Investigating the role of the Leishmania (Leishmania) major HASP and SHERP genes during metacyclogenesis in the sand fly vectors, Phlebotomus (Phlebotomus) papatasi and Ph. (Ph.) duboscqi Johannes Doehl PhD University of York Department of Biology Centre for Immunology and Infection September 2013 1 I’d like to dedicate this thesis to my parents, Osbert and Ulrike, without whom I would never have been here. 2 Abstract Leishmania parasites are the causative agents of a diverse spectrum of infectious diseases termed the leishmaniases. These digenetic parasites exist as intracellular, aflagellate amastigotes in a mammalian host and as extracellular flagellated promastigotes within phlebotomine sand fly vectors of the family Phlebotominae. Within the sand fly vector’s midgut, Leishmania has to undergo a complex differentiation process, termed metacyclogenesis, to transform from non-infective procyclic promastigotes into mammalian-infective metacyclics. Members of our research group have shown previously that parasites deleted for the L. (L.) major cDNA16 locus (a region of chromosome 23 that codes for the stage-regulated HASP and SHERP proteins) do not complete metacyclogenesis in the sand fly midgut, although metacyclic-like stages can be generated in in vitro culture (Sádlová et al. Cell. Micro.2010, 12, 1765-79). To determine the contribution of individual genes in the locus to this phenotype, I have generated a range of 17 mutants in which target HASP and SHERP genes are reintroduced either individually or in combination into their original genomic locations within the L. (L.) major cDNA16 double deletion mutant. All replacement strains have been characterized in vitro with respect to their gene copy number, correct gene integration and stage-regulated protein expression, prior to phenotypic analysis. HASPA1 was not detected in cultured promastigotes, but was expressed in mouse isolated amastigotes. Parasite mutant lines were passaged through susceptible BALB/c mice, during which HASPA2 gene containing mutant lines, in the absence of a HASPA1 gene, were shown not to develop lesions. Mouse-passaged parasites were used to infect the L. (L.) major specific sand fly vectors, Ph. (Ph.) papatasi and Ph. (Ph.) duboscqi. The progress of parasite metacyclogenesis was then monitored over twelve days, by midgut dissection and microscopy. Metacyclogenesis was not fully recovered in any of the replacement mutants tested. Surprisingly, HASPB protein expression could not be detected in the replacement mutants within the sand fly midgut, although HASPB protein was readily detected when the same parasite lines were cultured in vitro. The same was true for SHERP, although in situ expression was recovered in the presence of a HASPB gene, which itself did not expressed detectable HASPB protein levels. These observations suggest a requirement for one or multiple as-yet-unidentified regulatory component(s) for HASPB expression within the sand fly midgut and these are not required in culture. Quantitative PCR data suggested HASPB upregulation to be essential for metacyclogenesis completion, suggesting a sand fly specific function for HASPB. 3 Table of content Abstract 3 Table of content 4 List of figures 9 List of tables 12 List of appendices 13 Acknowledgment 14 Author’s declaration 15 1. CHAPTER I. – Introduction 16 1.1. The Leishmaniases 16 1.1.1. Epidemiology of the Leishmaniases 16 1.1.2. Clinical Manifestations of Leishmaniasis 18 1.1.3. Immunopathogenesis 20 1.1.4. Leishmania / Human Immunodeficiency Virus co-infection 25 1.1.5. Diagnosis of Leishmaniasis 25 1.1.6. Treatment 28 1.1.7. Vaccine development 29 1.2. Leishmania parasites 30 1.2.1. New World Model of Leishmania Origin 33 1.2.2. Old World model of Leishmania Origin 35 1.2.3. Separate origins of L. (Leishmania) spp. and L. (Viannia) spp. 35 1.2.4. Leishmania and its reservoirs 36 1.2.5. Leishmania lifecycle 36 1.2.5.1. Sand fly stage: Metacyclogenesis of L. (Leishmania) spp. 38 1.2.5.2. Sand fly stage: Metacyclogenesis of L. (Viannia) spp. 42 1.2.6. The Leishmania cell surface 42 1.2.6.1. Glycosylphosphatidylinositol-anchors 43 1.2.6.2. Lipophosphoglycans 43 1.3. Sand Fly Vectors 44 1.3.1. Sand fly Development 46 1.3.2. Structure of the sand fly alimentary canal 48 1.3.3. Midgut Physiology 53 1.4. Sand fly Vector and Leishmania Parasite 54 1.4.1. Manipulating host enzyme expression patterns 54 1.4.2. Evading the sand fly’s complement system 56 1.4.3. Peritrophic Matrix 58 1.4.4. Leishmania interactions with the sand fly alimentary canal 60 4 1.4.5. Inhibiting sand fly gut peristalsis 60 1.4.6. Promastigote secretory gel 61 1.4.7. The stomodeal valve 65 1.4.8. Bacterial midgut flora 65 1.4.9. Sexual reproduction of Leishmania 67 1.4.10. Transmission to a mammalian host 67 1.5. The Leishmania genome 68 1.5.1. Gene transcription 69 1.5.2. Gene expression regulation 71 1.6. The L. (L.) major cDNA16 locus 72 1.6.1. HASPA1, HASPA2 and HASPB 74 1.6.1.1. HASPA1 and HASPA2 properties 77 1.6.1.2. HASPB properties 77 1.6.2. SHERP1 and SHERP2 properties 78 1.7. Leishmania (Viannia) braziliensis Orthologous HASP Locus 79 1.8. Project aims 82 2. CHAPTER II. – Materials and Methods 83 2.1. In silico work 83 2.1.1. Databases 83 2.1.2. Primer design 83 2.1.3. The Basic Local Alignment Search Tool (Blast) 83 2.1.4. CLUSTAL Sequence alignments 83 2.1.5. Sequencing Data Analysis 85 2.1.6. Restriction Site Determination Tools 85 2.1.7. Other Computer Softwares 85 2.2. Leishmania manipulation 85 2.2.1. Leishmania species and strains used 85 2.2.2. Culture media and culture conditions 88 2.2.3. Splitting and passaging Leishmania parasites in vitro 88 2.2.4. Cryo-samples 89 2.2.5. Artificial mouse infection with L. (L.) major 89 2.2.5.1. L. (L.) major passage through BALB/c mice 89 2.2.5.2. Amastigote generation and isolation 89 2.2.6. Leishmania homologous recombination mutant generation 90 2.2.6.1. Transfection 90 2.2.6.2. Leishmania clone selection 91 2.2.6.3. Growing up parasite clones 91 2.2.7. Parasite measurements 91 5 2.2.8. Growth Assay 92 2.2.9. Osmotaxis Assay 92 2.3. DNA Manipulation Protocols 93 2.3.1. DNA sequencing and processing 93 2.3.2. Genomic DNA extraction 93 2.3.2.1. Phenol/chloroform gDNA extraction 93 2.3.2.2. Genomic DNA extraction by blood and tissue kit (Qiagen) 94 2.3.3. PCR amplifications 94 2.3.3.1. Conventional PCR 94 2.3.3.2. Long range PCR 94 2.3.3.3. Reverse transcriptase – PCR 97 2.3.3.4. Quantitative Real Time – PCR 97 2.3.4. PCR product purification 98 2.3.5. Plasmid construction 98 2.3.5.1. 3’ A-overhang addition 98 2.3.5.2. Restriction digests protocols 98 2.3.5.3. DNA ligation 100 2.3.5.4. Transformation of chemically competent E. coli cells 100 2.3.5.5. Plasmid extraction from cultured E. coli cells 102 2.3.6. Gel electrophoresis 102 2.3.7. DNA agarose gel extraction 102 2.3.8. Ethanol precipitation 102 2.3.9. Southern blot 103 2.3.10. Amplification of genomic DNA extracts 105 2.4. mRNA Manipulation Protocol 105 2.4.1. mRNA extraction from cultured parasites 105 2.4.2. mRNA extraction from midgut derived parasites 105 2.5. Protein studies 106 2.5.1. Protein extraction 106 2.5.2. Western / Immunoblotting 106 2.5.3. Ponceau S stain of immunoblot membranes 106 2.5.4. Promastigote secretory gel (PSG) extraction 107 2.5.5. PSG detection by Dot-blot 107 2.5.6. Biotinylation assay 107 2.6. Sand fly manipulation 108 2.6.1. Sand fly strains 108 2.6.2. Artificial sand fly infections 108 2.6.3. Sand fly midgut dissection and analysis 111 6 2.6.4. Gene regulation in culture 112 2.7. Microscopy 112 2.7.1. Giemsa stained gut slide analysis by light microscopy 112 2.7.2. Confocal microscopy 113 2.7.2.1. Fixed parasite antibody staining 113 2.7.2.2. Live / dead staining 113 3. Chapter III. – Generating Leishmania HASP and SHERP replacement mutants 115 3.1. Introduction 115 3.2. Recombinant construct generation 116 3.3. L. (L.) major HASP and SHERP gene(s) mutant generation 119 3.3.1. Screening the L. (L.) major cDNA16 mutant genes 123 3.3.1.1. PCR screen of L. (L.) major cDNA16 mutant genes 123 3.3.1.2. Southern blots of L. (L.) major cDNA16 genes mutant 123 3.3.1.3. qPCR screen for replacement gene copy number 127 3.3.1.4. Western blot time courses to assess the expression and stage- regulation of the replacement genes 134 3.3.1.5. Assessing HASPA1 expression in amastigotes 135 3.3.1.6. Assessing HASPB surface localization in vitro by biotinylation assay 138 3.4. Parasite passage through mice 142 3.5. Growth Assay 143 3.6. Conclusions 143 4. Chapter IV. – Investigating metacyclogenesis in the sand fly 148 4.1. Introduction 148 4.2. HASP and SHERP mutant development in the sand fly midgut 148 4.2.1. Artificial sand fly infections 148 4.2.2. Sand fly dissections 149 4.2.3. Assessing Leishmania forward migration in the sand fly vector 150 4.2.4. Assessing Leishmania infection loads in the sand fly vector 151 4.2.5.