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Genome Sequencing and in Silico Characterisation of Cladosporium Sphaerospermum

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Genome Sequencing and in Silico Characterisation of Cladosporium Sphaerospermum GENOME SEQUENCING AND IN SILICO CHARACTERISATION OF CLADOSPORIUM SPHAEROSPERMUM YEW SU MEI Malaya of FACULTY OF MEDICINE UNIVERSITY OF MALAYA UniversityKUALA LUMPUR 2017 GENOME SEQUENCING AND IN SILICO CHARACTERISATION OF CLADOSPORIUM SPHAEROSPERMUM YEW SU MEI Malaya of THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY FACULTY OF MEDICINE UNIVERSITY OF MALAYA KUALA LUMPUR University 2017 UNIVERSITY OF MALAYA ORIGINAL LITERARY WORK DECLARATION Name of Candidate: YEW SU MEI Registration/Matric No: MHA110037 Name of Degree: Doctor of Philosophy Title of Thesis: Genome Sequencing and in silico Characterisation of Cladosporium sphaerospermum Field of Study: MEDICAL MICROBIOLOGY I do solemnly and sincerely declare that: (1) I am the sole author/writer of this Work; (2) This Work is original; (3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title ofMalaya the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutesof an infringement of any copyright work; (5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained; (6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subject to legal action or any other action as may be determined by UM. Candidate’s Signature Date: UniversitySubscribed and solemnly declared before, Witness’s Signature Date: Name: Designation: ii ABSTRACT Dematiaceous fungi are a taxonomically diverse group of heterogeneous fungi that are present in the environment. Although rare, many species in this group are known to cause diseases in humans and animals. In this study, a collection of 75 fungal isolates were recovered from various clinical specimens. They were identified through morphology and molecular methods using the internal transcribed spacer (ITS)-based phylogenetic analysis. The complementation of the molecular approach enables the identification of 73 isolates at the species level. The most common species isolated were Cladosporium, Cochliobolus, and Neoscytalidium. Also, 16 isolates from the seven species identified have not been reported to cause human infections. Among the Cladosporium species that were isolated, the speciesMalaya C. sphaerospermum was most frequently isolated. This study was proceeded by whole genome analysis of the C. sphaerospermum, using the strain UM of843 as a study model to explore genetic information and its basic biology. C. sphaerospermum is a ubiquitous saprophytic fungus that has been documented to cause allergy and other opportunistic diseases in humans. Detail characterisation of this isolate has affirmed the identity of UM 843 and its ability to survive in high salt medium (up to 20% NaCl). The genome of UM 843 was generated by combining 500-bp and 5-kb insert libraries, containing 9,652 predicted gene models in the 26.89 Mb genome. Functional annotation of the predicted genesUniversity suggests this fungus has the capability to degrade carbohydrate and protein complexes. The abundance of carbohydrate active enzymes (CAZymes) assists in the degradation of hemicellulose and pectin, thus suggesting the precedence of this fungus towards soft plant tissues. Putative genes encoding peptidases from family A01, S09, and M36 that share similarity with Aspergillus peptidases responsible for lung tissue hydrolysis were also identified. UM 843 was also found to carry 16 putative secondary iii metabolite backbone genes, three of these are predicted to be responsible for the synthesis of melanin, siderophores, and cladosins. Besides siderophores, UM 843 contains gene encoding proteins that are involved in iron homeostasis via the reductive iron assimilation (RIA) process. In response to salinity, UM 843 was identified to encode 29 ion transporters and the synthesis of various compatible solutes. The analysis of sexual reproduction suggests that UM 843 is a heterothallic fungus. The genome of UM 843 revealed 28 putative genes associated with C. sphaerospermum allergy. Orthologous gene analysis on UM 843 with 22 other Dothideomycetes showed 125 genes uniquely present in UM 843. Of these, four genes were predicted to encode class 1 hydrophobins which may be allergens specific to Cladosporium. Further analysis showed that the mRNA of the four genes were detected by reverse transcription- polymerase chain reaction (RT-PCR). The genomic Malayaanalysis of C. sphaerospermum UM 843, can significantly contribute to the knowledge of the biology and allergenicity of this species which has not been studied previously.of University iv ABSTRAK Kulat Dematiaceous merupakan kumpulan kulat heterogenus pelbagai taksonomi hadir dalam persekitaran. Walaupun jarang ditemui, kebanyakan spesies dalam kumpulan ini dikenalpasti berupaya menyebabkan jangkitan pada manusia dan haiwan. Di dalam kajian ini, sebanyak 75 pencilan kulat daripada pelbagai spesimen klinikal telah dikaji. Kulat-kulat ini telah dikenalpasti secara penggunaan kaedah morfologi dan molekul melalui analisis filogenetik berasaskan penjarak transkripsi dalaman (ITS). Pendekatan kaedah pelengkap molekul ini telah berjaya mengenalpasti 73 pencilan pada peringkat spesies. Spesies yang paling lazim dikenalpasti adalah Cladosporium, Cochliobolus dan Neoscytalidium. Selain itu, sebanyak 16 pencilan daripada tujuh spesies yang telah dikenalpasti tidak pernah dilaporkanMalaya lagi menyebabkan jangkitan pada manusia. C. sphaerospermum merupakan spesies yang paling kerap dipencil di antara spesies Cladosporium yang telah dikenalpasti.of Kajian analisis keseluruhan genom C. sphaerospermum telah dilanjutkan dengan menggunakan strain UM 843 sebagai model kajian untuk meneroka maklumat genetik dan biologi asas pencilan tersebut. C. sphaerospermum merupakan kulat saprofitik ubiquitous yang sebelum ini telah dilaporkan menyebabkan tindak balas alahan dan penyakit oportunis pada manusia. Identiti pencilan UM 843 telah disahkan secara pencirian teliti dan ia dikenalpasti mempunyai keupayaan hidup di dalam media yang mengandungi kandungan garam tinggiUniversity (sehingga 20% NaCl). Genom UM 843 yang dihasilkan melalui gabungan perpustakaan selitan bersaiz 500-bp dan 5-kb, mengandungi sebanyak 9,652 gen model diramalkan hadir dalam 26.89 Mb genom tersebut. Hasil ramalan kefungsian anotasi gen-gen ramalan menunjukkan kulat ini mempunyai keupayaan untuk mengurai kompleks karbohidrat dan protein. Kulat ini dicadangkan memberikan keutamaan pada tisu-tisu lembut oleh kerana kehadiran kandungan enzim aktif karbohidrat (CAZymes) v yang tinggi berupaya membantu di dalam penguraian hemiselulosa dan pektin. Kehadiran gen-gen putatif mengekodkan kumpulan peptidase A01, S09 dan M36 yang mempunyai persamaan dengan peptidase Aspergillus yang berperanan dalam hidrolisis tisu peparu telah berjaya dikenal pasti. UM 843 juga didapati mempunyai 16 gen putatif pengkodan gen tulang belakang metabolit sekunder, di mana tiga daripada gen ramalan tersebut mempunyai peranan di dalam sintesis melanin, siderofor dan kladosin. Selain daripada siderofor, UM 843 mengandungi gen pengekodan protein yang terlibat dalam homeostasis besi melalui proses asimilasi penurunan besi (RIA). Sebanyak 29 gen yang terlibat sebagai pengangkut ion dan sintesis pelbagai bahan larut serasi yang berupaya memberikan gerak balas terhadap kandungan garam tinggi turut dikenalpasti. Hasil analisis pembiakan seksual menunjukkan UM 843 merupakan kulat heterotalik. Sebanyak 28 gen yang berkemungkinan berkaitanMalaya dengan tindak balas alahan C. sphaerospermum turut didapati hadir dalam genom UM 843. Manakala hasil analisis gen ortolog UM 843 bersama 22 kulat dariof kelas Dothideomycetes lain menunjukkan sebanyak 125 gen unik hadir di dalam UM 843. Antaranya adalah empat gen diramalkan mengekod hidrofobin kelas 1 yang bermungkinan merupakan alergen khusus kepada Cladosporium. Analisis lanjutan menunjukkan bahawa mRNA daripada empat gen tersebut dapat dikesan melalui tindak balas rantai polimerase transkripsi berbalik (RT-PCR). Analisis genom C. sphaerospermum UM 843 dijangka akan menyumbang kepada kefahaman biologi dan tindak balas alahan yang sebelum ini tidak pernahUniversity dikaji lagi. vi ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest gratitude to my supervisor, Professor Dr. Ng Kee Peng for his guidance, encouragement throughout my doctoral study, and giving me the freedom to venture into research on my own. I am also thankful for Professor Dr. Ngeow Yun Fong for her suggestion and discussion on my work. I would like to express my deepest appreciation to Dr. Kuan Chee Sian and collaborators from Codon Genomics Sdn Bhd (especially Dr. Yee Wai Yan and Lee Kok Wei), for their contribution to valuable feedback, assistance, advice and insightful discussion about the research. Malaya I would like to thank all my friends (especially Fan Suet Pin, Kang Wen Tyng, Theresa Yap and Lydia Ong) and fellow colleaguesof from UM (Na Shiang Ling, Shirley Liew, Toh Yue Fen) for their help and make my time
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