How should we sample snail microbiomes?

Background: The microbiome is increasingly recognized to shape many aspects of its host biology and is a key determinant of health and disease. The microbiome may influence transmission of pathogens by their vectors, such as mosquitoes or aquatic snails. We previously sequenced the bacterial 16S V4 ribosomal DNA of the hemolymph (blood) of Biomphalaria spp. snails, one of the vectors of the human blood fluke schistosome. We showed that snail hemolymph harbored an abundant and diverse microbiome. This microbiome is distinct from the water environment and can discriminate snail species and populations. As hemolymph bathes snail organs, we then investigated the heterogeneity of the microbiome in these organs. Results: We dissected ten snails for each of two different species (B. alexandrina and B. glabrata) and collected their organs (ovotestis, hepatopancreas, gut, and stomach). We also ground in liquid nitrogen four whole snails of each species. We sampled the water in which the snails were living (environmental controls). Sequencing the 16S V4 rDNA revealed organ-specific microbiomes. These microbiomes harbored a lower diversity than the hemolymph microbiome, and the whole-snail microbiome. The organ microbiomes tend to cluster by physiological function. In addition, we showed that the whole-snail microbiome is more similar to hemolymph microbiome. Conclusions: These results are critical for future work on snail microbiomes and show the necessity of sampling individual organ microbiomes to provide a complete description of snail microbiomes.

Plasmodium falciparum K13 mutations in Africa and Asia impact artemisinin resistance and parasite fitness.

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.

Rational approach to drug discovery for human schistosomiasis.

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.