A Drug Repurposing Approach Reveals Targetable Epigenetic Pathways in Plasmodium vivax Hypnozoites.

Radical cure of Plasmodium vivax malaria must include elimination of quiescent 'hypnozoite' forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase (DNMT) inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

Large interclone differences in melezitose secretion in the facultatively ant-tended black bean aphid Aphis fabae.

Many aphids are known to engage in a trophic mutualism with ants, whereby the aphids secrete sugary-rich honeydew which is collected by the ants for food, and the ants, in exchange, protect the aphids against natural enemies. Previous results, however, suggest that the production of some of the honeydew sugars, such as the ant-attractant trisaccharide melezitose, may induce an indirect cost to the aphids. This led us to believe that large differences in the nature of the secreted honeydew might exist, due to some clones capitalizing more or less on their mutualistic interaction with ants, or due to some "cheater" clones foregoing the production of particular sugars, instead taking advantage of the ant-attracting effect of other non sugar-deficient clones, co-occurring on the same plant. Here we present data on clonal variation in the composition of honeydew of the black bean aphid Aphis fabae which confirm this prediction. In particular, our results show that there was large interclone variation in the amount of glucose, melezitose and total sugar produced. The variation in the production of melezitose, however, showed particularly large differences, with 54% (7 out of 13) of the clones screened being virtually deficient for the production of this sugar, irrespective of whether the aphid colonies were ant-tended or not. The consequences of this finding in the context of the evolution and maintenance of the ant-aphid mutualism, as well as the adaptive benefits of oligosaccharide synthesis in aphids and other insects are discussed.

Levels of clonal mixing in the black bean aphid Aphis fabae, a facultative ant mutualist.

Aphids are a worldwide pest and an important model in ecology and evolution. Little is known, however, about the genetic structure of their colonies at a microgeographic level. For example, it remains largely unknown whether most species form monoclonal or polyclonal colonies. Here, we present the first detailed study on levels of clonal mixing in a nonsocial facultative ant mutualist, the black bean aphid Aphis fabae. In contrast to the earlier suggestion that colonies of this species are generally monoclonal, we found that across two subspecies of the black bean aphid, A. fabae cirsiiacanthoidis and A. fabae fabae, 32% and 67% of the aphid colonies were in fact polyclonal, consisting of a mix of up to four different clones, which resulted in an overall average relatedness within colonies of 0.90 and 0.79 in the two subspecies. Data further show that the average relatedness in A. f. cirsiiacanthoidis remained relatively constant throughout the season, which means that clonal erosion due to clonal selection more or less balanced with the influx of new clones from elsewhere. Nevertheless, relatedness tended to decrease over the lifetime of a given colony, implying that clonal mixing primarily resulted from the joining of pre-existing colonies as opposed to via simultaneous host colonisation by several foundresses. Widespread clonal mixing is argued to affect the ecology and evolution of the aphids in various important ways, for example with respect to the costs and benefits of group living, the evolution of dispersal and the interaction with predators as well as with the ant mutualists.