Human Genetic Variation in VAC14 Regulates Pathogen Entry and Risk of Infectious Disease by Monica I. Alvarez Department of Molecular Genetics and Microbiology Duke University Date:_______________________ Approved: ___________________________ Dennis Ko, Supervisor ___________________________ Soman Abraham ___________________________ Andrew Alspaugh ___________________________ Jörn Coers ___________________________ David Tobin Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University 2017 ABSTRACT Human Genetic Variation in VAC14 Regulates Pathogen Entry and Risk of Infectious Disease by Monica I. Alvarez Department of Molecular Genetics and Microbiology Duke University Date:_______________________ Approved: ___________________________ Dennis Ko, Supervisor ___________________________ Soman Abraham ___________________________ Andrew Alspaugh ___________________________ Jörn Coers ___________________________ David Tobin An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University 2017 Copyright by Monica I. Alvarez 2017 Abstract Human genetic variation can be leveraged to understand the subtleties of how common variants with small effect sizes can alter cellular phenotypes and ultimately affect susceptibility to pathogenic disease. By combining GWAS of different phenotypic scales and basic cell biology, we can answer how a particular SNP affects a disease. This body of work elucidates the biological mechanism of how a SNP in VAC14, which encodes a human scaffolding protein involved in phosphoinositide metabolism, alters susceptibility to Typhoid Fever and other pathogens. Using Hi-HOST (High-throughput Human in vitro Susceptibility Testing), a GWAS platform for cellular host-pathogen traits, we discovered that the ‘A’ allele of rs8060947 was associated with decreased VAC14 protein expression and increased Salmonella Typhi invasion. We experimentally confirmed the phenotype using RNAi to transiently decrease VAC14 protein expression in LCLs and Helas and saw increased Salmonella Typhi invasion. Further studies, using genetic and pharmacological manipulations were able to determine how VAC14 affects Salmonella Typhi invasion. CRISPR knockout VAC14 cells had a robust increase in invasion, and had increased cholesterol accumulation in the cell. Salmonella preferentially docks to cholesterol on the host plasma membrane as one of the first steps involved in invasion. Thus, increasing i cholesterol at the plasma membrane increased the number of docked bacteria and ultimately caused higher invasion percentages. To confirm the relevance of cholesterol and Salmonella Typhi beyond cell culture, we infected the swim bladder of Zebrafish with S. Typhi. Fish were pretreated with Ezetimibe, an FDA approved cholesterol-reducing drug, and then subsequently infected with S. Typhi. Fish treated with Ezetimibe, had decreased cholesterol staining by filipin, and had increased survival from S. Typhi infections. Additionally, because of the optically transparent nature of the zebrafish embryo we were able to image the fish 24hrs after infection and show that ezetimibe treated fish had higher bacterial clearance. In addition to the fish studies, a collaboration with Dr. Sarah Dunstan (University of Melbourne) was able to retrospectively determine that VAC14 had an effect on human susceptibility to typhoid fever. The ‘A’ allele for SNP rs8060947, which we showed had decreased VAC14 protein expression and increased S. Typhi invasion in cell culture, was found to be more common in people with typhoid fever, suggesting the ‘A’ allele increases human susceptibility to this disease. All together, we have shown that decreased VAC14 expression causes an increase in cellular cholesterol, leading to an increase in docking and invasion of Salmonella and ultimately increasing your chances of acquiring typhoid fever. ii The central role of cholesterol in entry of multiple pathogens led us to hypothesize that natural variation or experimental manipulation of VAC14 expression could play a role in pathogens beyond Salmonella. Here we show that its effects extend beyond bacteria to parasites. With cholesterol regulating entry of Plasmodium into hepatocytes, we hypothesized that increasing the amount of cellular cholesterol in hepatocytes will increase Plasmodium entry. These ideas are being tested in collaboration with Maria Toro and Dr. Emily Derbyshire (Duke University). However, unpublished human genetic data already support the idea that VAC14 regulates susceptibility to malaria infection. The same SNP associated with Salmonella invasion (rs8060947) is associated with malaria risk in African populations (Gavin Band and the MalariaGEN Consortium, personal communication). VAC14 may also affect pathogen entry through its role in regulation of endosomal trafficking. VAC14 forms a complex with the FIG4 phosphatase and PIKfyve kinase to modulate endosomal trafficking through the metabolism of PtdIns(3,5)P2. Recently, FIG4 and PIKfyve were found to be necessary for Ebola entry in a somatic cell genetic screen. Using our VAC14 CRISPR knockout cells we determined that cells mutated for VAC14 had a similar phenotype. Ebola virus-like-particle (VLP) entry decreased dramatically in cells lacking VAC14. While we discovered that VAC14 affects cellular cholesterol, its main reported function is to regulate endosomal trafficking. We iii hypothesize that lack of VAC14 interferes with proper endosomal maturation and thus prevents the Ebola VLP from reaching its intracellular receptor NPC1 and exiting into the cytoplasm. The common allele (A) that alters VAC14 expression is associated with decreased protein synthesis, and increased susceptibility to both Salmonella and Malaria infection. On the other hand, decreased VAC14 expression inhibits proper endolysosomal trafficking, inhibiting Ebola infection. These two mechanisms of affecting different infectious diseases may provide opposing forces in an example of balancing selection. iv Dedication To my husband, Luis, whose support and encouragement got me through these five years, and my daughter, Emma, who has become my reason to succeed. v Contents Abstract ............................................................................................................................................ i List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................ x Acknowledgements ..................................................................................................................... xi 1. Introduction ............................................................................................................................... 1 1.1 Human Genetic Variation and its role in Disease ........................................................ 1 1.2 High-throughput Human In Vitro Susceptibility Testing (Hi-HOST) ..................... 5 1.3 Salmonella ......................................................................................................................... 6 1.3.1 Typhoid Fever .............................................................................................................. 7 1.3.2 Invasion ....................................................................................................................... 10 1.4 VAC14 .............................................................................................................................. 12 1.4.1 Protein Complex ........................................................................................................ 13 1.4.1.1 FIG4 ...................................................................................................................... 14 1.4.1.2 PIKfyve ................................................................................................................ 14 1.4.2 Role of PtdIns(3,5)P2 .................................................................................................. 15 1.5 Cholesterol ....................................................................................................................... 15 1.5.1 Cholesterol Metabolism ............................................................................................ 16 2. Material and Methods ................................................................................................................ 18 2.1 Cell Biology ..................................................................................................................... 18 2.1.1 Cells ............................................................................................................................. 18 vi 2.1.2 Bacterial Infection ...................................................................................................... 18 2.1.3 RNAi experiments ..................................................................................................... 19 2.1.4 Western blotting ........................................................................................................ 20 2.1.5 Generation of VAC14 CRISPR mutant cells .........................................................