Dennis Chun-Yone Ko - US grants

DESCRIPTION (provided by applicant): Despite improvements in public health, advancements in vaccines, and the development of many classes of antibiotics, infectious disease is still responsible for over a quarter of all deaths worldwide. However, even for the most devastating of pandemics, history demonstrates a large variability in the severity and duration of infection. The long-term research goal of the candidate, Dr. Dennis Ko, is to determine the genetic basis for differences in susceptibility to bacterial infections. This knowledge is important for determining why some individuals are resistant to different infections and in developing therapies to decrease the mortality and morbidity of susceptible individuals. Experiments in this proposal will elucidate how human genetic differences contribute to variation in infection by the bacteria that causes typhoid fever, Salmonella typhi. The first aim is to identify human genetic differences that modify invasion of S. typhi into cultured cells. This is accomplished through a novel genetic association screen using cells obtained from hundreds of genotyped individuals. Genetic variants revealed by the screen will then be validated by measuring the effect of RNA interference on S. typhi invasion. The second aim is to determine how the identified genetic variants affect the cellular mechanism of S. typhi invasion. Fluorescence microscopy and flow cytometry will be used to determine if uptake of the bacteria or early establishment of an intracellular niche is being affected. Further experiments will include perturbation and assessment of molecules known to play roles in Salmonella invasion, including phosphoinositides, Rho-family GTPases, and secreted bacterial effectors. The third aim is to assess the relevance of identified differences on disease using mouse models and clinical association data. Mouse models will be used to determine if the identified genes affect establishment of infection and/or spread within the host. As a complement to these studies, the relevance of the genetic differences upon typhoid fever and other diseases will be addressed through case-control genetic association studies. The overall goal of these proposed experiments is discovery of both basic cell biological mechanism as well as human genetic variation important for health and disease. Dr. Ko, is a Life Sciences Research Foundation fellow in the laboratory of Dr. Samuel Miller. He received MD-PhD degrees from Stanford University. His thesis research on the lipid-storage disorder Niemann-Pick Type C provided a firm foundation in genetics and cell biology, and he has applied these disciplines to his current research on understanding human variation to bacterial infection. As a postdoctoral fellow, his efforts focused on developing a novel screening strategy for discovering genetic variants that modify Salmonella- induced cell death and determining how these variants affect human health. Dr. Ko plans to start an independent research lab studying susceptibility to S. typhi invasion, with the goal of expanding his focus to the importance of variation and adaptation on both sides of the host-pathogen relationship.

PROJECT SUMMARY Damage to reproductive organs as a result of inflammatory responses to STIs can lead to severe complications such as pelvic inflammatory disease, ectopic pregnancy and infertility. Multiple environmental factors including the infecting strain, dose and frequency of infections, and composition of the microbial communities in the urogenital tract contribute to the severity of disease manifestation and ensuing sequelae. Similarly, co-infection with other sexually transmitted pathogens may act synergistically to worsen disease. Finally, host genetics likely plays an important role in susceptibility to infection and ensuing immunopathologies. This project will address the role played by human genetic polymorphisms that regulate cellular interactions and responses to C. trachomatis alone or in the context of N. gonorrhoeae and vaginal microbiota components. We will identify and characterize genetic variants that affect STIs by applying two parallel and complementary approaches. First, we will use a discovery platform for genome-wide association studies (GWAS) of cellular traits called Hi-HOST (high throughput human in vitro susceptibility testing). Hi-HOST combines precise measurement of phenotypes in cells derived from hundreds of normal, genotyped individuals with genome-wide association to identify genetic differences that underlie the phenotypic variation. Furthermore, we propose to extend the Hi-HOST framework to examine how co-infection and the microbiota can act synergistically or antagonistically on the immune response and how human genetic differences can modulate these effects. Second, we will carry out GWAS of clinical traits and outcomes using the STING cohort. This dual approach will allow for study of human genetic variation in both the controlled experimental setting of identical infections with Hi-HOST and the more clinically relevant but complex setting of patients. We predict that overlap of SNPs identified by Hi-HOST and GWAS of the STING cohort will highlight human variation affecting both cellular infection phenotypes and clinical phenotypes and outcomes. Thus, we will determine not only which human genetic variants are associated with susceptibility to STIs but also intermediate phenotypes (such as cytokine levels, miRNA, and microbiota composition) that are likely responsible for the altered physiology. This will facilitate identification of biomarkers and possible drug targets, as well as specific genetic populations that might benefit most from targeted therapies.

? DESCRIPTION (provided by applicant): Salmonella enterica serovar Typhi causes typhoid fever, a severe infection that affects more than twenty million people every year. Much knowledge has been gained over the last twenty years concerning how Salmonella causes disease through manipulating host cellular pathways and how host cells respond. However, there is tremendous variation in who gets infected and the severity of infections. How host signaling pathways and responses are affected by human genetic variation to modulate susceptibility to Salmonella infection is poorly understood. This naturally occurring host genetic variation is an untapped resource that could reveal novel components of pathways, prediction of susceptibility, and possibly host-directed therapeutic targets. The long-term goal of this research is to understand how host genetic differences alter the responsiveness and susceptibility of cells to Salmonella infection and how this affects risk and severity of disease in people. In pursuit of this goal, genome-wide association studies (GWAS) of cellular Salmonella infection traits were carried out using a platform called Hi-HOST (High-throughput Human in vitro Susceptibility Testing). In Hi- HOST screens, precise measurements of infection readouts are conducted on cells derived from hundreds of genotyped individuals. Genome-wide association is then used to identify genetic differences that underlie variation in the cellular infection phenotypes. Using Hi HOST, three new regulators that affect Salmonella- induced cell death in cells and sepsis in human populations were identified and characterized. The objective of this application is to use Hi-HOST and subsequent studies to define and characterize host genetic differences that alter S. typhi invasion and early survival. The host genetic differences serve as the starting point for mechanistic studies to determine how host pathways involving macropinocytosis and early survival are altered. By integrating these cellular studies with animal models and genotyping of human typhoid fever cohorts, an in depth understanding will be achieved for how genetic differences contribute to who gets sick and why. In Aim 1, common human genetic variation that modulates S. typhi invasion will be identified and validated. In Aim 2, the impact of variation in S. typhi invasion on typhoid fever in human populations and Salmonella infections in animal models will be assessed. In Aim 3, mechanisms for how the newly identified regulators modulate S. typhi invasion will be characterized. Carrying out these Aims will result in the elucidation of new mechanisms in host-pathogen interactions and ultimately a broad understanding for how genetic differences determine which individuals are at risk for typhoid fever and possibly other diseases caused by pathogens which utilize similar invasion mechanisms. With rising concerns over resistance to broad-spectrum antimicrobials, this study may leverage our genetic differences to reveal new therapeutic targets focused instead on host modulation.

While the genomes of two people are >99% identical, the genetic differences encode amazing diversity in human traits and disease susceptibility. Understanding of the genetic architecture underlying human diversity in complex traits has been transformed by the application of genome-wide association studies (GWAS). However, GWAS are just the first step in understanding how genetic variants contribute to disease risk by impacting genes that encode components of cellular physiology. Elucidating this chain of causality from SNP to gene to cell biology to disease can serve to not only functionally validate the genetic association with disease but also reveal potential therapeutic targets. Therefore, there is a need for approaches that can facilitate identification of the cellular pathways regulated by human genetic variants associated with disease. The objectives of this application are to systematically reveal the shared genetic architecture connecting cellular physiology and disease susceptibility and to develop a database to facilitate hypothesis generation from these data. The experimental and computational tools are in place to carry this out successfully. GWAS of molecular and cellular traits have been generated by our lab and acquired from publicly available datasets. An extensively validated cellular GWAS platform called Hi-HOST (High-throughput human in vitro susceptibility testing) uses pathogens to stimulate fundamental cellular pathways. Hi-HOST combines precise measurement of cellular phenotypes in lymphoblastoid cell lines (LCLs) from nearly a thousand people with genome-wide association using 15 million genetic variants to identify genetic differences that underlie the phenotypic variation. With this platform, cellular responses of infectivity and replication, cytokine levels, and host cell death using 9 different pathogens have already been carried out and analyzed with family-based GWAS analysis on 148 cellular traits. Dozens of SNPs pass genome-wide significance have been incorporated into established workflows in the lab to validate their importance and determine mechanism. Additional datasets to expand our analyses beyond LCLs include metabolomics GWAS data from blood and urine and GWAS of immune cell levels. These molecular and cellular GWAS dataset will be integrated with human disease GWAS from the NHGRI/EMBL catalog. The central hypothesis is that the SNPs associated with each cellular phenotype and disease serve as a ?GWAS signature? that can be used to connect these different traits based on similarity and that the contribution of individual cellular traits to heritability of diseases can be estimated from comparing these signatures. Published methods developed by our own lab (CPAG (Cross-Phenotype Analysis of GWAS)), as well as by other investigators (LD-score regression) will be used to carry out these analyses. Thus, this project will integrate cellular and disease GWAS to create a re-interpretation of the human genome through the lens of cell biology. The project leverages existing datasets into a hypothesis generating engine for researchers looking to explore new diagnostic and therapeutic possibilities.

Abstract Pathogenic bacteria within a mammalian host are bombarded by intercellular, interspecies, and cross-kingdom signaling molecules and metabolites. For Salmonella enterica, multiple signals, including pH, bile, and short chain fatty acids, regulate Salmonella Pathogenicity Island I (SPI-1) to trigger invasion and inflammation in the gut. Recently, our lab demonstrated that the methionine-derived metabolite methylthioadenosine (MTA) is a previously unrecognized signal produced by the host and pathogen that suppresses SPI-1, flagellar motility, and in vivo virulence. These conclusions were drawn from experiments treating S. Typhimurium with MTA and by characterizing a mutant with increased endogenous MTA. This mutant lacks the master repressor of the methionine metabolism pathway, metJ. In addition to MTA?s effect on S. Typhimurium, MTA also modulates host responses and is released into plasma during S. Typhimurium infection in mice and during sepsis in humans. Thus, MTA can suppress S. Typhimurium virulence and its levels in the host undergo dramatic shifts during infection. However, it is unknown if and how host- or microbiota-produced MTA signals to S. Typhimurium at the crucial initial site of this host-pathogen conflict, the intestinal lumen. We hypothesize that MTA in the intestine is dynamically regulated by host, commensal microbiota, and pathogen, and that shifts in MTA levels are sensed by Salmonella to regulate virulence gene expression. Therefore, the first goal of this project is to characterize contributions and consequences of host- and microbial-derived MTA during S. Typhimurium infection. MTA levels in gut contents and tissue will be measured from germ-free and microbiome reconstituted mice at baseline and with S. Typhimurium infection. Furthermore, we will manipulate host MTA levels through exogenous MTA and the use of an inhibitor that specifically blocks host MTA metabolism. The second goal is to elucidate mechanisms of MTA suppression of S. Typhimurium virulence. We will test whether MTA modulates regulators of SPI-1 expression and if MTA facilitates broader gene expression changes through regulation of methylation. Completion of these aims will identify whether MTA acts as an interspecies signal in the gut and identify how MTA suppresses virulence. This work could improve clinical outcomes by demonstrating that existing methionine metabolism inhibitors can suppress S. Typhimurium virulence.

Salmonellae cause an estimated 150 million cases of gastroenteritis and 25 million cases of invasive disease (enteric fever and non-typhoidal bacteremia), leading to 300,000 deaths per year. Salmonella manipulates multiple host cellular pathways through secreted effector proteins, but the molecular functions of most effectors, especially in animals and humans, remain poorly understood. A thorough characterization of how Salmonella effectors function and the pathways they target is required to understand how effectors impact infection and their long-term consequences on chronic inflammatory conditions, such as inflammatory bowel disease (IBD). IBD is an immune-mediated disease and whether IBD is affected by previous Salmonella infection is unclear, with conflicting evidence from both epidemiological and mouse studies. To understand how acute Salmonella infections may impact long-term immune modulation, studies are needed to determine how specific Salmonella effectors target host immune regulatory pathways, whether these changes persist beyond infection, and if these changes modulate risk or severity of disease. Through comparative genomics of Salmonella serovars, we recently identified a novel prophage-encoded Type III secreted effector: Salmonella anti-inflammatory response activator (SarA). SarA is the only Salmonella effector demonstrated to be necessary and sufficient to activate STAT3 (signal transducer and activator of transcription-3), a key transcription factor that regulates immune cell proliferation, development, and autoimmune conditions including IBD. SarA-mediated manipulation of STAT3 reprograms transcription in host cells and increases virulence in mice. We hypothesize SarA has both direct effects on cells injected with the effector and secondary consequences due to STAT3 target genes (such as the anti-inflammatory cytokine IL-10) that may cause persistent changes after infection has cleared. Therefore, the objective of this application is to determine how SarA activates STAT3 and affects immune cell populations during and after infection. Based on preliminary data, we hypothesize that SarA directly binds STAT3 and cofactors to assemble a STAT3- activating platform. Activation of this pathway phosphorylates STAT3 in multiple cell types, but it is unknown which cells are targeted in vivo and what the consequences of this activation are during and after infection. Therefore, we propose Specific Aims to 1) determine how SarA drives STAT3 activation through biochemical and genetic approaches and 2) determine the effects of SarA on immune cell populations and long-term consequences for the host, including severity of intestinal inflammation in colitis. Following completion of these aims, we will have determined how SarA mediates STAT3 signaling and how/if these signaling events alter immune function during and after infection. Revealing these mechanisms could lead to new therapeutic strategies for treating salmonellosis, as well as other STAT3-dependent pathological conditions including autoimmune diseases, cancer, and other infectious diseases.

Salmonellae cause an estimated 150 million cases of gastroenteritis and 25 million cases of invasive disease (enteric fever and non-typhoidal bacteremia), leading to 300,000 deaths per year. Findings based on inbred mouse strains and rare human mutations indicate that the clinical presentations and outcomes of salmonellosis are influenced by the host?s genetic makeup. However, we do not understand the impact of common, naturally occurring human genetic differences on Salmonella infections. Our long-term goal is to leverage high- throughput cellular phenotyping of infection and natural genetic diversity to define molecular parameters of susceptibility to human infectious diseases. While genome-wide association studies (GWAS) of enteric fever and bacteremia identified genetic differences regulating susceptibility, such studies are limited by high variability in patient exposure, pathogen genetic diversity, and a limited understanding of the pathophysiology of identified variants. We developed a complementary cellular GWAS approach (Hi-HOST: High-throughput Human in vitrO Susceptibility Testing; http://h2p2.oit.duke.edu) to perform high resolution analysis of human genetic differences that impact host-pathogen traits while enabling experimental dissection of these traits. In the previous funding period, we focused on human genetic variants that regulate Salmonella invasion of host cells. We identified a regulatory variant in VAC14 that modulates levels of plasma membrane cholesterol and thus limits the docking of the Salmonella SPI-1 type III secretion system to host cells. Humans with the high-invasion allele of VAC14 have increased risk of typhoid fever and bacteremia. The objectives of this application are to define and characterize human genetic differences that alter the full spectrum of Salmonella host-pathogen interactions and assess their impact on disease. Building from our unique resource of Hi-HOST cellular GWAS of Salmonella invasion, replication, and host cytokine levels, we have identified SNPs mediating susceptibility to infection phenotypes that will undergo experimental validation and mechanistic studies. We will determine how regulatory SNPs affecting ARHGEF26 and MCOLN2 expression impact Salmonella invasion and replication and the role these genes play during infection in mice. For ARHGEF26, a Rho GEF that stimulates Salmonella-induced membrane ruffling, our association data and experimental evidence indicate SNPs regulate ARHGEF26 expression to increase invasion dependent on the Salmonella effector sopB. For MCOLN2, we hypothesize that this divalent cation channel is a novel factor that regulates intracellular replication by modulating nutrient access, metal toxicity, and/or immune cell polarization. Using Arhgef26 and Mcoln2 mice, we will elucidate how altered expression of these genes regulates bacterial burden and immune response during infection. Finally, we will test whether SNPs identified by Hi-HOST are associated with enteric fever and bacteremia in humans. Revealing these mechanisms could lead to new therapeutic strategies for salmonellosis and other diseases impacted by the same genetic variants.

Project Summary/Abstract It is estimated that 50-100 million people (~5% of the global population) died from the 1918 influenza pandemic. While influenza infections usually do not cause such severe disease, ~30 million are infected every year in the United States alone (2014-2015). However, there are broad differences in influenza susceptibility and severity, with outcomes from asymptomatic infections (~16%) to death (0.2% in 2014-2015). These differences arise from the complex interplay of exposure, environment, influenza genetics, and human genetics. The overall goal of my lab is to understand how human genetic diversity regulates susceptibility and severity of infections. Famous examples of genetic differences that profoundly impact susceptibility include sickle cell allele protection against malaria and CCR5 deletion protection against HIV. Such genetic differences can lead to insights on pathogenesis, drug targets (e.g. CCR5 inhibitors), and more personalized care. For influenza, common genetic variation has been most convincingly shown to influence flu severity at a single locus (IFITM3) that regulates a single step (cytosolic entry) in the complex influenza life cycle. We hypothesize that other human genetic differences affect influenza infection and can be identified through measuring inter-individual variation in cellular infection phenotypes. To facilitate identification of SNPs that affect cellular infection phenotypes, we developed and validated a cell-based GWAS approach called Hi-HOST. SNPs identified as important for influenza infection by Hi-HOST can then be examined for relevance in human infection using already completed human flu challenge studies and population-based studies. We propose that the intersection of human subject and cell line data facilitates discovery of novel pathways and genetic determinants of susceptibility. This project will generate a high resolution analysis of how human genetic variants impact transcription, cellular phenotypes, and human disease following influenza exposure. We will accomplish this through 1) identifying human SNPs that confer resistance/susceptibility to cellular and molecular phenotypes of flu infection, including entry, replication, cell death, cytokine levels, and host transcriptional responses, 2) determining the impact of SNPs on host transcription during influenza challenge of healthy volunteers, and 3) integrating the generated cellular and human challenge datasets to generate and test hypotheses linking transcriptional response and cellular susceptibility. Understanding these differences could lead to new diagnostic approaches in identifying at-risk individuals and novel therapeutic strategies for treatment.