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Identifying Host Genetic Factors Controlling Susceptibility to Blood-Stage Malaria in Mice

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Identifying Host Genetic Factors Controlling Susceptibility to Blood-Stage Malaria in Mice Identifying host genetic factors controlling susceptibility to blood-stage malaria in mice Aurélie Laroque Department of Biochemistry McGill University Montreal, Quebec, Canada December 2016 A thesis submitted to McGill University in partial fulfilment of the requirements of the degree of Doctor of Philosophy. © Aurélie Laroque, 2016 ABSTRACT This thesis examines genetic factors controlling host response to blood stage malaria in mice. We first phenotyped 25 inbred strains for resistance/susceptibility to Plasmodium chabaudi chabaudi AS infection. A broad spectrum of responses was observed, which suggests rich genetic diversity among different mouse strains in response to malaria. A F2 intercross was generated between susceptible SM/J and resistant C57BL/6 mice and the progeny was phenotyped for susceptibility to P. chabaudi chabaudi AS infection. A whole genome scan revealed the Char1 locus as a key regulator of parasite density. Using a haplotype mapping approach, we reduced the locus to a 0.4Mb conserved interval that segregates with resistance/susceptibility to infection for the search of positional candidate genes. In addition, I pursued the work on the Char10 locus that was previously identified in [AcB62xCBA/PK]F2 animals (LOD=10.8, 95% Bayesian CI=50.7-75Mb). Pyruvate kinase deficiency was found to protect mice and humans against malaria. However, AcB62 mice are susceptible to P. chabaudi infection despite carrying the protective PklrI90N mutation. We characterized the Char10 locus and showed that it modulates the severity of the pyruvate deficiency phenotype by regulating erythroid responses. We created 4 congenic lines carrying different portions of the Char10 interval and demonstrated that Char10 is found within a maximal 4Mb interval. Exome sequencing in this region identified genetic variants in 13 genes while RNA sequencing revealed 14 genes differentially expressed between CBA/Pk and Char10C congenic mice. Overall, my ii thesis work uncovered and precised the role of genetic factors controlling susceptibility/resistance to P. chabaudi infection in mice and contributes to better understand the genetic and physiological basis of host response to blood-stage malaria. iii RÉSUMÉ Cette thèse examine les facteurs génétiques contrôlant la réponse de l’hôte à la malaria chez la souris. Dans un premier temps, nous avons phénotypé 25 souches de souris pour déterminer leur résistance ou susceptibilité à l’infection par P. chabaudi chabaudi AS. Un spectre de réponses important a été observé, suggérant une diversité génétique riche en réponse à la malaria. Un croisement F2 a été généré entre les souris susceptibles SM/J et les résistantes C57BL/6. Le phenotypage, le génome scan et l’analyse de linkage ont révélé le locus Char1 comme régulateur clé du niveau de parasitémie dans les [SM/JxC57BL/6]F2. Une analyse d’haplotypes dans Char1 a permis de réduire le locus à un intervalle de 0.4Mb pour la priorisation de gènes candidats. J’ai aussi poursuivi le travail sur le locus Char10 qui avaient été identifié dans un croisement [AcB62xCBA/PK]F2 (LOD=10.8, 95% Bayesian CI=50.7- 75Mb). Il avait été précédemment démontré que la déficience en pyruvate kinase protège les souris et les hommes contre la malaria. Cependant, les souris AcB62, qui portent la mutation protectrice PklrI90N s’avèrent être susceptibles à l’infection par P. chabaudi. Nous avons caractérisé le locus Char10 et avons montré qu’il module la sévérité de la déficience en pyruvate kinase en régulant la réponse erythroïde. Nous avons créé 4 lignées congéniques portant des portions différentes de la région Char10 et avons démontré que le gène responsable de l’effet Char10 se trouve dans un intervalle maximal de 4Mb. Le séquençage d’exome dans cette région a permis d’identifier des variants génétiques dans 13 gènes et le séquençage d’ARN a révélé iv que 14 gènes étaient différentiellement exprimés entre les lignées CBA/Pk et Char10C. En résumé, ma thèse aura permis de découvrir et de préciser le rôle de facteurs génétiques contrôlant la résistance/susceptibilité à l’infection par P. chabaudi chez la souris et contribue à l’avancée des connaissances génétiques et des mécanismes physiologiques entourant la réponse de l’hôte à la malaria. v PREFACE The work described in chapter 2, 3 and 4 of this thesis has been published as follows: Chapter 2: Laroque A., Min-Oo G., Tam M., Radovanovic I., Stevenson M.M. and Gros P. Genetic control of susceptibility to infection with Plasmodium chabaudi chabaudi AS in inbred mouse strains. Genes and Immunity. 2012; 13: 155-163. Chapter 3: Laroque A., Min-Oo G., Tam M., Ponka P, Stevenson M.M. and Gros P. The mouse Char10 locus regulates severity of pyruvate kinase deficiency and susceptibility to malaria. 2016. Submitted to PLoSOne. Chapter 4: Laroque A., Min-Oo G., Tam M., Stevenson M.M. and Gros P. The Char10 locus: a genetic modifier of the malaria-protective effect of pyruvate kinase deficiency. 2016. In preparation. In addition, parts of Chapter 1 have been adapted from: Laroque A., Bongfen S.E., Berghout J. and Gros P. Genetic and genomic analyses of host-pathogen interactions in malaria. Trends in Parasitology. 2009; 9: 417-422. vi CONTRIBUTION OF AUTHORS Chapter 2: I performed most of the work presented in this chapter. Helpful assistance was provided by the following individuals. Gundula Min-Oo initiated the survey of the 25 inbred strains. She performed the first screening. I took over the 1st and performed a 2nd screening. I smeared the mice and phenotyped them for survival and parasitemia (Table 2.1 and Figure 2.1). Susan Gauthier generated the [SM/JxC57BL/6]F2 animals. I phenotyped the F2 animals for survival by monitoring disease and taking blood smears for the 250 mice each day during 21 days. Mifong Tam injected all mice and counted the parasitemia levels in the F2 cross. I prepared DNA from tail clips, genotyped all mice for additional microsatellite markers and performed the linkage analysis (Figure 2.3). I performed the haplotype analysis (Figure 2.4). Irena Radovanovic performed the EMMA scan (Figure 2.S1). Mifong Tam provided guidance and assistance with flow cytometry (Figure 2.S2). I wrote the first draft of the manuscript and generated all tables and figures. Dr Mary M. Stevenson provided feedback on the article as well as on the experimental design throughout the project. Chapter 3: I performed most of the work presented in this chapter. Helpful assistance was provided by the following individuals. I generated the Char10C congenic line (which was initiated by Gundula Min-Oo). Susan Gauthier helped with the breeding. I phenotyped mice for peak parasitemia vii and reticulocyte levels. I performed the hematology and the histology characterization (Figure 3.2 and Figure 3.3). Marc Mikhael and Dr Ponka performed the iron measurements in the plasma (Figure 3.4). I determined the iron measurements in the tissues (Figure 3.3). Mifong Tam performed the biotin labeling assay (Figure 3.5). I did the flow cytometry experiments (Figure 3.6) and, after being trained by Mifong Tam, the CFUe assay (Figure 3.7). I wrote the first draft of the manuscript and generated all tables and figures. Dr Mary M. Stevenson provided feedback on the article as well as on the experimental design throughout the project. Chapter 4: I performed most of the work presented in this chapter. Helpful assistance was provided by the following individuals. Gundula Min-Oo did the linkage analysis of the [AcB62xCBA/Pk]F2 cross, while Mifong Tam helped with the phenotyping (Figure 4.1 and Figure 4.2). Gundula Min-Oo and I generated 4 congenic lines. I phenotyped them for both peak parasitemia and reticulocyte levels and refined the Char10 interval (Figure 4.3). I extracted the genomic DNA from ear biopsies for the exome sequencing and analysed the data (Table 4.1). For the RNA sequencing, I extracted the RNA and analysed the results with guidance from David Langlais (Table 4.2). I wrote the manuscript. My supervisor: Dr. Philippe Gros, provided guidance and advice throughout my PhD, reviewed the results and edited all manuscripts. viii TABLE OF CONTENT ABSTRACT ................................................................................................................... ii RÉSUMÉ ..................................................................................................................... iv PREFACE .................................................................................................................... vi CONTRIBUTION OF AUTHORS ................................................................................ vii LIST OF FIGURES ..................................................................................................... xii LIST OF TABLES ...................................................................................................... xiv ACKNOWLEDGEMENTS ........................................................................................... xv RATIONALE AND OBJECTIVES .............................................................................. xvi Chapter 1 : Introduction and literature review .............................................................. 1 1.1 Introduction: historical perspectives and prevalence of malaria .......................... 2 1.2. The parasite ........................................................................................................ 6 1.2.1. The Plasmodium life cycle ........................................................................ 6 1.2.1.1. Pre-erythrocytic stage
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