Mollusc Biomphalaria Glabrata to Understand Host: Parasite Interactions
Total Page：16
File Type：pdf, Size：1020Kb
Genomic analysis of the fresh water mollusc Biomphalaria glabrata to understand host: parasite interactions A thesis submitted for the degree of Doctor of Philosophy By Edwin Chukwuemeka Odoemelam Biosciences, School of Health Sciences and Social Care Brunel University September 2009 Abstract The fresh water mollusc Biomphalaria glabrata is the intermediate host for the trematode parasite Schistosoma mansoni, this parasite is responsible for the human disease Schistosomiasis. The significance of B. glabrata in the transmission of schistosomiasis is such that it has been selected for complete genome sequencing. The Biomphalaria glabrata embryonic cell line is an important resource for researchers investigating the interaction between the snail and parasite. The genome of the Bge cells was analysed at the chromosomal level, using DAPI karyotyping. The karyotype revealed extensive aneuploidy, whereby a modal chromosome complement of 63 and 67 was observed in two isolates of the Bge cells, which exceeds B. glabrata’s 2n=36 chromosome number. Indeed, in addition to characterising the Bge cell chromosomes, a method was established for mapping single copy B. glabrata genes onto the chromosomes from the Bge cells using fluorescence in situ hybridisation. Despite the Bge cell’s inherent aneuploidy, the four genes mapped onto diploid homologous chromosomes. This methodology will be an important resource for the genome sequencing consortium. The interphase nucleus is an organised organelle, whereby chromosomes and gene loci have been shown to be located non-randomly and hence it is hypothesised that the organisation of the interphase nucleus is pertinent to the function of the genome. Since there is no data on how the genes of the snail genome behaves in interphase, it was assessed in the Bge cells line. Again, this is important for the sequencing initiative, but also for evolutionary biology. Radially distributed chromosome territories were observed in the nuclei of the Bge cells. The territory position was organised according to territory size, with small chromosome territories positioned towards the interior and large territories intermediately located. In addition, four B. glabrata genes were positioned non-randomly in the interphase nuclei of the Bge cells, again emphasising organised positioning of the genome. With co-culture of S. mansoni miracidia with the Bge cells there is up regulation of specific genes known to be involved in the host response to parasite. These genes are dramatically relocated within the interphase nuclei, implying that these are specific parasite induced nuclear events. An analysis of the genomic distribution of specific histone modified chromatin in the interphase nuclei of B. glabrata, revealed different nuclear distribution of modified i chromatin. Indeed, a statistically significant difference in these patterns was observed between juvenile and adult snails, indicating developmental differences in the organisation of the snails’ genome. These differences maybe relevant to the snails’ resistance/susceptibility to the parasite. ii Declaration I hereby state that the work presented in this thesis has been performed by me unless stated otherwise. Edwin C. Odoemelam iii Acknowledgements I would like to begin my acknowledgements by thanking Dr Joanna Bridger; my supervisor, colleague and friend, for her guidance throughout my PhD. Her scientific erudition as well as her unabated passion for academic research has permeated my life for the last 4 years and for that I am eternally indebted. I would like to thank the following collaborators from the Biomedical research institute (BRI, USA). I would like to thank Dr Matty Knight, Dr Nithya Raghavan, Dr Fred Lewis and Andre Miller for supplying the B. glabrata snails, the Bge cells and the B. glabrata BAC genes. I’d especially like to thank Dr Matty Knight for her helpful advice and kind input. I’d like to extend the hand of gratitude to Dr Wannaporn Ittiprasert for her RT-PCR work on the B. glabrata ferritin and actin genes. I would like to thank Prof. Wendy Bickmore and Dr Paul Perry for the 2D FISH erosion analysis script. I express my gratitude to my colleagues from the Brunel institute for the environment (ife). I would like to express a particular thank you to Steve Pash and Alice Baynes for their helpful comments and assistance pertaining to snail husbandry. I would like thank Dr David Tree (2nd supervisor), who has played an integral role in my development as a scientist. For their assistance in microscopy, I would like to acknowledge Dr Ian Kill and Dr Julio Masabanda. I reserve a special thank you to Ishita Mehta, Lauren Elcock, Dr Helen Foster and Dr Karen Meaburn for providing perspicacious insight, support and assistance throughout my PhD. Finally, I would like to thank Stella Odoemelam, Stella-Jane Odoemelam, Sam Taffs, Peter Bingham, Inesa Bingham and Julia Giannetti. Thank you all for your dependable friendships! iv Abbreviations 2D Two- dimensional 3D Three-dimensional BB02 Non susceptible B. glabrata snail Bp Base pair BSA Bovine serum albumin BrdU Bromodeoxyuridine BS90 Brazilian isolate resistant B. glabrata snail CHO Chinese hamster ovary cells Cy3 Cyanine 3 DAPI 4, 6 di-amidino-2-phenylindole DNA Deoxyribonucleic acid DOP-PCR Degenerate oligonucleotide primed polymerase chain reaction ES Embryonic stem cells EST Expressed sequence tags FISH Fluorescence in situ hybridisation FITC Fluorescein isothiocyanate FBS Foetal bovine serum FUrd 5-fluoro-2’-deoxyuridine G0 Quiescence or senescence, exit from cell cycle G1 Cell cycle gap 1 G2 Cell cycle gap 2 H3Me2K9 Histone dimethylation modification to lysine 9 H3Me3K4 Histone trimethylation modification to lysine 4 H4Me3K20 Histone trimethylation modification to lysine 20 HAT Histone acetyltransferase HCl Hydrochloric acid HDAC Histone deacetylase Hr Hour HSA Homo sapien autosome HU Hydroxyurea v ICD Interchromatin domain compartment Kb Kilobase (one thousand DNA base pairs) Mb Megabase (106 DNA base pairs) MEF Mouse embryonic fibroblast cells MHC Major histocompatibility complex Min Minute mRNA Messenger RNA n Sample size NaCl Sodium chloride NBF Neutral buffered formalin NE Nuclear envelope NCS New born calf serum NMRI Lab derived susceptible B. glabrata snail NORs Nucleolar organiser regions PBS Phosphate buffered saline RNA Ribonucleic acid RNAi RNA interference ROS Reactive oxygen species RT Room temperature RT-PCR Reverse transcription-polymerase chain reaction S Svedberg units SSC Saline sodium citrate Sec Second SEM Standard error of the mean (SD/square root of the sample size) S-phase DNA synthesis cell cycle stage TBS Tris-buffered saline solution UV Ultraviolet light v/v Volume/volume (ml of solution/100ml) w/v Weight/volume (g of substance/100ml) Xa Active chromosome X Xi Inactive chromosome X vi Table of contents Abstract..............................................................................................................i Declaration.......................................................................................................iii Acknowledgements..........................................................................................iv Abbreviations....................................................................................................v Table of contents............................................................................................viii Table of figures.............................................................................................xvii Table of tables................................................................................................xxi 1. Introduction..................................................................................................1 1.1 Schistosomiasis.............................................................................................................2 1.1.1 Public Health Importance.................................................................................2 1.1.2 Trematode schistosome parasites......................................................................3 1.1.3 Schistosome lifecycle........................................................................................4 1.1.4 Human pathogenesis.........................................................................................6 1.1.5 Diagnosis and Treatment..................................................................................8 1.2 The role of Biomphalaria glabrata in the spread of Schistosoma mansoni.................9 1.2.1 The Molluscan Phylum.....................................................................................9 1.2.2 Gastropoda......................................................................................................10 1.2.3 Biomphalaria glabrata....................................................................................11 1.2.4 Susceptibility traits..........................................................................................12 1.3 Biomphalaria glabrata embryonic (Bge) cell line......................................................14 1.4 The Chromosomes of Pulmonate snails......................................................................16 1.4.1 Snail cytogenetics...........................................................................................16 1.4.2 Biomphalaria glabrata chromosomes.............................................................17 1.5 Molecular analysis of B. glabrata responses to trematode parasites..........................23 1.5.1