GENE EXPRESSION IN THE MICROFILARIAE OF BRUGIA PAHANGI RICHARD DAVID EMES A thesis submitted for the degree of Doctor of Philosophy Department of Veterinary Parasitology, Faculty of Veterinary Medicine, Glasgow University June 2000 © Richard D Ernes 2000 ProQuest Number: 13818964 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13818964 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 GLASGOW UNIVERSITY LIBRARY 1184 3 COPH \ To Mum and Dad With love and thanks. List of Contents Page List of Contents i Declaration xi Acknowledgements xii Abbreviations xiv List of Figures xvi Abstract xxii Chapter 1 Introduction. Page 1.1 The parasite. 1 1.1.1 Filarial nematodes. 1 1.1.2 Life cycle. 2 1.2 The human disease. 4 1.2.1 Clinical spectrum of disease. 4 1.2.2 Diagnosis and treatment of lymphatic filariasis. 6 1.2.3 Control of lymphatic filariasis. 8 1.3 The microfilariae. 9 1.3.1 Periodicity of the mf. 9 1.3.2 Non-continuous development of filarial nematodes. 11 1.3.3 The microfilarial sheath. 17 1.3.4 Modulation of the host immune response by the mf. 18 1.4 Cloning stage-specific genes from filarial nematodes. 19 1.4.1 The filarial genome project. 21 1.4.2 Determining gene function in parasitic nematodes. 25 1.4.3 Cloning of differentially expressed genes. 28 1.5 Aims of the project. 30 Chapter 2 Materials and methods. 2.1 Maintenance of parasite life cycle. 31 2.1.1 Maintaining susceptible mosquitoes. 31 2.1.2 Maintaining B. pahangi parasites. 31 2.1.3 Recovery of infective L3 stage B. pahangi. 32 2.1.4 Recovery of mammalian-derived larvae. 32 2.2 Molecular biology techniques. 33 2.2.1 Standard PCR protocol. 33 2.2.2 "Hot Start" PCR. 33 2.2.3 PCR mix for 100 pi reaction (Taq polymerase). 34 2.2.4 Agarose gel electrophoresis 35 2.2.5 Preparation of restriction fragments for 36 use as size markers in gel electrophoresis. 2.2.6 Purification of DNA from agarose gels. 36 2.2.6.1 Purification by Spin-X columns. 36 2.2.6.2 Purification of DNA from agarose gels 37 by QIAquick gel extraction kit (Qiagen). 2.2.7 Ligation and transformation. 37 2.2.8 In-gel Ligation. 39 2.2.9 Plasmid miniprep. 40 2.2.10 Glycerol stocks of bacterial cultures. 41 2.2.11 Restriction digestion of DNA. 41 2.2.12 Genomic DNA isolation. 41 2.2.13 Preparation of genomic DNA for Southern blotting. 43 ii 2.2.14 DEPC treatment of solutions used for RNA work. 44 2.2.15 RNA extraction. 44 2.2.16 Preparation of RNA for northern blotting. 45 2.2.17 Southern and northern blotting. 46 2.2.18 First strand cDNA synthesis 46 2.2.19 Random (High Prime) labelling and purification of 47 DNA probes. 2.2.20 Automated sequencing. 48 2.2.21 Genotypes of bacterial strains used. 50 2.2.22 Analysis of mmc-1 5' upstream region. 50 2.2.22.1 Screening of a B. pahangi genomic library. 50 2.2.22.2 Cloning of mmc-1 upstream region by PCR. 51 2.3 Production of mammalian-derived mf cDNA library. 52 2.3.1 Generation of mammalian-derived mf cDNA 52 2.3.2 Ligation of cDNA to predigested Uni-ZAP XR 53 vector and packaging of library. 2.3.3 Calculation of titre and percentage recombinants 54 of the primary library 2.3.4 Amplification of primary library. 56 2.4 Production of mammalian-derived and 56 vector-derived mf cDNAs. 2.4.1 Mf exsheathment and purification. 56 2.4.2 Preparation of cDNA probes. 57 2.5 Screening of mammalian-derived mf cDNA library. 58 iii 2.5.1 Plating the mammalian-derived mf cDNA library. 58 2.5.2 Differential screening of the mammalian-derived 58 mf cDNA library. 2.5.3 In vivo excision of the pBluescript phagemid. 60 from the Uni-Zap vector. 2.6 Analysis of gene expression by semi-quantitative 60 RT-PCR. 2.6.1 Ethidium Bromide plate assay for quantitation 61 ofcDNA. 2.6.2 Preparation of cDNA panel from different 61 life-cycle stages for RT-PCR analysis. 2.6.3 Titration of RT-PCR amplified products. 62 2.6.4 Semi-quantitative RT-PCR analysis. 63 2.7 5’ RACE 64 2.8 Culture of B. pahangi parasites in vitro. 65 2.9 Production and purification of MBP-MMC-1 66 fusion protein. 2.9.1 Cloning of MMC-1 downstream of the MalE gene. 66 2.9.2 Subcloning of MMC-1 into pMal-p2. 67 2.9.3 Transformation of Topp competent cells. 67 2.9.4 Large scale expression of MBP-MMC-1 68 2.9.5 Affinity purification of the MBP -MMC-1 69 fusion protein. iv 2.9.6 Digestion of MBP-MMC-1 fusion protein 70 and purification of recombinant MMC-1. 2.10 Raising anti-MMC-1 immune sera. 70 2.11 Immunochemical techniques. 71 2.11.1 SDS polyacrylamide gel electrophoresis (PAGE) 71 2.11.2 SDS-sample cocktail extracts. 74 2.11.3 Molecular weight markers. 74 2.11.4 Staining SDS-Polyacrylamide gels with 75 silver nitrate. 2.11.5 Western blotting. 75 2.11.6 Metabolic labelling of mf and immunoprecipitation 77 of MMC-1. 2.12 Culture of mf for collection of E/S products. 78 2.13 Effect of MMC-1 IgG on parasite development 79 in mosquitoes. 2.13.1 Isolation of IgG from immunised rabbit serum. 79 2.13.2 Feeding mosquitoes. 79 2.13.3 Assessing the development of parasites. 79 2.14 Fluorescent localisation of MMC-1. 80 2.14.1 Staining whole parasites. 80 2.14.2 Staining of permeabilised parasites. 81 2.15 Analysis of immune response to MMC-1 antigen. 82 2.15.1 Preparation of soluble extracts. 82 2.15.2 Protein assay the Bradford method. 83 v 2.15.3 Animals and infection protocols. 83 2.15.4 Preparation of spleen cells. 84 2.15.5 Proliferation assay. 84 2.15.6 Analysis of cytokine production by ELISA. 85 2.15.7 IgG responses to MMC-1 86 2.15.8 Human IgG responses to MMC-1 87 Chapter 3 Production and screening of a mammalian-derived mf cDNA library. 3.1 Introduction. 88 3.2 Results. 90 3.2.1 Artificial exsheathment of mf. 90 3.2.2 Agarose pad purification of exsheathed mf. 91 3.2.3 Comparison of different media for the culture 92 of B. pahangi mf. 3.2.4 Comparison of cDNA produced from mammalian- 94 derived and vector-derived mf. 3.3 Screening of a B. malayi mf cDNA library. 95 3.4 Construction of a B. pahangi cDNA library. 96 3.4.1 Characterisation of the B. pahangi mf cDNA library. 97 3.4.2 Differential screening of the B. pahangi 98 cDNA library. 3.4.3 Confirmation of differential expression by 100 reverse northern. 3.4.4 Analysis of differentially expressed cDNAs. 101 3.4.5 Selection of genes for further study. 101 3.5 Discussion. 104 Chapter 4 Analysis of vmc-2. 4.1 Introduction. Ill 4.2 Results 111 4.2.1 Nucleotide sequence information. 111 4.2.2 Generation of gene specific probes. 114 4.2.3 Comparison of vmc-2 cDNA and genomic 114 PCR fragments. 4.2.4 Northern blot analysis of vmc-2. 115 4.2.5 Attempts to obtain a full length transcript of vmc-2. 116 4.2.6 Southern blot analysis of high molecular weight DNA. 119 4.2.7 The temporal expression pattern of vmc-2 in vivo. 120 4.2.7.1 Semi-quantitative RT-PCR. 120 4.2.7.2 Titration of gene products produced by RT-PCR. 121 4.2.7.3 Test of life-cycle stage cDNAs. 122 4.2.7.4 Semi-Quantitative RT-PCR of vmc-2. 122 4.3 Discussion. 124 vii Chapter 5 Analysis of mmc-1. 5.1 Introduction. 131 5.2 Results. 132 5.2.1 The mmc-1 clone. 132 5.2.2 Identifying homologues of mmc-1. 133 5.2.3 Is mmc-1 SL1 trans- spliced ? 135 5.2.4 Comparison of cDNA and genomic 137 sequence of mmc-1 5.2.5 Northern blot analysis of mmc-1. 137 5.2.6 Southern blot analysis of ramc-1. 138 5.2.7 Zooblot analysis of mmc-1. 139 5.2.8 Temporal expression of mmc-1 in vivo. 141 5.2.9 Expression of mmc-1 in mf in utero. 142 5.2.10 Expression of mmc-1 in mf in vitro. 143 5.2.10.1 Does temperature influence mmc-1 expression? 144 5.2.10.2 Does the presence of FCS or glucose affect 145 the expression of mmc-1 ? 5.3 Attempts to isolate the upstream region of mmc-1. 146 5.3.1 Screening of a Brugia pahangi genomic DNA library.