IDENTIFYING A ROLE FOR HEAT SHOCK PROTEINS IN SCHISTOSOMA MANSONI by KENJI ISHIDA Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology CASE WESTERN RESERVE UNIVERSITY August 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Kenji Ishida candidate for the degree of Doctor of Philosophy Committee Chair Michael Benard Committee Member Ronald Blanton Committee Member Christopher Cullis Committee Member Claudia Mieko Mizutani Committee Member Emmitt R. Jolly Date of Defense April 26, 2017 *We also certify that written approval has been obtained for any proprietary material contained therin. Table of Contents Table of Contents Table of Contents ............................................................................................................. iii List of Figures .................................................................................................................. vii List of Abbreviations ...................................................................................................... viii Abstract ........................................................................................................................... xiv Chapter 1: Introduction ................................................................................................... 1 1.1 Purpose ...................................................................................................................... 1 1.2 Schistosomiasis ......................................................................................................... 1 1.3 Geographic distribution ............................................................................................ 2 1.4 Parasite lifecycle ....................................................................................................... 3 1.5 Clinical manifestation and pathophysiology ............................................................. 5 1.6 Diagnosis................................................................................................................... 6 1.7 Treatment .................................................................................................................. 8 1.8 Immunology .............................................................................................................. 9 1.9 Host invasion by schistosome cercariae ................................................................. 11 1.9.1 Host identification and cercarial honing .......................................................... 11 1.9.2 Penetration of host skin and key morphological features of cercariae ............ 12 1.10 Heat shock response .............................................................................................. 14 1.11 Heat shock genes in schistosomes ........................................................................ 17 1.12 Thesis objective .................................................................................................... 19 1.13 Study rationale ...................................................................................................... 19 1.14 Summary of findings............................................................................................. 20 1.15 References ............................................................................................................. 21 iii Table of Contents Chapter 2: Immunolocalization of anti-Hsf1 to the acetabular glands of infectious schistosomes suggests a non-transcriptional function for this transcriptional activator ........................................................................................................................... 29 2.1 Abstract ................................................................................................................... 30 2.2 Author Summary ..................................................................................................... 30 2.3 Introduction ............................................................................................................. 31 2.4 Materials and Methods ............................................................................................ 35 2.4.1 Animals and parasites ...................................................................................... 35 2.4.2 Preparation of schistosomal RNA .................................................................... 35 2.4.3 Cloning ............................................................................................................. 36 2.4.4 Yeast transformation and modified yeast one-hybrid ...................................... 36 2.4.5 Electrophoretic mobility shift assay (EMSA) .................................................. 37 2.4.6 Comparison of protein sequences .................................................................... 39 2.4.7 Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) ...... 39 2.4.8 Recombinant protein purification .................................................................... 40 2.4.9 Custom antibody production ............................................................................ 41 2.4.10 Western blotting ............................................................................................. 42 2.4.11 Immunohistochemistry ................................................................................... 42 2.4.12 Imaging .......................................................................................................... 43 2.5 Results ..................................................................................................................... 44 2.5.1 Schistosome Hsf1 is a transcriptional activator. .............................................. 44 2.5.2 SmHsf1 recognizes the heat shock DNA binding element from the schistosome HSP70 promoter. ....................................................................................................... 47 2.5.3 SmHSF1 is expressed across schistosome developmental stages. ................... 51 2.5.4 A polyclonal antibody detects the SmHsf1 protein. ......................................... 52 iv Table of Contents 2.5.5 Antibody raised against SmHsf1 localizes to the acetabular glands of S. mansoni cercariae. ..................................................................................................... 54 2.6 Discussion ............................................................................................................... 57 2.7 Acknowledgements ................................................................................................. 61 2.8 Supporting information ........................................................................................... 62 2.9 References ............................................................................................................... 64 Chapter 3: Hsp70 may be a molecular regulator of schistosome host invasion ........ 71 3.1 Abstract ................................................................................................................... 72 3.2 Author summary ...................................................................................................... 73 3.3 Introduction ............................................................................................................. 74 3.4 Methods................................................................................................................... 79 3.4.1 Phylogenetic analysis of Hsp70 ....................................................................... 79 3.4.2 Animals and parasites ...................................................................................... 79 3.4.3 Parasite observation and treatments ................................................................. 79 3.5 Results ..................................................................................................................... 80 3.5.1 Schistosome Hsp70 is highly conserved .......................................................... 80 3.5.2 Establishment of cercarial swimming in culture .............................................. 81 3.5.3 Treatment of cercariae with human skin lipid, linoleic acid, and PES ............ 83 3.5.4 Treatment of cercariae with other Hsp70 modulators ...................................... 86 3.5.5 Treatment of cercariae with Hsp90 inhibitors .................................................. 88 3.5.6 Treatment of cercariae with other compounds ................................................. 89 3.6 Discussion ............................................................................................................... 93 3.7 Acknowledgments ................................................................................................... 98 3.8 Supporting information ........................................................................................... 99 v Table of Contents 3.9 References ............................................................................................................. 101 Chapter 4: Discussion ................................................................................................... 109 4.1 References ............................................................................................................. 119 List of References .......................................................................................................... 123 vi List