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Understanding Functional Mechanisms of Genetic Susceptibility to Mycobacterial

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Understanding Functional Mechanisms of Genetic Susceptibility to Mycobacterial Understanding functional mechanisms of genetic susceptibility to mycobacterial infection Ali M. Alisaac Department of Medicine University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy Darwin College 2017 Abstract Title: Understanding functional mechanisms of genetic susceptibility to mycobacterial infection Name: Ali M. Alisaac Tuberculosis remains a major public health problem and one of the leading causes of death worldwide. Human genetic factors determine susceptibility to M. tuberculosis (M. tb) infection and predispose to clinical TB. Genome-wide association studies (GWAS) aim to discover human genes associated with susceptibility to TB. Recently, a GWAS conducted by our lab identified a new TB-associated gene ASAP1 that encodes an Arf GTPase-activating protein (GAP). ASAP1 is known to be involved in regulation of actin and membrane remodeling. My Ph.D. included three projects. In my first project, I used RNAi and CRISPR-Cas9 technologies to study the role of ASAP1 in dendritic cells and macrophages, cells that play critical roles during mycobacterial infection. I demonstrated that in these cells ASAP1 is essential for migration and phagocytosis of mycobacteria. I characterized proteins that ASAP1 interacts with during mycobacterial infection. Finally, I found that the ASAP1- mediated pathway regulates expression of a large number of the immune response genes. These findings emphasize the important role of ASAP1 in mycobacterial infection and explain its involvement in TB pathogenesis. In my second project, I was involved in a large study conducted by our laboratory that characterized transcriptional responses to M. tb infection in macrophages from a cohort of 144 healthy subjects. We used RNA-Seq to study transcriptomes of the infected and non- infected macrophages and identified differentially expressed genes. We also genotyped DNA polymorphisms of these subjects and studied the association between genetic variants and levels of gene expression, which allows us to identify expression quantitative trait loci (eQTLs), i.e., DNA polymorphism that affect gene expression. In particular, we identified an eQTL located in the TLR10-TLR1-TLR6 gene cluster. In non-infected macrophages, a group of polymorphisms in this region was associated in cis with the level of expression of TLR1, but not of the other two TLR genes. In M. tb-infected macrophages the same polymorphisms were associated in trans with levels of expression of 37 genes. This network includes essential immune response proteins, including multiple cytokines and chemokines. The discovery of this TLR1-driven network will help to better understand mechanisms of macrophage responses to mycobacterial infection. Our study also identified a DNA polymorphism located upstream of the ARHGAP27 gene, regulating its expression in infected and non-infected macrophages. In our GWAS this polymorphism was associated with TB risk, which implicated ARHGAP27 in TB pathogenesis. The ARHGAP27 protein is a Rho-GAP involved in the endocytic pathway. In my third project, I used CRISPR technology to establish the ARHGAP27-knockout macrophage cell model and characterized the function of ARHGAP27, showing that it is involved in cell migration and phagocytosis of mycobacteria. Taken together, my studies highlighted functional mechanisms implicating TB-associated GAP proteins ASAP1 and ARHGAP27 in mycobacterial infection and TB pathogenesis. I would like to dedicate this thesis to my lovely wife Tagreed Alisaac, my children Renaad, Mohammed, and Ajeebah, and my loving parents, borthers, and sisters for their love and support during my Ph.D. study. Declaration I hereby declare that except where specific reference is made to the work of others, the contents of this dissertation are original and have not been submitted in whole or in part for consideration for any other degree or qualification in this, or any other University. This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration, except where specifically indicated in the text. This dissertation contains less than 65,000 words including appendices, bibliography, footnotes, tables and equations and has less than 150 figures. Ali M. Alisaac 2017 Acknowledgments I would like to acknowledge and thank colleagues who have supported and helped me during my Ph.D. First and foremost, I would like to express my great gratitude to my supervisor Dr. Sergey Nejentsev for giving me the opportunity to be one of his students, his support, and guidance throughout my research projects. I would also like to thank Mrs. Mailis Maes, Ms. Emma Goss, and Mr. James Curtis who worked with me as a team and for their help and suggestions. I would like to express my sincerest thanks to Dr. Davide Eletto for introducing me to cloning and helping me with CRISPR-Cas9 genome editing. I would express thanks to Dr. Olivier Papapietro for his comments and help for protein interaction analysis. I would like to thank Dr. Changxin Wu who trained me on tissue culture. My sincere thanks also go to Dr. Delphine Cuchet- Lourenco for being my mentor at the beginning of my P.hD and her help with confocal microscopy experiments. Finally, I would express a deep sense of gratitude to my family, especially to my wife, who has always supported, encouraged, and stood by me through all the good and bad times. Contents Contents .................................................................................................................................... xi List of Figures .......................................................................................................................... xv List of Tables .......................................................................................................................... xix Abbreviation ........................................................................................................................... xxi Chapter 1 Introduction .......................................................................................................... 1 Microbiology ............................................................................................................... 1 Prevalence ................................................................................................................... 3 Treatment and drug resistance..................................................................................... 4 Vaccine ........................................................................................................................ 5 Pathogenesis of TB...................................................................................................... 6 Molecular mechanisms of host-pathogen interaction................................................ 11 Genetic susceptibility to tuberculosis ........................................................................ 12 Lübeck disaster .................................................................................................. 13 Mendelian susceptibility to mycobacterial disease (MSMD) ............................ 13 Twin studies ....................................................................................................... 14 Animal models ................................................................................................... 15 Genome-wide association studies ...................................................................... 16 Molecular function of GTPases, GTPase-Activating Proteins (GAPs) and Guanine nucleotide Exchange Factors (GEFs) ................................................................ 28 Genetic regulation of gene expression in M. tb-infected macrophages. ............ 29 Aim and objectives .................................................................................................... 37 Chapter 2 Materials and Methods ....................................................................................... 39 Mycobacteria preparation .......................................................................................... 39 Cell lines .................................................................................................................... 39 Ethics ......................................................................................................................... 40 Cell preparation and infection ................................................................................... 40 RNA extraction ......................................................................................................... 41 qRT-PCR ................................................................................................................... 42 Immunofluorescence for confocal microscopy ......................................................... 43 DNA extraction and genotyping ............................................................................... 44 Microarray sample preparation ................................................................................. 44 Library preparation .................................................................................................... 45 Bioinformatics analysis of the transcriptome data and eQTLs in macrophages ....... 46 Bioinformatics analysis of the transcriptome data in THP-1 cells ............................ 47 siRNA transfection .................................................................................................... 47 Lentiviral particles production .................................................................................
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