SummaryHeterochromatin plays a central role in the process of immune evasion, pathogenesis, and transmission of the malaria parasite Plasmodium falciparum during blood stage infection. Here, we use ChIP sequencing to demonstrate that sporozoites from mosquito salivary glands expand heterochromatin at subtelomeric regions to silence blood-stage-specific genes. Our data also revealed that heterochromatin enrichment is predictive of the transcription status of clonally variant genes members that mediate cytoadhesion in blood stage parasites. A specific member (here called NF54varsporo) of the var gene family remains euchromatic, and the resultant PfEMP1 (NF54_SpzPfEMP1) is expressed at the sporozoite surface. NF54_SpzPfEMP1-specific antibodies efficiently block hepatocyte infection in a strain-specific manner. Furthermore, human volunteers immunized with infective sporozoites developed antibodies against NF54_SpzPfEMP1. Overall, we show that the epigenetic signature of var genes is reset in mosquito stages. Moreover, the identification of a strain-specific sporozoite PfEMP1 is highly relevant for vaccine design based on sporozoites.

From a research program aimed at the design of new chemical entities followed by extensive screening on various models of infectious diseases, an original series of 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidines endowed with notable antiviral properties were found. Using a whole cell measles virus replication assay, we describe here some aspects of the iterative process that, from 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)pyrimidine, led to 2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-5-ethylpyrimidine and a 4000-fold improvement of antiviral activity with a subnanomolar level of inhibition. Moreover, recent precedents in the literature describing antiviral derivatives acting at the level of the de novo pyrimidine biosynthetic pathway led us to determine that the mode of action of this series is based on the inhibition of the cellular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this pathway. Biochemical studies with recombinant human DHODH led us to measure IC50 as low as 13 nM for the best example of this original series when using 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (coenzyme Q1) as a surrogate for coenzyme Q10, the cofactor of this enzyme.

Experimental studies of Plasmodium parasites that infect humans are restricted by their host specificity. Humanized mice offer a means to overcome this and further provide the opportunity to observe the parasites in vivo. Here we improve on previous protocols to achieve efficient double engraftment of TK-NOG mice by human primary hepatocytes and red blood cells. Thus, we obtain the complete hepatic development of P. falciparum, the transition to the erythrocytic stages, their subsequent multiplication, and the appearance of mature gametocytes over an extended period of observation. Furthermore, using sporozoites derived from two P. ovale-infected patients, we show that human hepatocytes engrafted in TK-NOG mice sustain maturation of the liver stages, and the presence of late-developing schizonts indicate the eventual activation of quiescent parasites. Thus, TK-NOG mice are highly suited for in vivo observations on the Plasmodium species of humans.

SummaryDormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state‐of‐the‐art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual Plasmodium cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA‐seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up‐ or down‐regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi and P. vivax, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. Together, this work not only provides a primary experimentally‐derived list of molecular markers of hypnozoites but also identifies transcriptional and posttranscriptional regulation of gene expression as potentially being key to establishing and maintaining quiescence.

Plasmodium vivax is the most widespread human malaria parasite, in part because it can form latent liver stages known as hypnozoites after transmission by female anopheline mosquitoes to human hosts. These persistent stages can activate weeks, months, or even years after the primary clinical infection; replicate; and initiate relapses of blood stage infection, which causes disease and recurring transmission. Eliminating hypnozoites is a substantial obstacle for malaria treatment and eradication since the hypnozoite reservoir is undetectable and unaffected by most antimalarial drugs. Importantly, in some parts of the globe where P. vivax malaria is endemic, as many as 90% of P. vivax blood stage infections are thought to be relapses rather than primary infections, rendering the hypnozoite a major driver of P. vivax epidemiology. Here, we review the biology of the hypnozoite and recent discoveries concerning this enigmatic parasite stage. We discuss treatment and prevention challenges, novel animal models to study hypnozoites and relapse, and hypotheses related to hypnozoite formation and activation. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes.

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