laboratory work.

Lastly, special appreciation goes to my parents, Channy, sisters and brother for their

encouragement, patient and cares that brought me to the end of my study.

I certify that Examination Committee met on _______________________ to conduct

the final examination of Jee Jap Meng on his Doctor of Philosophy thesis entitled

“Cytotoxicity Mechanism of a Fungal Inhibitor from a Soil-Derived Streptomyces

sp.” in accordance with the Universiti Pertanian Malaysia (Higher Degree) Act 1980

and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The

Committee recommends that the candidate be awarded the relevant degree. Members

of the Examination Committee are as:

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______________________________

(Chairperson)

________________________________

(Internal Examiner I)

________________________________

(Internal Examiner II)

_______________________________

(Independent Examiner)

_______________________

Professor / Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as

fulfilment of the requirement for the degree of Doctor of Philosophy. The members

of the Supervisory Committee are as follows:

Seow Heng Fong, Ph.D.

Professor

Faculty of Medicine and Health Sciences


(Member)

Chong Pei Pei, Ph.D.

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Associate Professor

Faculty of Medicine and Health Sciences


(Member)

Tan Wen Siang, Ph. D.

Professor

Faculty of Biotechnology and Biomolecular Sciences


(Member)

______________________________

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduate Studies


Date: 25 November 2010

DECLARATION

I declare that the thesis is my original work except for quotations and citations,

which have been duly acknowledged. I also declare that it has not been previously

and is not concurrently submitted for any other degree at Universiti Putra Malaysia

or other institutions.

__________________________

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JEE JAP MENG

Date: 30 July 2010

TABLE OF CONTENTS

Page

ABSTRACT I

ABSTRAK III

ACKNOWLEDGEMENTS V

DECLARATION

VIII

LIST OF TABLES

XIII

LIST OF FIGURES XIV

LIST OF ABBREVIATIONS XVI

CHAPTER

1 GENERAL INTRODUCTION 1

2 LITERATURE REVIEW 5

2.1 Fungi 5

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2.1.1 Medically Important Fungi and Mycoses 6

2.1.2 Candida and Candidissis 7

2.1.3 A Shift towards Non-albicans Species 8

2.1.4 Aspergillus species 9

2.2 Antifungal Agents and Mode of Actions 9

2.2.1 Polyenes 11

2.2.2 Azoles 12

2.2.3 Echinocandins 14

2.2.4 Flucytosine 17

2.3 Actinomycetes 18

2.3.1 Secondary Metabolites-Antibiotics 19

2.3.2 Actinomycetales and Streptomyces as

Major Antibiotics Producers 20

2.3.3 Streptomyces as Antifungal Producer 24

2.4 Challenges in Antifungal Drug Research 25

2.4.1 Are Virulence Factors Good Target of Antifungals? 25

2.4.2 Antifungal Resistance 27

2.5 Cycloheximide 30

2.5.1 Chemical Properties of Cycloheximide 30

2.5.2 Toxicity of Cycloheximide 31

2.5.3 Application of Cycloheximide 31

2.6 Scanning and Transmission Electron Microscopy 33

2.7 Yeast cDNA Microarray in Studies of Mode of Action

of Antifungal Compounds 35

3 16S rRNA GENE ANALYSIS FOR MOLECULAR

PHYLOGENETIC TAXONOMY OF H7372

3.1 Introduction 38

3.2 Materials and methods 40

3.2.1 H7372 Culture maintenance 40

3.2.2 DNA Extraction and PCR Amplification of

16S rRNA Gene 40

3.2.3 Microbial Strains for Phylogeny Studies 41

3.2.4 Phylogenetic Tree Construction 44

3.3 Results 46

3.3.1 Culture Morphology 46

3.3.2 Phylogeny of H7372 47

3.4 Discussion 52

3.4.1 Phylogeny of H7372 52

3.4.2 Novel Strain Status of H7372 53

3.5 Conclusion 54

4 ISOLATION AND IDENTIFICATION OF BIOACTIVE

COMPOUNDS FROM ORGANIC EXTRACTS OF H7372

4.1 Introduction 55

4.2 Materials and Methods 57

4.2.1 Culture Stock of H7372 57

4.2.2 Fermentation of H7372 and Preparation of

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Crude Acetone Extract 57

4.2.3 Preparation of Crude Acetone Extract 57

4.2.4 HPLC Isolation of Bioactive Compound 57

4.2.5 Identification of Active Fraction 58

4.2.6 Determination of Fraction Purity 59

4.2.7 NMR and HRESIMS Analysis of Active Compound 59

4.3 Results 60

4.3.1 Isolation and Determination of Active Compound 60

4.3.2 Structural Analysis 65

4.4 Discussion 69

4.4.1. HPLC Isolation of Active Compound 69

4.4.2. Structure Analysis of J5 70

4.5 Conclusion 72

5 CHARACTERISATION OF THE GROWTH INHIBITION

PROPERTIES OF J5 (CYCLOHEXIMIDE) 5.1 Introduction 73


5.2.1 Test Strains for MIC Determination 74

5.2.2 MIC Determination 75

5.2.3 Time-kill Kinetic Study 76

5.2.4 Viable Cell Count 76

5.3 Results 77


5.3.2 Time-kill Kinetics 81

5.4 Discussion 84


5.4.2 Time-kill Kinetics 87

5.5 Conclusion 90

6 EVALUATING THE TOXICITY OF J5 (CYCLOHEXIMIDE)

IN C. GLABRATA BY ELECTRON MICROSCOPY

6.1 Introduction 91


6.2.1 IC50 of J5 92

6.2.2 J5 Treatment 92

6.2.3 Sample Preparation for Electron Microscopy 92

6.2.4 Scanning Electron Microscopy 93

6.2.5 Transmission Electron Microscopy 93

6.3 Results 95

6.3.1 IC50 of J5 95

6.3.2 Scanning Electron Microscopy 97

6.3.3 Transmission Electron Microscopy

100

6.4 Discussion

107

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6.4.1 Length of J5 Exposure

107

6.4.2 The Effects of J5 on Cell Envelope

108

6.4.3 Highly Affected J5-Treated Cells

111

6.4.4 Other Possible Intracellular Activity of J5

112

6.5 Conclusion

112

7 GENE EXPRESSION PROFILLING FOR ANALYSIS

OF THE TOXICITY OF J5 (CYCLOHEXIMIDE)

7.1 Introduction

113

7.2 Materials and Methods

115

7.2.1 J5 Treatment

115

7.2.2 RNA Isolation

115

7.2.3 cDNA Conversion, Labelling and Microarray Analysis

117

7.3 Results

118

7.4 Discussion

136

7.4.1 Genes Associated with the Plasma Membrane

136

7.4.2 Genes Associated with Mitochondria Maintenance

and Biogenesis

143

7.4.3 Genes Associated with Glucose and Nitrogen

Metabolisms

146

7.4.4 Genes Associated with Protein Synthesis

148

7.5 Conclusion

150

8 GENERAL DISCUSSION AND CONCLUSION

151

REFERENCES 162

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