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Molecular Characterisation of Virulence in Entamoeba Histolytica

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Molecular Characterisation of Virulence in Entamoeba histolytica Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Kanok Preativatanyou April 2015 Acknowledgement s My sincerest gratitude and thanks go to Prof. Neil Hall and Prof. Steve Paterson for their supervision, knowledge, care and support throughout this PhD. I am very delighted to have been given the opportunity to learn how to think integratively about Biology and explain wisely in an evolutionary sense. Over the past four years, Neil has taught me different ways of how to comprehensively approach and solve the problems, changing my learning attitude into the better direction than ever. This makes me very impressed and I will be very fortunate if I have a chance to collaborate with them in the long run! Heartfelt thanks also go to Dr. Gareth Weedall who has provided me with a lot of knowledge, guidance, inspiration as well as warm encouragement. He often advises me to analyse the data carefully and professionally, upgrading my research skills and performance. Every time I can answer his difficult questions, I get more self confident and relaxed. I have always been very grateful for all his special attention and warm kindness. Absolutely, I can say that Gareth has contributed a lot of success to me in this PhD! To Dr. Xuan Liu and Dr. Yongxiang Fang, I really appreciate their wonderful Linux command lines and statistical scripts. As Xuan said ‘You are in the Big Data Era’, this sentence really motivated me to explore more about Bioinformatics. Thanks for their teachings and great encouragement! In addition, I wish to express sincere gratitude to Dr. Linda D’Amore, Dr. John Kenny, Dr. Lisa Olohan, Dr. Margaret Hughes and all CGR members. They gave me a clear understanding of RNA Biology and provided me with good practices in RNA-Seq library preparation and NanoString analysis. I admire their professional laboratory skills very much! Particular thanks and appreciation go to Dr. Graham Clark of the London School of Hygiene and Tropical Medicine for his kind advice and help on amoeba cultures and high- quality figures. I have learned a lot from his collection of published papers! In addition, sincere thanks also go to my PhD thesis examining committee: Dr. Alistair Darby of the University of Liverpool and Dr. Mark Van Der Giezen of the University of Exeter for revising my dissertation and giving me nice suggestions! For all of my friends, I would like to convey my special thanks to Dr. Ian Wilson for his kind support and help in writing some perl scripts; Dr. Ian Goodhead for his qPCR software demonstration; Dr. Jennifer Kelly and Sarah Forrester for their warm encouragement and advice on grammar corrections. i I wish to gratefull y acknowledge the support from Dr. Padet Siriyasatien of the Department of Parasitology, Faculty of Medicine, Chulalongkorn University , who encouraged me to write up and offered some photomicrographs included in my introductory chapter. Also, appreciation and gratitude are extended to the Faculty of Medicine and Chulalongkorn University for their sponsorship and great support throughout the years! Finally, I would like to dedicate all my knowledge in this thesis to my parents, grandparents and beloved family. They gave a life and all things to me. Inexpressibly, I am extremely grateful to them and promise that I will take care of them until my last breath. ii Abstract Entamoeba histolytica is a parasitic protozoan that infects the human digestive tract. Infection results from ingestion of cyst-contaminated food or water. To date, E. histolytica infection remains a major worldwide public health problem in worldwide endemic areas. The spectrum of clinical manifestations ranges from asymptomatic carrier to mucous and bloody diarrhea or even extraintestinal amoebiasis, usually amoebic liver abscess. Several molecular studies have been carried out to reveal novel aspects of E. histolytica infection. However, the studies focused on genomic-wide analysis comparing between E. histolytica strains are still limited. Thus, the aims of this project are to comprehensively study the comparative analysis of the whole and small RNA transcriptomes amongst nonvirulent and virulent strains of laboratory cultured E. histolytica trophozoites as well as to integrate such transcriptomic findings with the genomic data for advanced understanding of the molecular pathogenesis and virulence in amoebiasis. In this study, genome-wide transcriptome analysis using Illumina RNA-Seq technology can illustrate significant expression differences between nonvirulent and virulent E. histolytica strains. Differential gene expression analysis between nonvirulent Rahman strain and other three virulent strains (i.e. PVBM08B, HM-1:IMSS and IULA:1092:1) reveals that transcripts involved in host cell killing and mucosal invasion, nucleic acid interaction and response to oxidative stress are notably upregulated in the virulent trophozoites. InterProScan results show the upregulation of genes encoding proteolysis-related domains and the co-upregulation of cytoskeleton and actin-modulating domains in the virulent strains. Also, process ontologies related to protein degradation, cellular biosynthesis, DNA metabolism, repair and recombination, mitotic cell division, actin dynamics and response to stress are highly enriched as a core metabolism in the virulent strains, indicating the rapid growing and active metabolic state are the main drivers of virulence. However, the striking underrepresentation of ontologies involved in signaling and regulatory processes was observed in the virulent parasites. It could be inferred that reduced regulation of sensing and correctly responding to the environmental stimuli potentially enable the parasites to become virulent and subsequently cause the invasive infection. Also, NanoString validation reveals the spectrum of virulence-associated gene expression among these four strains, reflecting their different degrees of virulence. Gene copy number variation (CNV) is widespread among the genomes of the E. histolytica strains, reflecting genomic plasticity and variability in gene family content. Herein, this present data show that patterns of CNV contribute to differential expression profiles, therefore it can be extrapolated that differences in gene copy number between genomes could contribute to the variation in phenotypic attributes, including virulence, among E. histolytica strains. Also, genome plasticity can also be seen in Trypanosomes and Leishmania, suggesting that CNV is a potentially important mechanism in generating genetic diversity and regulating gene expression levels in almost exclusively asexual parasite group. For small RNA transcriptomics, the size-fractionated sRNA sequencing data demonstrate the inverse relationship between antisense sRNA abundance and target gene expression levels, strongly suggesting the sRNA-mediated regulation. Differential sRNA regulation in virulence-associated gene expression was found among strains, indicating that sRNA-mediated post-transcriptional regulation may be important in shaping the parasite virulence. In addition, this study identified the novel putative miRNA from the sRNA sequencing data using the biogenesis-based bioinformatic analysis and qPCR validation, implying that miRNA potentially play a regulatory role in E. histolytica. In summary, it can be inferred that genomic plasticity and sRNA-mediated regulation are important mechanisms of virulence modulation in E. histolytica. iii Table of Content s Acknowledgements .................................................................................................................................. i Abstract...................................................................................................................................................... iii Table of Contents .................................................................................................................................... iv Figure Legends ........................................................................................................................................ ix Table Legends ......................................................................................................................................... xv List of abbreviations .......................................................................................................................... xvii Chapter One: Introduction ..................................................................................................... 1 1.1 Amoebiasis ......................................................................................................................................................... 1 1.2 The life cycle of Entamoeba histolytica ........................................................................................................ 2 1.3 Mechanisms of pathogenesis ..................................................................................................................... 4 1.4 Genomic structure and organisation ...................................................................................................... 6 1.5 Closely related Entamoeba species relevant to human amoebic research ............................. 8 1.6 Differential virulence of amoebiasis across E. histolytica strains .............................................12 1.7 Treatment of amoebiasis ...........................................................................................................................17
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