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Abstract Background While genome-wide associations studies (GWAS) have successfully elucidated the genetic architecture of complex human traits and diseases, understanding mechanisms that lead from genetic variation to pathophysiology remains an important challenge. Methods are needed to systematically bridge this crucial gap to facilitate experimental testing of hypotheses and translation to clinical utility. Results Here, we leveraged cross-phenotype associations to identify traits with shared genetic architecture, using linkage disequilibrium (LD) information to accurately capture shared SNPs by proxy, and calculate significance of enrichment. This shared genetic architecture was examined across differing biological scales through incorporating data from catalogs of clinical, cellular, and molecular GWAS. We have created an interactive web database (interactive Cross-Phenotype Analysis of GWAS database (iCPAGdb)) to facilitate exploration and allow rapid analysis of user-uploaded GWAS summary statistics. This database revealed well-known relationships among phenotypes, as well as the generation of novel hypotheses to explain the pathophysiology of common diseases. Application of iCPAGdb to a recent GWAS of severe COVID-19 demonstrated unexpected overlap of GWAS signals between COVID-19 and human diseases, including with idiopathic pulmonary fibrosis driven by the DPP9 locus. Transcriptomics from peripheral blood of COVID-19 patients demonstrated that DPP9 was induced in SARS-CoV-2 compared to healthy controls or those with bacterial infection. Further investigation of cross-phenotype SNPs associated with both severe COVID-19 and other human traits demonstrated colocalization of the GWAS signal at the ABO locus with plasma protein levels of a reported receptor of SARS-CoV-2, CD209 (DC-SIGN). This finding points to a possible mechanism whereby glycosylation of CD209 by ABO may regulate COVID-19 disease severity. Conclusions Thus, connecting genetically related traits across phenotypic scales links human diseases to molecular and cellular measurements that can reveal mechanisms and lead to novel biomarkers and therapeutic approaches. The iCPAGdb web portal is accessible at http://cpag.oit.duke.edu and the software code at https://github.com/tbalmat/iCPAGdb .

Leishmaniasis is a neglected tropical disease with diverse outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune-mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling to identify a putative CXCL10-binding site on GP63. The putative CXCL10 binding site is in a region of gp63 under significant positive selection, and it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. Mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis sequence at the putative binding site impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panamensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles. Our results demonstrate how parasite genetic diversity can contribute to variation in immune responses to Leishmania spp. infection that may play critical roles in the outcome of infection. Author summary Leishmaniasis is a neglected tropical disease caused by Leishmania parasites and spread by the bites of infected sand flies. Most cases of leishmaniasis present as self-healing sores that are resolved by a balanced immune response. Other cases of leishmaniasis involve spread to sites distant from the original bite, including damage of the inner surfaces of the mouth and nose. These cases of leishmaniasis involve an excessive immune response. Leishmania parasites produce virulence factor proteins, such as GP63, to trick the immune system into mounting a weaker response. GP63 specifically degrades signaling proteins that attract and activate certain immune cells. Here, we demonstrate that Leishmania parasite species have evolved to differ in their ability to degrade signaling proteins. In Leishmania species known to cause more immune-mediated tissue damage, the GP63 virulence factor has evolved to not degrade specific immune signaling proteins, thus attracting, and activating more immune cells. Our results demonstrate how diversity among Leishmania parasite species can contribute to variation in immune responses that may play critical roles in the outcome of infection.

Clearance of intracellular pathogens, such as Leishmania (L.) major, depends on an immune response with well-regulated cytokine signaling. Here we describe a pathogen-mediated mechanism of evading 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. We found that this transcriptionally independent suppression of CXCL10 is mediated by 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 homologs, 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. Ultimately, we propose CXCL10 suppression is a convergent mechanism of immune evasion, 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.ImportanceLeishmaniasis, an infectious disease that annually affects over one million people, is caused by intracellular parasites that have evolved to evade the host's attempts to eliminate the parasite. Cutaneous leishmaniasis results in disfiguring skin lesions if the host immune system does not appropriately respond to infection. A family of molecules called chemokines coordinate recruitment of the immune cells required to eliminate infection. Here, we demonstrate a novel mechanism that Leishmania (L.) spp. employ to suppress host chemokines: a Leishmania-encoded protease cleaves chemokines known to recruit T cells that fight off infection. We observe that other common human intracellular pathogens, including Chlamydia trachomatis and Salmonella enterica, reduce levels of the same chemokines, suggesting a strong selective pressure to avoid this component of the immune response. Our study provides new insights into how intracellular pathogens interact with the host immune response to enhance pathogen survival.

Eukaryotic parasites in the genus Leishmania place approximately 350 million people per year at risk of disease. In addition to their global health significance, Leishmania spp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR-based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we developed a sybr-green based quantitative touchdown PCR assay for a highly conserved and single-copy putative RNA-binding protein, DRBD3. With primers that share greater than 90% sequence identity across all sequenced Leishmania spp., we demonstrate that this assay has a lower limit of detection of 100 fg of parasite DNA for Leishmania major, L. donovani, L. venezuelensis, and L. panamensis. Using C57BL6/J mice, we used this assay to monitor parasite burden over 1 month of infection with two strains of L. major (Seidman and Friedlin), and L. venezeuelensis. These characteristics rival the sensitivity of previously reported qPCR based methods of parasite quantitation while amplifying a stable, single copy gene. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.

Sulfadoxine-resistant Plasmodium falciparum undermines malaria prevention with sulfadoxine/pyrimethamine. Parasites with a highly resistant mutant dihydropteroate synthase (dhps) haplotype have recently emerged in eastern Africa; they negated preventive benefits of sulfadoxine/pyrimethamine, and might exacerbate placental malaria. We explored emerging lineages of dhps mutant haplotypes in Malawi, the Democratic Republic of the Congo, and Tanzania by using analyses of genetic microsatellites flanking the dhps locus. In Malawi, a triple-mutant dhps SGEG (mutant amino acids are underlined) haplotype emerged in 2010 that was closely related to pre-existing double-mutant SGEA haplotypes, suggesting local origination in Malawi. When we compared mutant strains with parasites from the Democratic Republic of the Congo and Tanzania by multiple independent analyses, we found that SGEG parasites were partitioned into separate lineages by country. These findings support a model of local origination of SGEG dhps haplotypes, rather than geographic diffusion, and have implications for investigations of emergence and effects of parasite drug resistance.

Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor-α gene (IL7R) in MS-risk. Based on evolutionary and functional arguments, we postulated that DDX39B enhances immune tolerance thereby decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified Forkhead Box P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized type of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

Leishmaniasis is a neglected tropical disease with diverse outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune-mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling to identify a putative CXCL10-binding site on GP63. The putative CXCL10 binding site is in a region of gp63 under significant positive selection, and it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. Mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis sequence at the putative binding site impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panamensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles. Our results demonstrate how parasite genetic diversity can contribute to variation in immune responses to Leishmania spp. infection that may play critical roles in the outcome of infection.