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SummaryGenes 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 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 ForkheadBox 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 subclass 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 infection outcomes ranging from self-healing lesions, to progressive non-healing lesion, to metastatic spread and destruction of mucous membranes. Although resolution of 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 of disease.Leishmaniagenetic 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 theLeishmania (L.)subgenus and theViannia (V.)subgenus, which is associated with severe immune mediated pathology such as mucocutaneous leishmaniasis. WhileLeishmania (L.)subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63(gp63)to suppress the type-1 associated host chemokine CXCL10,L. (V.) panamensisdid not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we usedin silicomodeling of the primary amino acid sequence and protein crystal structures to identify a putative CXCL10-binding site on GP63. We found the putative CXCL10 binding site to be located in a region ofgp63under significant positive selection and that it varies from theL. majorwild-type sequence in allgp63alleles identified in theL. (V.) panamensisreference genome. We determined that the predicted binding site and adjacent positively selected amino acids are required for CXCL10 suppression by mutating wild-typeL. (L.) major gp63to theL. (V.) panamensisallele and demonstrating impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably,Vianniaclinical isolates confirmed thatL. (V.) panemensisprimarily encodes non-CXCL10-cleavinggp63alleles. In contrast,L. (V.) braziliensishas an intermediate level of activity, consistent with this species having more equal proportions of both alleles at the CXCL10 binding site, possibly due to balancing selection. Our results demonstrate how parasite genetic diversity can contribute to variation in the host immune response toLeishmaniaspp. infection that may play critical roles in the outcome of infection.

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.

Susceptibility to infectious diseases is determined by a complex interaction between host and pathogen. For infections with the obligate intracellular bacterium Chlamydia trachomatis, variation in immune activation and disease presentation are regulated by both host genetic diversity and pathogen immune evasion. Previously, we discovered a single nucleotide polymorphism (rs2869462) associated with absolute abundance of CXCL10, a pro-inflammatory T-cell chemokine. Here, we report that levels of CXCL10 change during C. trachomatis infection of cultured cells in a manner dependent on both host and pathogen. Linear modeling of cellular traits associated with CXCL10 levels identified a strong, negative correlation with bacterial burden, suggesting that C. trachomatis actively suppresses CXCL10. We identified the pathogen-encoded factor responsible for this suppression as the chlamydial protease- or proteasome-like activity factor, CPAF. Further, we applied our modeling approach to other host cytokines in response to C. trachomatis and found evidence that RANTES, another T-cell chemoattractant, is actively suppressed by Chlamydia. However, this observed suppression of RANTES is not mediated by CPAF. Overall, our results demonstrate that CPAF suppresses CXCL10 to evade the host cytokine response and that modeling of cellular infection parameters can reveal previously unrecognized facets of host–pathogen interactions.

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 asLeishmania (L.) major, depends on a well-regulated adaptive T cell response. Here we describe a pathogen-encoded mechanism to alter T cell recruitment by suppressing CXCL10, a chemokine that recruits CD4+ and CD8+ T cells by signaling through the CXCR3 receptor.L. majorsuppresses CXCL10 through the virulence factor and protease, glycoprotein-63(gp63).GP63 cleaves CXCL10 after amino acid A81, impairing T-cell chemotaxisin vitro.GP63 from either extracellular promastigotes or intracellular amastigotes is capable of cleaving CXCL10. Consistent with CXCL10 cleavage during infection, we observed GP63-mediated impairment of activated CD8+ T-cells in the draining lymph nodes of C57BL/6JWTmice. Correspondingly, in C57BL/6JWTmice,gp63deletion 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. Finally,Salmonella entericaandChlamydia trachomatisinfection also suppress CXCL10, demonstrating convergent evolution of this immune evasion strategy in diverse intracellular pathogens. Understanding mechanisms of CXCL10 evasion may facilitate the development of novel therapeutic strategies to treat intracellular pathogens.Author SummaryLeishmaniasis is an infectious disease that affects over one million people annually. It is caused by intracellular parasites that have evolved to evade the host’s attempts to eliminate the parasite. The most common form of disease, cutaneous leishmaniasis, causes disfiguring skin lesions if the host immune system does not appropriately respond to the infection. A family of molecules called chemokines are critical for the recruitment of specific subsets of immune cells required to eliminate infection. Here, we demonstrate a novel mechanism that the parasiteLeishmania (L.) majoremploys to disrupt the immune response by interfering with chemokine signaling. By this strategy,L. majoruses a protease to cleave chemokines known to recruit T-cells required for fighting off infection. We show thatL. majorcleavage of these immune signals changes the recruitment of CD8+ T-cells and alters disease progression in mice. Finally, we observe that multiple common human intracellular pathogens, includingChlamydia trachomatisandSalmonella enterica, interfere with 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 and exacerbate disease outcomes.

Pathogens have been a strong driving force for natural selection. Therefore, understanding how human genetic differences impact infection-related cellular traits can mechanistically link genetic variation to disease susceptibility. Here we report the Hi-HOST Phenome Project (H2P2): a catalog of cellular genome-wide association studies (GWAS) comprised of 79 infection-related phenotypes in response to 8 pathogens in 528 lymphoblastoid cell lines. Seventeen loci surpass genome-wide significance for infection-associated phenotypes ranging from pathogen replication to cytokine production. We combined H2P2 with clinical association data from patients to identify a SNP near CXCL10 as a risk factor for inflammatory bowel disease. A SNP in the transcriptional repressor ZBTB20 demonstrated pleiotropy, likely through suppression of multiple target genes, and was associated with viral hepatitis. These data are available on a web portal to facilitate interpreting human genome variation through the lens of cell biology and should serve as a rich resource for the research community.

Eukaryotic parasites in the genusLeishmaniaplace approximately 350 million people per year at risk of disease. In addition to their global health significance,Leishmaniaspp. 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 sequencedLeishmaniaspp., we demonstrate that this assay has a lower limit of detection of 100 fg of parasite DNA forLeishmania major,L. donovani,L. venezuelensis, andL. panamensis. Using C57BL6/J mice, we used this assay to monitor parasite burden over 1 month of infection with two strains ofL. major(Seidman and Friedlin), andL. 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.

Parasites with increased resistance to sulfadoxine might undermine malaria control measures., 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.

Pregnancy-associated malaria (PAM) is an important, preventable cause of poor birth outcomes in malaria-endemic areas in sub-Saharan Africa [1]. Intermittent preventive therapy with antimalarials during pregnancy (IPTp) is a critical tool for reducing the incidence of PAM-attributable adverse birth outcomes [2]. Sulfadoxine-pyrimethamine (SP) is the most commonly administered drug for IPTp owing to its long half-life, favorable side-effect profile, and safety during pregnancy. However, Plasmodium falciparum strains harboring mutations associated with SP resistance are increasingly prevalent throughout sub-Saharan Africa. The outcome of antimalarial therapy results from complex interactions between parasite pathogenicity, host immunity, drug characteristics, and parasite susceptibility to drug therapy. Recent in vitro studies have suggested that the removal of susceptible parasites from heterogeneous parasitemias by antimalarial therapy may facilitate the relative overgrowth of the remaining resistant parasites and therefore potentiate their pathogenicity [3]. A recent study in Tanzania documented increases in placental inflammation that were associated with SP receipt, suggesting that SP may potentiate placental pathology when applied to partially susceptible infections [4]. However, the effect of these placental findings upon birth outcomes was unclear. Given the prevalence of SP resistance and the current lack of appropriate, safe alternatives for use as IPTp, it is critical to explore the associations between IPTp-SP, SP resistance, and delivery outcomes in further contexts to inform malaria control policies. Malawi adopted a policy of IPTp with SP in 1993, and despite the subsequent development of widespread SP resistance [5], a sustained decline in the prevalence of PAM was observed from 1997 to 2005 [6]. In view of the evidence from Tanzania [4], we hypothesized that the emergence of SP-resistant parasites would modify the effect of IPTp-SP and thereby potentiate placental inflammation and parasite densities and worsen birth outcomes. The Queen Elizabeth Central Hospital Epidemiology of Resistance in Pregnancy-Associated Malaria (QuEERPAM) study [7] was a serial cross-sectional analysis in which we explored the relationships between IPTp-SP, the presence of resistant parasites at delivery, and multiple measures of adverse delivery outcome, including parasite densities, placental histology, maternal hemoglobin concentration, and birth weight.

In the Democratic Republic of the Congo (DRC), artesunate-amodiaquine is first-line therapy for falciparum malaria; little is known about the prevalence of molecular markers of parasite drug resistance. Across the DRC, we genotyped 166 parasites in Plasmodium falciparum chloroquine resistance transporter (pfcrt) using polymerase chain reaction (PCR) and sequencing. Of these parasites, 73 (44%) parasites were pure wild-type CVMNK, 55 (31%) parasites were chloroquine-resistant CV IET , 35 (21.1%) parasites were mixed CVMNK and CV IET , and 3 parasites were other genotypes. Ninety-two infections (55.4%) harbored the pfcrt K76 T substitution that is highly correlated with chloroquine failure. The amodiaquine-resistant S VMN T haplotype was absent. Geographically, pfcrt haplotypes were not clearly clustered. Chloroquine accounted for 19.4% of antimalarial use, and amodiaquine accounted for 15.3% of antimalarial use; there were no associations between drug use and mutant haplotype prevalence. In the DRC, our molecular survey indicates that resistance to chloroquine is substantial but that resistance to amodiaquine is absent. These contrasting findings highlight the need for molecular surveillance of drug resistance to inform malaria control policies.

Understanding the spatial clustering of Plasmodium falciparum populations can assist efforts to contain drug-resistant parasites and maintain the efficacy of future drugs. We sequenced single nucleotide polymorphisms (SNPs) in the dihydropteroate synthase gene (dhps) associated with sulfadoxine resistance and 5 microsatellite loci flanking dhps in order to investigate the genetic backgrounds, genetic relatedness, and geographic clustering of falciparum parasites in the Democratic Republic of the Congo (DRC). Resistant haplotypes were clustered into subpopulations: one in the northeast DRC, and the other in the balance of the DRC. Network and clonal lineage analyses of the flanking microsatellites indicate that geographically-distinct mutant dhps haplotypes derive from separate lineages. The DRC is therefore a watershed for haplotypes associated with sulfadoxine resistance. Given the importance of central Africa as a corridor for the spread of antimalarial resistance, the identification of the mechanisms of this transit can inform future policies to contain drug-resistant parasite strains.

Sulfadoxine-pyrimethamine (SP) has been widely deployed in Africa for malaria control and molecular evidence of parasite drug-resistance is prevalent. However, the temporal effects on the selection of Plasmodium falciparum are not well understood. We conducted a retrospective serial cross-sectional study between 1997 and 2006 to investigate changes in drug-resistant malaria among pregnant women delivering at a single hospital in Blantyre, Malawi. P. falciparum parasites were genotyped for parasite clone multiplicity and drug-resistance mutations, and the strength of selection upon mutant genotypes was quantified. Five mutations in the dihydrofolate reductase and dihydropteroate synthase genes began at moderate frequencies and achieved fixation by 2005; the frequency of the highly-SP-resistant “quintuple mutant” haplotype increased from 19% to 100%. The selective advantage of alleles and haplotypes were quantified with selection coefficients: Selection was positive on all mutant alleles and haplotypes associated with SP resistance, and the relative fitness of the quintuple mutant haplotype was 0.139 (95% C.I. 0.067 – 0.211), indicating a substantial positive selective advantage. Mutations that confer higher levels of resistance to SP did not emerge. SP-resistant haplotypes were rapidly selected for and fixed in P. falciparum populations infecting pregnant women while SP was widely deployed in Malawi. These results underscore the pressing need for new preventive measures for pregnancy-associated malaria and provide a real-world model of the selection landscape malaria parasites.