Browsing by Author "Ko, Dennis C"

Background

Results

Conclusions

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 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.

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 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.

Human genetic diversity can have profound effects on health outcomes upon exposure to infectious agents. For infections with Chlamydia trachomatis (C. trachomatis), the wide range of genital and ocular disease manifestations are likely influenced by human genetic differences that regulate interactions between C. trachomatis and host cells. Using a cellular GWAS, we leveraged this diversity in cellular responses to demonstrate the importance of variation at the Toll-like Receptor 1 (TLR1), TLR6, and TLR10 gene locus to cytokine production in response to C. trachomatis. We determined that a single nucleotide polymorphism (SNP), rs1057807, located in a region that forms a loop with the TLR6 promoter, is associated with increased expression of TLR1, TLR6, and TLR10 and secreted levels of ten C. trachomatis-induced cytokines. Production of these C. trachomatis-induced cytokines is primarily dependent on MyD88 and TLR6 based on experiments using inhibitors, blocking antibodies, RNAi, and protein overexpression. Population genetic analyses further demonstrated that the mean IL-6 response of cells from two European populations were higher than the mean response of cells from three African populations and that this difference was partially attributable to variation in rs1057807 allele frequency. In contrast, a SNP associated with a different pro-inflammatory cytokine (rs2869462 associated with the chemokine CXCL10) exhibited an opposite response, underscoring the complexity of how different genetic variants contribute to an individual’s immune response. This multidisciplinary study has identified a long-range chromatin interaction and genetic variation that regulates TLR6 to broaden our understanding of how human genetic variation affects the C. trachomatis-induced immune response.The identification of host factors involved in infectious disease pathogenesis have the potential to lead to the clinical development of therapeutics or biomarkers of disease progression. Through the identification and characterization of a SNP associated with C. trachomatis-induced cytokine responses, we have elucidated a role for TLR6 in the host immune response to C. trachomatis. Further characterization of this genetic locus and the involvement of TLR6 in C. trachomatis infection will determine the clinical relevance of this association. As C. trachomatis is a disease with a wide range of clinical outcomes that are likely influenced by human genetic differences, other genetic loci beyond the TLR locus are also likely involved in C. trachomatis pathogenesis and host response. To broaden the tools available to study the role of human genetic variation in C. trachomatis cellular infection traits beyond the TLR10/TLR1/TLR6 locus, we developed single-cell Hi-HOST (scHi-HOST), a cellular single-cell RNA-sequencing-based variation of the original Hi-HOST cellular GWAS screen and applied the platform to C. trachomatis infection. Whereas Hi-HOST is powerful and has proved successful, the technique is time- and labor-intensive. scHi-HOST decreases the amount of time and labor required for cellular GWAS screening and introduces a cellular phenotype not assayed in Hi-HOST: host gene expression. As scHi-HOST replicates the original Hi-HOST phenotypes and introduces a new cellular phenotype measurement, this screen serves as a replication screen for previously identified associations and a discovery screen for new associations.

As the current SARS-CoV-2 pandemic highlights, infectious diseases outcomes range from asymptomatic cases to prolonged morbidity and death. We can predict these outcomes using risk factors like age and BMI; however, we currently lack the capacity to generate more fine-grained, or even individualized, outcome predications. In my thesis, I increased mechanistic insight into how Salmonella enterica (S. enterica) generates an equally diverse set of outcomes by unraveling how genetic diversity in both Salmonellae and humans impact infection.

S. enterica is a very diverse pathogen with thousands of serovars. In their core genome, all S. enterica serovars carry two molecular syringes called type-three secretions systems (T3SS), which inject bacterial proteins called effectors into host cells. These effectors create a hospitable niche inside host cells for the replicating bacteria. Outside of the core genome, each serovar carries hundreds to thousands of different genes, which means each serovar packs a unique genetic toolset. We discovered that only a handful of non-typhoidal serovars carry an effector called SarA—an effector that activates the host transcription factor STAT3 to render host cells and mice more permissive to S. enterica replication through an anti-inflammatory transcriptional program.

Following this discovery, I determined that SarA activates STAT3 by mimicking the active cytokine co-receptor gp130. Specifically, a 40 amino acid stretch in SarA is homologous to the STAT3-binding portion of gp130’s cytoplasmic tail. We dubbed this stretch the GBS (gp130-binding of STAT3) sequence. By generating chimeric gp130-SarA proteins, I determined that the two GBS are functionally interchangeable. In fact, the SarA GBS binds STAT3 with even greater affinity when measured with isothermal titration calorimetry. This results in SarA driving prolonged and more robust STAT3 signaling than cytokine-gp130 signaling. This continues a reoccurring theme in bacterial pathogenesis: effectors evolve to be more effective than their mammalian counterparts. This is due to selective pressure driving effectors toward supraphysiological responses that enable replication and dissemination, whereas host signaling is pressed toward a measured and regulated response that promotes homeostasis.

In complementary studies exploring how human genetics contribute to variable outcomes, we infected hundreds of genotyped human cell lines from around the world and measured S. enterica serovar Typhi (S. Typhi) replication inside these human cells as a proxy for virulence in a whole person. We then associated this quantitative outcome with more than 10 million genetic markers in the genotyped cell lines to identify specific loci that associate with significantly more or less intracellular S. Typhi replication. One of these loci regulates expression of the divalent cation channel gene MCOLN2. By deleting the MCOLN2 gene, I confirmed mucolipin-2 (MCOLN2 or TRPML2) is a host factor that reduces S. Typhi replication inside human immune cells. Further, MCOLN2-/- mice have increased Salmonella burden.

To determining how mucolipin-2 restricts S. Typhi replication, I used dual RNA- seq of host and bacterial transcripts, which allows the intracellular S. Typhi to serve as a probe that reports how intracellular conditions change when MCOLN2 is removed. These results indicated that mucolipin-2 reduces Mg2+ availability to the bacteria. Repleting Mg2+ during in vitro infection only increases S. Typhi replication when mucolipin-2 is present, demonstrating that Mg2+ addition overcomes MCOLN2- dependent restriction.

Identifying these variable bacterial and host factors can improve the targeting of therapeutics—especially as next generation sequencing becomes more common clinically. In the long-term, elucidating how these genetic variants modulate the outcomes of Salmonella infection teaches us how severe outcomes occur, and hopefully, how to avoid or mitigate them.

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.

Salmonella enterica is an important foodborne pathogen that utilizes secreted effector proteins to manipulate host pathways to facilitate survival and dissemination. Different S. enterica serovars cause disease syndromes ranging from gastroenteritis to typhoid fever and vary in their effector repertoire. Knowledge of this effector repertoire is still incomplete. The effector protein encoded by stm2585 had previously been identified as relevant to in vivo infection, but the mechanism of action was unknown. We leveraged S. enterica’s natural genetic diversity to identify stm2585, here designated sarA (Salmonella anti-inflammatory response activator), as a Salmonella effector that induces production of the anti-inflammatory cytokine IL-10 in lymphoblastoid cell lines (LCLs) through activating STAT3 to promote virulence. RNA-seq of cells infected with either ∆sarA or wild-type S. Typhimurium revealed that SarA activated STAT3 transcriptional targets. Consistent with this, SarA was necessary and sufficient for STAT3 phosphorylation, STAT3 inhibition blocked IL-10 production, and physical interaction of SarA and STAT3 was identified by co-immunoprecipitation. These effects of SarA contributed to intracellular replication in vitro and bacterial load at systemic sites in mice.

In order to further characterize SarA’s mechanism, we searched for interactions with host proteins. A yeast-two-hybrid screen revealed that SarA bound the cytoplasmic region of IL-7Rα. Co-expression of FLAG-SarA and IL-7Rα in HeLas resulted in phosphorylation of IL-7Rα and interaction of IL-7Rα with FLAG-SarA, as confirmed by co-immunoprecipitation of FLAG-SarA. In LCLs, siRNA against IL-7Rα moderately inhibited SarA’s ability to induce IL-10. However, IL-7Rα’s endogenous ligands IL-7 and TSLP did not mimic SarA’s effect, and IL-7Rα knockout mice did not provide ΔsarA with any benefit versus wild-type in competitive index, so IL-7Rα’s contribution to SarA’s activation of STAT3 is still unclear.

Mass spectrometry of proteins which co-immunoprecipitated with SarA confirmed interaction with STAT3 and revealed several other interacting proteins. Mass spectrometry also indicated that SarA contained phosphorylated residues, including a phosphorylated tyrosine in a previously reported STAT3 binding motif. Mutation of this phosphorylated tyrosine to phenylalanine on a bacterial expression plasmid greatly reduced SarA’s ability to induce IL-10 in LCLs. The same mutant on a mammalian expression plasmid showed less activation and no co-immunoprecipitation of STAT3. This indicated that SarA’s interaction with STAT3 was at least partially dependent on that tyrosine, and suggested that SarA was directly binding STAT3 at this site.

Our results demonstrate the power of using comparative genomics for identifying effectors and that S. enterica has evolved mechanisms for activating an important anti-inflammatory pathway. We also characterize SarA as a potential basis for immunomodulatory therapy.

Leishmaniasis is a neglected tropical disease with diverse disease outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. With no vaccines available and treatment options limited by significant costs and side effects, understanding the pathophysiology of leishmaniasis is paramount. However, the complex interactions between parasites in the Leishmania genus and their hosts make this task challenging. This dissertation dissects host and pathogen regulation of a chemokine, CXCL10, that can provide protection against leishmaniasis. First, a novel mechanism by which Leishmania parasites suppress CXCL10 by proteolytic cleavage is identified. Second, variation in CXCL10 between Leishmania spp. that cause different forms of disease is characterized. Third, CXCL10 signaling is studied in a murine model by using small molecule agonists to circumvent parasite immune evasion.

The first goal of this project was to identify how Leishmania parasites are able to suppress CXCL10, a chemokine with diverse antimicrobial functions, including T cell recruitment. Infection with L. major in a human monocyte cell line induced robust CXCL10 transcription without increasing extracellular CXCL10 protein concentrations. CXCL10 suppression was found to require the virulence factor and protease, glycoprotein-63 (gp63). Specifically, GP63 cleaves CXCL10 after amino acid A81 at the base of a C-terminal alpha-helix. Cytokine cleavage by GP63 demonstrated specificity, as GP63 cleaved CXCL10 and its homologues, which all bind the CXCR3 receptor, but not distantly related chemokines, such as CXCL8 and CCL22. Further characterization demonstrated that CXCL10 cleavage activity by GP63 was produced by both extracellular promastigotes and intracellular amastigotes. Crucially, CXCL10 cleavage impaired T cell chemotaxis in vitro, indicating that cleaved CXCL10 cannot signal through CXCR3. Consistent with CXCL10 cleavage during infection, we observed GP63-mediated impairment of activated CD8+ T-cells in the draining lymph nodes of C57BL/6JWT mice. Correspondingly, in C57BL/6JWT mice, gp63 deletion resulted in slower lesion development and a smaller maximum lesion size. However, infection in C57BL/6Jcxcr3-/- mice revealed the delay to lesion development required CXCR3 signaling. Ultimately, we propose CXCL10 suppression is a convergent mechanism of immune evasion by intracellular pathogens, as Salmonella enterica and Chlamydia trachomatis also suppress CXCL10. This commonality suggests that counteracting CXCL10 suppression may provide a generalizable therapeutic strategy against intracellular pathogens.

Although cutaneous leishmaniasis is a classic example of type-1 immunity leading to well controlled self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania diversity in both pathogen sensing by the host and immune evasion by the parasite contributes to variation in the polarization and robustness of the immune response. Here, I demonstrate that unlike other Leishmania (L.) spp., L. Viannia (V.) panamensis is unable to suppress CXCL10 due to reduced cleavage activity by GP63. Similarly, other parasites in the Viannia subgenus, which cause the immunopathologic mucocutaneous form of leishmaniasis, have reduced CXCL10 cleavage capacity. To understand how this altered phenotype emerged, we used protein-protein interaction modeling of the primary amino acid sequence and known crystal structures to identify a putative CXCL10 binding site on GP63. The putative CXCL10 binding site, which varies between the Leishmania and Viannia subgenera, was found to be in a region under significant positive selection. Finally, we experimentally confirmed that the predicted binding site and adjacent positively selected amino acids are involved in CXCL10 suppression by demonstrating a specific reduction in CXCL10 cleavage after mutagenesis of the wild-type L. (L.) major gp63 to the L. (V.) panamensis allele at the binding site. These results demonstrate how genetic diversity of parasites contribute to variation in the host immune response to Leishmania spp. Unraveling additional molecular mechanisms whereby parasite genetic diversity contributes to heterogeneity of host immune response will be vital to understanding variation in the pathophysiology leishmaniasis.

Because CXCL10 contributes to the recruitment and activation of immune cell subsets that confer protection against leishmaniasis, it has the potential to serve as a host-directed therapy for leishmaniasis. However, the consequences of stimulating its receptor, CXCR3, during infection remains uncertain, due to reported protective and exacerbating effects of CXCR3 ligands in different infection contexts and the discovery here that Leishmania spp. cleave CXCR3 ligands. In the final chapter, small molecule CXCR3 agonists were leveraged to circumvent parasite chemokine cleavage and interrogate how specific downstream CXCR3 signaling patterns impact disease outcome in C57BL6/J mice. A G-protein-biased agonist (VUF11418) decreased lesion size, but this beneficial impact was due to an off-target effect. Unexpectedly, both a CXCR3 -arrestin-biased agonist (VUF10661) and a CXCR3 antagonist (AMG487) increased the size of the cutaneous lesion, despite having opposite effects on T-cell recruitment in vitro. We next determined whether -arrestin-biased CXCR3 signaling increased lesion size due to excess recruitment of anti-parasitic immune cell types or due to altered polarization and activation of the inflammatory cells recruited. Despite not improving outcomes in C57BL6/J mice, the observed increase in inflammation by CXCR3 stimulation in response to L. major highlights the importance of understanding the host, pathogen, and environmental factors that determine when CXCR3 signaling is protective or pathologic after Leishmania spp. infection.

Together these studies highlight the complexity of host interactions with Leishmania parasites. Future work is needed to elucidate the clinical relevance of the mechanisms and diversity of CXCL10 suppression described here. However, this work demonstrates how parasite diversity can be leveraged to unravel the molecular basis for pathogenesis and inform the design of novel treatment strategies for leishmaniasis.

If nothing else, the 2019 Coronavirus pandemic has made it abundantly clear that understanding the mechanisms of infectious disease is imperative to the survival of our species. While the last fifty years of developments in molecular biology has accelerated our ability to study microbial pathogens, limitations in pathogen tropism, microbial survival in laboratory conditions, uneven sampling of human cohorts across geographical and socioeconomic lines, and heterogeneous complexity during human infection have limited our ability to study complex mechanisms of human susceptibility to infectious disease. In this work, I build on recent developments in utilizing High-throughput Human in vitro Susceptibility Testing (Hi-HOST) to not only (a) identify novel sites in the human genome that contribute to natural variation in infectious disease susceptibility based on highly quantifiable cellular phenotypes, but (b) use these sites as a springboard to understand the entire, complex host-pathogen interaction. From this perspective, I paired the model pathogen Salmonella enterica and the Hi-HOST system to identify that natural variation in the mammalian gene arhgef26 contributes to susceptibility to Salmonella invasion. I used this finding as a starting point to fully explore the role of ARHGEF26 during infection, redefining its role in invasion, inflammation, and its interaction with host and bacterial proteins during the process. Similarly, I used prior Hi-HOST findings that methionine metabolism influences the host response to Salmonella enterica serovar Typhimurium (S. Typhimurium) as a launching point to investigate the impacts of host and bacterial metabolism on the virulence of S. Typhimurium. I found that the metabolite methylthioadenosine is a potent inhibitor of S. Typhimurium type III secretion, motility, and invasion. Finally, I mechanistically explain some of these findings by linking methionine metabolism to DNA methylation using a novel approach to integrate the Salmonella Typhimurium methylome and transcriptome. In sum, these findings demonstrate the ability for cellular GWAS to serve as a launching point to understand complex host-pathogen interactions.