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Functional Genomic Screening of Nematocida Parisii Host-Exposed Proteins

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Functional Genomic Screening of Nematocida Parisii Host-Exposed Proteins Functional Genomic Screening of Nematocida parisii host-exposed proteins By Eashwar Mohan A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Eashwar Mohan 2021 Functional Genomic Screening of Nematocida parisii host-exposed proteins Eashwar Mohan Master of Science Department of Molecular Genetics University of Toronto 2021 Abstract Microsporidia are a divergent group of obligate, intracellular pathogens that are relatively poorly understood. The Caenorhabditis elegans intestinal infecting Nematocida parisii, has been shown to secrete a diverse arsenal of proteins to their cells upon infection, labelled “host-exposed” proteins. These secreted proteins may be serving roles as effector proteins, and the model organism Saccharomyces cerevisiae has proven to be a reliable system for the study of bacterial effectors. This uses yeast to study microsporidian host-exposed proteins. I have generated a pipeline to clone N. parisii genes into yeast expression vectors and demonstrated reliability by generating a gene set of 97 N. parisii host-exposed genes along with additional controls. Screening this gene collection identified 23 toxic genes, demonstrating that this strategy may help further understanding of microsporidian infection biology by identifying novel microsporidian effector proteins and learning more about the rules of these proteins and their expression in yeast. ii Table of Contents ABSTRACT……………………………………………………………………………………...ii TABLE OF CONTENTS……………………………………………………………………….iii LIST OF TABLES……………………………………………………………………………….v LIST OF FIGURES……………………………………………………………………………..vi LIST OF ABBREVATIONS………………………………………………………………...…vii 1. INTRODUCTION 1.1. Microsporidia……………………………………………………………………………1 1.1.1. Microsporidia History, Evolution and Host Range………………………………..1 1.1.2. Microsporidia Genetic and Cellular Biology……………………………………...2 1.1.3. Microsporidian Life Cycle………………………………………………………...4 1.1.4. Infection and Clinical Information………………………………………………...6 1.1.5. Nematocida parisii………………………………………………………………...7 1.2. Pathogen Effector Proteins……………………………………………………………..8 1.2.1. Pathogen Effector Proteins………………………………………………………..9 1.2.2. Microsporidia Host-Exposed Proteins…………………………………………...10 1.3. Leveraging Yeast to Study Effector Proteins………………………………………...11 1.3.1. Usage and Applications of Yeast to Study Bacterial Effector Proteins………….11 1.4. High-throughput Cloning Techniques………………………………………………..12 1.5. Thesis Rationale………………………………………………………………………..13 2. MATERIALS AND METHODS 2.1. N. parisii Gene Amplification and Cloning…………………………………………...14 2.1.1. N. parisii Spore Prep and Genomic DNA Extraction……………………………14 2.1.2. Two-Step PCR Primer Design…………………………………………………...15 2.1.3. Two-Step PCR Protocol………………………………………………………….16 2.1.4. Gateway Cloning Protocols……………………………………………………...17 2.2. Bacterial/Yeast Strains and Culturing………………………………………………..17 2.2.1. Bacterial and Yeast Strains………………………………………………………17 2.2.2. Media Formulations……………………………………………………………...18 2.2.3. Transformation Protocols and Strain Generation………………………………..18 2.3. Yeast Growth Assays…………………………………………………………………..19 2.3.1. Spot Dilution Assays…………………………………………………………….19 2.3.2. Arrayed Liquid Growth Assays………………………………………………….20 2.4. LASSO Protocol………………………………………………………………………..20 2.4.1. LASSO Probe Synthesis…………………………………………………………20 2.4.2. Blunt-end Intramolecular Ligation………………………………………………21 2.4.3. Large Volume Intramolecular Ligation …………………………………………22 2.4.4. OPool Probe Capture…………………………………………………………….22 3. RESULTS 3.1. Cloning and Screening of N. parisii Host-Exposed Factors…………………………23 3.1.1. Host-Exposed Gene Pilot and Development of the Two-Step PCR System…….24 iii 3.1.2. Yeast Growth Pilot Screen of the 3 N. parisii genes…………………………….31 3.2. N. parisii Host-Exposed Protein Screening List and Composition………………….34 3.2.1. Choosing Genes and Designing Primers…………………………………………34 3.2.2. Functional Genomic Screening of N. parisii host-exposed genes……………….37 3.3. Future Directions………………………………………………………………………40 3.4. LASSO Cloning………………………………………………………………………...41 3.4.1. LASSO Cloning Procedure………………………………………………………43 3.4.2. Implementation and Progress Using the Published LASSO Protocol…………...43 3.5. Modifications to the LASSO Protocol………………………………………………...45 3.5.1. Blunt-End Ligation………………………………………………………………45 3.5.2. Large Volume Ligation Reactions……………………………………………….47 3.5.3. Oligo Pool Mediated Gene Capture……………………………………………...50 3.6. Future Directions………………………………………………………………………52 4. DISCUSSION 4.1. Yeast as a tool to study microsporidian effector proteins……………………………….53 4.2. High-throughput LASSO-based gene capture remains a challenge…………………….56 4.3. Thesis Summary………………………………………………………………………...57 5. REFERENCES……………………………………………………………………………...58 6. APPENDICES………………………………………………………………………………67 iv List of Tables Table 1. Two-Step PCR reaction components and parameters …………………………………...16 Table 2. List of Bacteria and Yeast source strains ………………………………………………..18 Table 3. List of source Plasmids …………………………………………………………………19 Table 4. Gene Cloning Pilot Summary Table …………………………………………………….28 Table 5. Toxic N. parisii Genes Summary ………………………...……………………………..39 Table 6. Blunt-end Ligation Protocols and Results……...…………………………………….….46 v List of Figures Figure 1. Diagram of a microsporidian spore ……………………………………………………...4 Figure 2. Microsporidian Life Cycle ………………………………………………………………6 Figure 3. PCR Results for HE Pilot Screen ………………………………………………………26 Figure 4. Two-Step PCR Primer Diagram ……………………………………………………….29 Figure 5. Two-Step PCR Development Results ………………………………………………….30 Figure 6. Streaking Assay of Pilot Gene Transgenic Yeast Lines ………………………………32 Figure 7. Spot Dilution Assay of Pilot Gene Transgenic Yeast Lines ……………………………33 Figure 8. Cloning and Screening Pipeline for N. parisii HE Genes ………………………………34 Figure 9. Screen Control Gene Growth Curves …………………………………………………..37 Figure 10. Normalized Growth Values for N. parisii Genes……………………………………...38 Figure 11. LASSO Probe Creation and Gene Capture Diagram ………………………………...44 Figure 12. Blunt-end Ligation Results …………………………………………………………...47 Figure 13. Large Volume Ligation Results ………………………………………….…………...48 Figure 14. Inverse PCR Results ………………………………………………………………….49 Figure 15. N. parisii Opool LASSO Probe Capture Results………………………………….......51 Figure 16. K12 Opool LASSO Probe Capture Results…………………………………………...52 vi List of Abbreviations Amp Ampicillin APE A Plasmid Editor APX Ascorbate peroxidase ATP Adenosine triphosphate bp Base pairs Carb Carbenicillin cDNA Complementary DNA Gal Galactose gDNA Genomic DNA Glu Glucose HE Host-Exposed HK Hexokinase Kan Kanamycin LASSO Long Adapter Single Strand Oligonucleotide Mb Megabases PCR Polymerase Chain Reaction PNK Phosphonucleokinase ProK Proteinase K RNA-Seq RNA Sequencing RTH Round-the-horn site directed mutagenesis SP Signal Peptide Tm Melting Temperature TMD Transmembrane Domain tRNA Transfer RNA Ura Uracil vii Introduction: 1.1: Microsporidia In our world, there are thousands of pathogenic microbes that cause great harm to human health and wellbeing. These pathogens have evolved their lifestyles from many distinct evolutionary lineages and phylogenetic taxa1, with many developing to fit the niche of intracellular pathogens2–4. These pathogens are marked by their ability to invade host cells as an ecological niche, using the host cell as an environment to replicate. Intracellular pathogens can be facultative or obligate, with the latter having an indispensable requirement for a host cell to reproduce2,3,5–7. An interesting class of obligate, intracellular pathogens that have a profound effect on human health and interests are microsporidia. 1.1.1: Microsporidia History, Evolution and Host Range Microsporidia were first investigated by Louis Pasteur, who was studying a “pepper disease” spreading in silkworms 150 years ago8,9. The scientific consensus on the origins and classification of microsporidia would be a topic of discussion for many years. In 1867, the causative agent for this disease was determined to be a microscopic parasite, which was named as Nosema bombycis and was classified as a schizomycete fungi9. Later, further study would place Nosema into its own new group, Microsporidia, although the taxanomic classification of this group would be altered many times in the coming future. Microsporidia have a highly unique method of infection, which would lead to their removal from fungi and placement in Sporozoa, a group of protists characterized by forming spores9. In the 1980s, it was proposed that some eukaryotes arose before the endosymbiosis of the mitochondrion and this theory would influence the understanding of microsporidian evolution. Microsporidia lacked mitochondria and molecular evidence, such as the short and almost prokaryotic ribosomal RNAs, would lead to the belief that microsporidia were early diverging eukaryotes9. Further phylogenetic analysis on other microsporidian genes would raise doubts about the latest origin theory. Tubulin genes of microsporidia showed high conservation with fungi and despite the lack of mitochondria, microsporidia do have mitochondrial genes. This information would place microsporidia as a member of fungi, and although learning the exact relationships between microsporidia and its fungal relatives is ongoing, microsporidia are believed to be a distinctly evolved member of fungi9. 1 Currently, there are over 1400 identified microsporidia species that are divided into
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