Differential transcriptome profiling identifies Plasmodium genes encoding pre‐erythrocytic stage‐specific proteins

Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials.host-parasite interactions | malaria | protein trafficking | P. falciparum

Section: Eef Elimination and Pvm-resident Protein Mislocalization Arementioning

confidence: 99%

Section: Plasmodium Growth Is Inhibited By Torin2 Throughout Liver Stagementioning

confidence: 99%

Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins

Hanson

Ressurreição

Buchholz

et al. 2013

“…The most prominent members of this family, named up-regulated in infective sporozoites (UIS) 3 and 4, are expressed exclusively in sporozoites and liver stages (21,22). Depletion of these proteins leads to developmental…”

mentioning

confidence: 99%

Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development

Cunha

Nyboer

Heiß

et al. 2017

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wildtype parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies. malaria | Plasmodium liver stages | host-parasite interaction | ApoH | EXP-1

“…The TRAP promoter used here should prove useful to induce SSR in midgut sporozoites before infection of mosquito salivary glands, for example, to assess the role in salivary gland invasion of parasite molecules that are common to sporozoites and RBC stages. Another promoter that should prove useful is one active specifically in sporozoites located inside salivary glands, for example, the promoter of the spect gene (26) or of genes identified by differential expression screens between sporozoites and RBC stages (27). SSR induction in salivary gland sporozoites, the most highly motile and invasive stage of the parasite, should open the way to a functional analysis of the parasite surface motor, which cannot be studied by using conventional gene-targeting techniques.…”

Section: Discussionmentioning

confidence: 99%

Conditional mutagenesis using site-specific recombination in Plasmodium berghei

Carvalho

Thiberge

Sakamoto

et al. 2004

Reverse genetics in Plasmodium, the genus of parasites that cause malaria, still faces major limitations. Only red blood cell stages of this haploid parasite can be transfected. Consequently, the function of many essential genes in these and subsequent stages, including those encoding vaccine candidates, cannot be addressed genetically. Here, we establish conditional mutagenesis in Plasmodium by using site-specific recombination and the Flp͞FRT system of yeast. Site-specific recombination is induced after cross-fertilization in the mosquito vector of two clones containing either the target sequence flanked by two FRT sites or the Flp recombinase. Parasites that have undergone recombination are recognized in the cross progeny through the expression of a fluorescence marker. This approach should permit to dissect the function of any essential gene of Plasmodium during the haploid phase of its life, i.e., during infection of salivary glands in the mosquito and infection of both the liver and red blood cells in the mammal.A ll symptoms and complications of malaria are caused by the multiplication of Plasmodium parasites inside the red blood cells (RBC) of a host. The parasite is transmitted between two mammalian hosts through mosquitoes, typically Anopheles, during blood feeding (Fig. 1). It is ingested as sexual forms (gametocytes), and fertilization occurs rapidly in the lumen of the mosquito midgut. It is then inoculated to a new mammalian host as haploid sporozoites, which transform inside hepatocytes into forms that invade RBC.Genome manipulation is an essential tool for understanding key events in the Plasmodium life cycle in molecular terms. Stable transfection of the parasite (1, 2) and modification of its genome by homologous recombination (3-5) are now common procedures. The genome, however, can be manipulated only in RBC stages of the parasite, which are the only stages that can be produced in large amounts and subjected to selection. An important limitation of the current technology is that loss-offunction mutations cannot be selected in genes that play a role in parasite invasion of, or multiplication inside RBC, which are required for selection. Although evidence can be gained that a gene is important for the RBC cycle when its disruption cannot be selected, as has been reported for the leading vaccine candidates merozoite surface protein 1 (MSP-1) (6) and apical membrane antigen 1 (AMA-1) (7) and many other proteins (8), the actual function of the protein cannot be studied in the RBC or subsequent stages. Likewise, an increasing proportion of Plasmodium proteins are recognized as being produced at more than just one parasite stage, and gene inactivation in RBC stages can only reveal their earlier role in the cycle. Therefore, conditional procedures are needed for inactivating or activating genes at will during the Plasmodium life cycle.Site-specific recombination (SSR) offers an effective way to inactivate a gene in a temporally defined manner. Two SSR systems have been widely used in eukaryotes, Cre͞...