Publications

Aquatic snails, the intermediate hosts of schistosomes, harbor a diverse unexplored microbiome. We speculate that this may play a critical role in host-parasite interactions. We summarize our current knowledge of snail microbiomes and highlight future research priorities.

Classical malaria parasite genetic crosses involve isolation, genotyping, and phenotyping of progeny parasites, which is time consuming and laborious. We tested a rapid alternative approach-bulk segregant analysis (BSA)-that utilizes sequencing of bulk progeny populations with and without drug selection for rapid identification of drug resistance loci. We used dihydroartemisinin (DHA) selection in two genetic crosses and investigated how synchronization, cryopreservation, and the drug selection regimen impacted BSA success. We detected a robust quantitative trait locus (QTL) at kelch13 in both crosses but did not detect QTLs at four other candidate loci. QTLs were detected using synchronized, but not unsynchronized progeny pools, consistent with the stage-specific action of DHA. We also successfully applied BSA to cryopreserved progeny pools, expanding the utility of this approach. We conclude that BSA provides a powerful approach for investigating the genetic architecture of drug resistance in Plasmodium falciparum.

Schistosoma mansoni, a snail-borne, blood fluke that infects humans, was introduced into the Americas from Africa during the Trans-Atlantic slave trade. As this parasite shows strong specificity to the snail intermediate host, we expected that adaptation to South American Biomphalaria spp. snails would result in population bottlenecks and strong signatures of selection. We scored 475,081 single nucleotide variants in 143 S. mansoni from the Americas (Brazil, Guadeloupe and Puerto Rico) and Africa (Cameroon, Niger, Senegal, Tanzania, and Uganda), and used these data to ask: (i) Was there a population bottleneck during colonization? (ii) Can we identify signatures of selection associated with colonization? (iii) What were the source populations for colonizing parasites? We found a 2.4- to 2.9-fold reduction in diversity and much slower decay in linkage disequilibrium (LD) in parasites from East to West Africa. However, we observed similar nuclear diversity and LD in West Africa and Brazil, suggesting no strong bottlenecks and limited barriers to colonization. We identified five genome regions showing selection in the Americas, compared with three in West Africa and none in East Africa, which we speculate may reflect adaptation during colonization. Finally, we infer that unsampled populations from central African regions between Benin and Angola, with contributions from Niger, are probably the major source(s) for Brazilian S. mansoni. The absence of a bottleneck suggests that this is a rare case of a serendipitous invasion, where S. mansoni parasites were pre-adapted to the Americas and able to establish with relative ease.

Both theory and experimental data from pathogens suggest that the production of transmission stages should be strongly associated with virulence, but the genetic bases of parasite transmission/virulence traits are poorly understood. The blood fluke Schistosoma mansoni shows extensive variation in numbers of cercariae larvae shed and in their virulence to infected snail hosts, consistent with expected trade-offs between parasite transmission and virulence. We crossed schistosomes from two populations that differ 8-fold in cercarial shedding and in their virulence to Biomphalaria glabrata snail hosts, and determined four-week cercarial shedding profiles in F0 parents, F1 parents and 376 F2 progeny from two independent crosses in inbred snails. Sequencing and linkage analysis revealed that cercarial production is polygenic and controlled by five QTLs (i.e. Quantitative Trait Loci). These QTLs act additively, explaining 28.56% of the phenotypic variation. These results demonstrate that the genetic architecture of key traits relevant to schistosome ecology can be dissected using classical linkage mapping approaches.

Mass drug administration with praziquantel (PZQ) monotherapy is considered the mainstay for control and elimination of the parasites causing schistosomiasis in humans. This drug shows imperfect cure rates in the field, and parasites showing reduced PZQ response can be selected in the laboratory, but the extent of resistance in Schistosoma mansoni populations is unknown. We examined the genetic basis of the variation in response in a PZQ-selected S. mansoni population (SmLE-PZQ-R) in which 35% of the parasitic worms survive high-dose PZQ (73 micrograms per milliliter) treatment. We used genome-wide association to map loci underlying PZQ response and identified a transient receptor potential (Sm.TRPMPZQ) channel (Smp_246790) within the major chromosome 3 peak that is activated by nanomolar concentrations of PZQ. The PZQ response showed recessive inheritance and marker-assisted selection of parasites at a single Sm.TRPMPZQ SNP that produced populations of PZQ-enriched resistant (PZQ-ER) and PZQ-enriched sensitive (PZQ-ES) parasites, exhibiting >377-fold difference in PZQ response. The PZQ-ER parasites survived treatment in rodents at higher frequencies compared with PZQ-ES, and resistant parasites exhibited 2.25-fold lower expression of Sm.TRPMPZQ relative to sensitive parasites. Specific chemical blockers of Sm.TRPMPZQ enhanced PZQ resistance, whereas Sm.TRPMPZQ activators increased sensitivity. We surveyed Sm.TRPMPZQ sequence variations in 259 parasites from different global sites and identified one nonsense mutation that resulted in a truncated protein with no PZQ binding site. Our results demonstrate that Sm.TRPMPZQ underlies variation in PZQ responses in S. mansoni and provides an approach for monitoring emerging PZQ-resistant alleles in schistosome elimination programs.

The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failures across Southeast Asia. In Africa, K13-propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing of point mutations in ferredoxin or mdr2, earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, -6, -7a, -7b, and -8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Human schistosomiasis is a debilitating, life-threatening disease affecting more than 229 million people in as many as 78 countries. There is only one drug of choice effective against all three major species of Schistosoma, praziquantel (PZQ). However, as with many monotherapies, evidence for resistance is emerging in the field and can be selected for in the laboratory. Previously used therapies include oxamniquine (OXA), but shortcomings such as drug resistance and affordability resulted in discontinuation. Employing a genetic, biochemical and molecular approach, a sulfotransferase (SULT-OR) was identified as responsible for OXA drug resistance. By crystallizing SmSULT- OR with OXA, the mode of action of OXA was determined. This information allowed a rational approach to novel drug design. Our team approach with schistosome biologists, medicinal chemists, structural biologists and geneticists has enabled us to develop and test novel drug derivatives of OXA to treat this disease. Using an iterative process for drug development, we have successfully identified derivatives that are effective against all three species of the parasite. One derivative CIDD-0149830 kills 100% of all three human schistosome species within 5 days. The goal is to generate a second therapeutic with a different mode of action that can be used in conjunction with praziquantel to overcome the ever-growing threat of resistance and improve efficacy. The ability and need to design, screen, and develop future, affordable therapeutics to treat human schistosomiasis is critical for successful control program outcomes.