Novel Methods and Mechanisms of Human Genetic Susceptibility to Infectious Disease

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2023

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Abstract

Understanding the complex interactions between humans and their pathogens is key to the development of effective therapeutic strategies for infectious diseases. One approach to gain insight into host-pathogen interactions is to leverage natural human genetic variation. Traditionally, researchers have employed clinical GWAS (genome-wide association studies) of infected individuals to identify genetic variants that confer susceptibility to infection phenotypes. However, standard clinical GWAS approaches are hampered by issues with sampling, variation in exposure, and difficulty obtaining appropriately matched controls. In this thesis, I have leveraged cellular and molecular GWAS of lymphoblastoid cell lines (LCLs) to uncover mechanisms of immune suppression by Chlamydia trachomatis and identify novel regulators of influenza infection.Previously, our lab developed Hi-throughput Human in vitrO Susceptibility Testing (Hi-HOST) to connect human genetic variation to infectious disease phenotypes measured by flow cytometry and immunoassays from cellular infection of LCLs. Applying Hi-HOST to Chlamydia trachomatis, an obligate intracellular bacterium, revealed a genome-wide significant association between rs2869462 and levels of a pro-inflammatory chemokine, CXCL10, measured in assay supernatants before and after infection. Curiously, we noticed wide variation in induction of CXCL10 that was not associated with rs2869462. Leveraging flow cytometric measurements of infected cells in a multivariate linear model revealed that the most highly infected LCLs showed a high degree of suppression of CXCL10. This indicated to us that C. trachomatis may be actively suppressing CXCL10 induction. We experimentally identified chlamydial protease-like activity factor (CPAF) as responsible for suppression of CXCL10. Applying our multivariate modeling to a panel of 17 other cytokines revealed a similar signature of suppression for RANTES. However, this phenotype was not mediated by CPAF, indicating some degree of specificity of CPAF activity. To further refine Hi-HOST with higher resolution phenotypes and integrated eQTL (expression quantitative trait loci) analyses all in a single infection, we developed single-cell Hi-HOST (scHi-HOST). scHi-HOST leverages single-cell RNA-sequencing of pooled LCLs to simultaneously identify alleles associated with gene expression and susceptibility to influenza A virus (IAV). scHi-HOST identified a common missense variant in ERAP1, rs27895, as associated with viral burden in LCLs. I confirmed this association experimentally using RNAi, overexpression and small molecule inhibition of ERAP1 in vitro. Finally, we performed analysis of human flu challenge and found that volunteers with the risk allele of rs27895 had increased viral burden and worse symptoms over the course of their infection, indicating that our cellular findings may translate to human flu susceptibility as well. Finally, to identify strain-specific susceptibility alleles, I applied scHi-HOST to six diverse strains of IAV. Analyses of these data suggested that infection with CA09 (the strain responsible for the 2009 “Swine Flu” pandemic, A/California/04/2009), produced distinct infection phenotypes and a distinct set of associated genetic variants relative to other strains. I identified rs7144228, an eQTL for HSP90AA1, as significantly associated with CA09 infection, but not any other IAV strain. rs7144228 is specific to populations with African ancestry and contributes more broadly to population differences observed during IAV infection of LCLs. I also identified rs113816500, a SNP intronic to CTSH, as associated with all six strains of IAV, and therefore is a conserved host factor that influenza exploits to increase viral burden. This study suggests that susceptibility to infection is not only dependent on the genotype of the affected individual but is also dependent on the genetic background of the virus.

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Schott, Benjamin (2023). Novel Methods and Mechanisms of Human Genetic Susceptibility to Infectious Disease. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/27625.

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