Understanding functional mechanisms of genetic susceptibility to mycobacterial infection
University of Cambridge
Doctor of Philosophy (PhD)
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Alisaac, A. (2018). Understanding functional mechanisms of genetic susceptibility to mycobacterial infection (Doctoral thesis). https://doi.org/10.17863/CAM.25239
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.
susceptibility to tuberculosis, ASAP1 assaociated with TB risk, ARHGAP27 associated with TB risk, TLR1-driven network genes in Mycobacterium tuberculosis infected human macrophages
Al Baha University and Ministry of Education in Saudi Arabia.
This record's DOI: https://doi.org/10.17863/CAM.25239
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