COMPARATIVE GENOME ANALYSIS AND EVOLUTIONARY STUDY OF HUMAN PATHOGENIC YERSINIA SPECIES TAN SHI YANG Malaya of FACULTY OF DENTISTRY UNIVERSITY OF MALAYA UniversityKUALA LUMPUR 2017 COMPARATIVE GENOME ANALYSIS AND EVOLUTIONARY STUDY OF HUMAN PATHOGENIC YERSINIA SPECIES TAN SHI YANG Malaya of THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY FACULTY OF DENTISTRY UniversityUNIVERSITY OF MALAYA KUALA LUMPUR 2017 UNIVERSITY OF MALAYA ORIGINAL LITERARY WORK DECLARATION Name of Candidate: TAN SHI YANG (I.C/Passport No: Registration/Matric No: DHA130012 Name of Degree: Doctor of Philosophy Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”): COMPARATIVE GENOME ANALYSIS AND EVOLUTIONARY STUDY OF HUMAN PATHOGENIC YERSINIA SPECIES Field of Study: BIOINFORMATICS 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 of the Work and its authorship have been acknowledged in this Work; (4) I do not have any actual knowledge nor doMalaya I ought reasonably to know that the making of this work constitutes 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”), whoof 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 Yersinia is a Gram-negative bacterial genus that includes serious pathogens such as the Yersinia pestis which causes plague, and Yersinia pseudotuberculosis and Yersinia enterocolitica which cause gastrointestinal infections. The remaining species are generally considered to be non-pathogenic to humans. While their virulence mechanisms are well-characterized, the evolution of Yersinia pathogens are not well-understood. To understand the evolution of Yersinia pathogens and Yersinia enterocolitica subspecies, an exhaustive evolutionary and comparative genome studies on a total of 86 Yersinia genomes using different bioinformatics approaches were performed. Based on phylogenetic and the gene gain-and-loss analyses, Yersinia enterocolitica and Yersinia pseudotuberculosis-Yersinia pestis were determined as belonging to different phylogroups and have acquired different set of metabolismMalaya genes, suggesting that the evolution of human pathogenic Yersinia species is most probably triggered by ecological specialization. Besides, pairwise sequenceof comparisons showed that the ail virulence gene of Yersinia enterocolitica had higher sequence identities to the ail gene family (consists of both ail gene and homologs in the same family) of Yersinia pseudotuberculosis-Yersinia pestis compared to its own ail homolog, suggesting that the ail gene might have been duplicated in the latter species and then transferred laterally to Yersinia enterocolitica. Taken all together, it is proposed that the evolution of Yersinia is not in parallel, but rather accompanied by the gene gain-and-loss, gene duplication and lateralUniversity gene transfer. This contradicts finding of previous study that suggested the human pathogenic Yersinia species might have evolved in parallel to acquire the same virulence determinants. On the other hand, phylogenetic tree and gene gain-and-loss analyses in this study showed that Yersinia enterocolitica strains could be demarcated into three distinct phylogroups, iii with each of them acquiring different sets of putative metabolism genes. This postulates that ecological specialization might have triggered subspeciations in Yersinia enterocolitica species and lead to the emergence of highly pathogenic, low pathogenic and non-pathogenic subspecies, instead of two subspecies as previously reported. Data gathered in this study also suggest that the lateral gene transfer between subspecies in Yersinia enterocolitica might not be extensive as the gene content-based phylogenetic tree highly resembled supermatrix tree. Further virulence gene analyses showed that the ail gene was pseudogenized in the non-pathogenic subspecies, probably causing the loss of pYV virulence plasmid and pathogenicity in this subspecies. To facilitate the ongoing and future research of Yersinia, YersiniaBase, a robust and user- friendly Yersinia resource and comparative analysis platform for analysing Yersinia genomic data was developed. The AJAX-basedMalaya real-time searching system was implemented to smooth the process of searchingof genomic data in large databases. YersiniaBase also has in-house developed tools: (1) Pairwise Genome Comparison tool for comparing two user-selected genomes; (2) Pathogenomics Profiling Tool for comparative virulence gene analysis of Yersinia genomes; (3) YersiniaTree for constructing phylogenetic tree of Yersinia. Successful applications of these useful tools was demonstrated in this study. Overall,University this study provides better insights in elucidating the evolution of human pathogenic Yersinia and subspeciation in Yersinia enterocolitica. Lastly, the YersiniaBase will offer invaluable Yersinia genomic resource and analysis platform for the analysis of Yersinia in the future. iv ABSTRAK Yersinia adalah genus bakteria Gram-negatif yang terdiri dari patogen penting seperti Yersinia pestis yang menyebabkan wabak, dan Yersinia pseudotuberculosis serta Yersinia enterocolitica yang menyebabkan jangkitan di usus. Spesies Yersinia yang lain tidak patogenik kepada manusia. Walaupun mekanisme kevirulenannya telah difahami, evolusi pathogen Yersinia masih kurang difahami. Untuk memahami evolusi patogen Yersinia dan subspesies Yersinia enterocolitica, kajian menyeluruh ke atas evolusi dan perbandingan genom dalam kalangan 86 genom Yersinia telah dijalankan menggunakan pelbagai pendekatan bioinformatik. Berdasarkan analisis filogenetik dan gen gain-and- loss, Yersinia enterocolitica dan Yersinia pseudotuberculosis-Yersinia pestis didapati telah ditempatkan dalam phylogroup yang berlainan dalam pokok filogenetik, dan turut memiliki gen metabolisme yang berbeza. Ini mencadangkanMalaya bahawa evolusi patogen Yersinia telah dicetuskan oleh pengkhususan ekologi. Di samping itu, perbandingan pasangan jujukan gen menujukkan gen ailof daripada Yersinia enterocolitica mempunyai identiti jujukan gen yang lebih tinggi kepada keluarga gen ail daripada Yersinia pseudotuberculosis-Yersinia pestis berbanding dengan homolog ail sendiri. Ini mencadangkan gen ail mungkin telah diduplikasi dalam spesies kedua dan dipindahkan ke Yersinia enterocolitica. Hasil penemuan ini mencadangkan bahawa evolusi dalam Yersinia adalah tidak selari, tetapi dicetuskan oleh gen gain-and-loss, duplikasi gendan pemindahan gen secara lateral. Ini bercanggah dengan hasil kajian sebelum ini yang mana evolusiUniversity patogen Yersinia dikatakan selari untuk mendapatkan kevirulenan gen yang serupa Selain itu, analisis pokok filogenetik dan gen gain-and-loss hasil kajian ini menujukkan strain Yersinia enterocolitica boleh dibahagikan kepada tiga phylogroup dengan setiapnya memiliki set gen metabolisme yang berbeza. Pengkhususan ekologi telah v dicadangkan sebagai penyebab yang membawa kepada kemunculan subspecies dengan kepatogenan tinggi, kepatogenan rendah dan tidak patogenik, dan bukan dua subspesies seperti yang dilaporkan sebelum ini. Data kajian juga mencadangkan bahawa pemindahan gen secara lateral di antara subspesies Yersinia enterocolotica mungkin tidak menyeluruh kerana pokok filogenetik kandungan gennya hampir menyamai pokok filogenetik supermatrix. Analisa lanjut ke atas gen virulen menunjukan gen ail telah tersingkir dalam subspesies yang tidak patogenik dan mungkin menyebabkan kehilangan plasmid virulen pYV dan kepatogenan dalam kalangan subspecies ini. Untuk memudahkan penyelidikan Yersinia pada masa kini dan akan datang, YersiniaBase iaitu satu platform untuk sumber dan perbandingan genom Yersinia telah dibangunkan Sistem carian real-time berasaskan AJAX ini di implementasikan untuk melancarkan pencarian data dalam pangkalan dataMalaya yang lebih besar. YersiniaBase dibangunkan dengan alat seperti (1) ofPairwise Genome Comparison tool yang membandingkan dua genom Yersinia (2) Pathogenomics Profiling Tool yang membandingkan gen virulen Yersinia (3) YersiniaTree yang membina pokok filogenetik Yersinia. Kejayaan aplikasi system carian ini telah dipamerkan dalam kajian ini. Kesimpulannya, kajian ini memberikan pemahaman yang lebih baik dalam menjelaskan evolusi Yersinia yang patogen kepada manusia dan pembahagian subspesies dalam YersiniaUniversity enterocolitica. Akhir sekali, YersiniaBase menawarkan sumber genom yang berharga untuk Yersinia dan satu platform bagi analisa Yersinia pada masa akan datang. vi ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my family members, especially grandparents and parents for their love, spiritual support and motivation of my Ph.D.