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Research on the Diversity and the Biomedical Potential of Marine Fungi in Hawaii a Thesis Submitted to the Graduate Division Of

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Research on the Diversity and the Biomedical Potential of Marine Fungi in Hawaii a Thesis Submitted to the Graduate Division Of lJN/VERS/"fY OF HAW.';''' LIBRARY RESEARCH ON THE DIVERSITY AND THE BIOMEDICAL POTENTIAL OF MARINE FUNGI IN HAWAII A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MICROBIOLOGY AUGUST 2007 By Mingxiao He Thesis Committee: Dr. Guangyi Wang, Chairperson Dr. Paul Patek Dr. Philip Williams We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope and quality as a thesis for the degree of Master of Science in Microbiology. THESIS COMMITTEE Chairperson 11 ACKNOWLEDGEMENTS I am extremely grateful to my advisor, the other professors in my committee, and people in my lab for their help in my research and my life. I especially want to thank Dr. Guangyi Wang. Without his guidance and funding, I would never have been able to have this project done; I also want to thank Dr Paul Patek, for teaching me the techniques of cell culture and cytotoxic assay, for allowing me to perform cytotoxicity assays in his laboratory, and for advising me in this project, and to thank Dr Philip Williams for his help and suggestion regarding natural products screening. I would also like to thank all the current and former lab members for their valuable help in teaching me new techniques, making suggestions for the experiments, and ordering materials and instruments for the project. I would also want to show my gratitude to Dr Susan Ayin in Microbiology Department and Stephanie Christensen in Post 07 for proofreading my thesis writing. Without their help, I could not have finished my thesis. Lastly, I would like to thank my new friends in US and myoid friends in China, without their comfort and support, life would have been harder here. Again, please allow me to express my gratitude to all those who have helped me in the past two years. iii ABSTRACT This study was designed to survey the diversity of marine fungi in Hawaiian waters using molecular biological methods and to do preliminary screening for biological isolates with pharmaceutical potential based on biological activities. 106 marine fungal isolates were isolated from nine algae species from eight different locations around the coast of the Hawaii Island in Hawaii. These fungal isolates were classified based on the ribosomal intemal transcribed spacer regions, and were found to represent 10 orders including 57 species of Ascomycota, and 2 orders including 3 species ofBasidiomycota. Two isolates, Haw 3BII and Haw 7A3, representing long phylogenetic distances from known sequences in Genbank, are potential new species. Anti-bacterial assays were performed with the crude extracts obtained from the cultures of 70 algae associate fungi in Hawaii. In total, eight extracts shown inhibitory activity to Bacillus subtilis and four to Staphylococcus aureus, respectively. None inhibited activity to the gram negative bacteria Pseudomonas aeruginosa and E.coli K12. Anti-tumoral assays using murine cell lines BCN (non-tumorgenic) and L88 (tumorgenic) were performed with the crude extracts of the cultures of 70 algae associated fungi and 58 sponge-associated fungi. In total, four extracts, one from algae- associated fungi and three from sponge-associated fungi, showed strong inhibition of L88 and slight or no inhibition of BeN. Those isolates will be further studied for their potential in drug discovery. iv TABLES OF CONTENTS Acknowledgement ...............................................................................iii Abstract .............................................................................................iv List of Tables .................................................................................... viii List of Figures ...................................................................................ix Lists of Abbreviations ........................................................................... x Chapter 1. Introduction and literature review ................................................ 1 1.1 Definition of Marine fungi ............................................................ .1 1.2 Historical perspective ................................................................... 1 1.3 Marine fungal research in Hawaii.. ................................................. 3 1.4 Identification and taxonomy of fungal species ...................................... 3 1.5 Molecular DNA targets for fugal identification, diversity and phylogeny studies .....................................................................................5 1.6 PCR Primers to amplify the ITS regions ............................................8 1.7 Pharmacological activities of the secondary metabolites from marine fungi .. 10 1.7.1 History ............................................................................ 10 1.7.2 Anti-bacterial activity ...........................................................11 1.7.3 Anti-viral activity ............................................................... 13 1.7.4 Anti-tumor and cytotoxic activity ............................................. 14 1.7.5 Anti-protozoal activity ......................................................... 15 1.7.6 Antifungal activity .............................................................. 16 v Chapter 2. Survey for the biodiversity of marine fungi on algal substrates around the coast of the Island of Hawaii (Big Island) .................................... 18 2.1. Materials and methods ............................................................. .18 2. I. I Sampling of marine algae .................................................... I 8 2.1.2 Extraction of Genomic DNA from fungal isolates ........................ 18 2.1.3 Marine Fungi isolation ...................................................... 18 2.1.4 PCR amplification of ribosomal internal transcribed spacer regions ... 19 2.1.5 Sequencing of the PCR products and analyses ............................ 19 2. 2 Results ..............................................................................20 2.2.1 Results of PCR. sequencing and BLAST analyses ......................20 2.2.2 ClassifYing of selected sequences in phylogenetic trees ................24 2. 3 Discussions ...........................................................................28 Charter 3. Biological activity assays of the Hawaiian marine fungal isolates ......... 33 3.1 Materials and Methods ............................................................... 33 3.1.1 Culturing of marine fungi.. ....................................................33 3.1.2 Culture extracts ................................................................33 3.1.3 Bacterial strains and murine cell lines .......................................33 3.1.4 Anti -bacterial assay .............................................................34 3.1.5 Cytotoxicity assay .............................................................. 35 3.2 Results ..................................................................................35 3.2.1 Antibacterial assay .............................................................35 3.2.2 Cytotoxicity assay .............................................................. 37 vi 3. 3 Discussions ...........................................................................33 Appendix 1...................................................................................... 42 Appendix 2 .....................................................................................43 Appendix 3 ......................................................................................45 Appendix 4 ......................................................................................47 References ......................................................................................50 vii LIST OF TABLES Table Page 1. Common ITS primers and their sequences .............................................9 2. BLAST results of ITS sequences of fungal isolates from algae collected in the Island of Hawaii ..................................................................... 21 3 Isolates which shown antibacterial activity against B. subtilis strain ...............36 4. Isolates which shown antibacterial activity against S. aureus strain ...............37 5. Isolates which shown cytotoxic activities ............................................. .38 viii LIST OF FIGURES Figure Page I. The number of distinct fungal genera based on the marine source ............ 2 2. Schematic map of a repeat unit of eukaryotic rDNA .............................7 3. Internal transcribed spacers (ITS) region primers .................................9 4. The distribution of new compounds reported from marine-derived fungi is shown as a function of the fungal source ..........................................11 5. Neighbor-Joining tree of algae-derived fungi in the Island of Hawaii based on ITS sequences .................................................................. 24 6. Neighbor-Joining tree of Group I .................................................25 7. Neighbor-Joining tree of Group 2 ................................................ 26 8. Neighbor-Joining tree of Group 3 .................................................27 9. Neighbor-Joining tree of Group 4 .................................................28 10. Inhibition zones of B. subtilis ...................................................... 36 II. Inhibition zones of S. aureus ...................................................... 36 12. Morphology of BeN cell line
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