Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane 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.The population at risk for developing malaria is vast, comprising some 3.3 billion people particularly in sub-Saharan Africa and Southeast Asia, with mortality estimates ranging from 655,000 to 1,200,000 (1). Widespread resistance has limited the therapeutic utility of most existing antimalarial drugs, and artemisinin, the highly efficacious cornerstone of artemisinin combination therapies, appears to be at risk for the same fate (2). The need for new antimalarial chemotherapeutic strategies is thus acute.Plasmodium spp., the causative agents of malaria, have a complex life cycle with alternating motile-nonreplicative and sessile-replicative forms in both mammal and mosquito. In the mammalian host, Plasmodium invades and replicates inside two very distinct cell types: hepatocytes and red blood cells (RBCs). In mammals, the Plasmodium life cycle is initiated by a motile sporozoite that invades a hepatocyte, where it resides for 2–14 d, multiplying into >10,000 merozoites in a single cycle (3). Once released into the bloodstream, each of these motile merozoites will infect an RBC and, within 1–3 d, generate 10–30 new merozoites, which will contribute to the continuous cycle of blood stage infection that causes the symptoms, morbidity, and mortality of malaria.These two stages of mammalian infection, despite taking place in distinct cell types and having an orders-of-magnitude difference in parasite replication, do share common features. In both, the motile “zoite” invades the host cell through formation of a parasitophorous vacuole (PV). Both stages grow and replicate exclusively within the confines of the PV, and the parasitophorous vacuole membrane (PVM), which is populated with parasite proteins, constitutes the physical host–parasite interface throughout development. Unlike the vacuoles of many intracellular pathogens including Leishmania, Chlamydia, Mycobacteria, and Legionella (4, 5), the Plasmodium vacuole, like that of Toxoplasma gondii, does not fuse with host lysosomes and is not acidified (6). This is not unsurprising in the context of Plasmodium development in an RBC, which lacks endomembrane system trafficking and, indeed, lysosomes. The highly polarized hepatocyte, however, has extensive vesicular transport networks (7) and can target intracellular pathogens residing in a vacuole (8), suggesting that the exoerythrocytic form (EEF) may need to resist host cell attack.Although the PVM is thought to be critical for Plasmodium growth in both the hepatocyte and the RBC contexts, its cellular roles remain elusive. The importance of several Plasmodium PVM-resident proteins, however, has been conclusively demonstrated in both blood and liver stages. Attempts to generate exported (exp)1 and Plasmodium translocon of exported protein (ptex)150 knockout parasites in Plasmodium falciparum failed (9, 10), revealing that these are both essential proteins for the blood stage, whereas Plasmodium berghei and Plasmodium yoelii mutants lacking up-regulated in sporozoites (uis)3 or uis4 fail to complete liver stage development (11, 12). These PVM-resident proteins, and thus the PVM itself, are performing functions that are crucial for Plasmodium growth, but delineating the functions of individual PVM-resident proteins has proven as difficult as identifying the cellular processes mediated by the PVM.The one process in which both the centrality of the PVM is known and evidence for the participation of specific PVM proteins exists is the export of parasite proteins to the RBC. A cohort of parasite proteins that are involved in extensive physiological and structural modifications of the infected RBC (iRBC) is exported into the iRBC cytoplasm and beyond (13). Five proteins have been identified as components of PTEX, the proposed export machinery at the iRBC PVM (9). Although liver stage protein export has been shown for the Circumsporozite (CS) protein (14) and PTEX components are expressed in P. falciparum EEFs (15), a role for parasite protein export into the hepatocyte remains speculative; the host hepatocyte may not require the extensive structural remodeling that the iRBC does.Conversely, however, the hepatocyte, with its extensive vesicular transport network, intuitively constitutes a more hostile host environment than the RBC, and there is evidence that the liver stage PVM may play a crucial role in preventing host cell-mediated parasite killing, as it does in Toxoplasma gondii (16). Support for a protective role for the liver stage PVM comes from knockout parasites that fail in the earliest steps of PVM formation and remodeling. Sporozoites lacking the p52/p36 gene pair invade hepatocytes successfully, but fail in PVM formation (17, 18) and are severely reduced in abundance midway through liver stage development. Parasites lacking slarp/sap (19, 20), a regulator of early liver stage development, fail to express UIS4 and exported protein 1 (EXP-1), along with other parasite proteins, and are also eliminated at the beginning of infection.Acquisition of resources from the host-cell environment, an unambiguous requirement for an obligate intracellular parasite like Plasmodium, is a function ascribed to the PVM in both mammalian stages. The PVM allows the free passage of molecules (21, 22), presumably through proteinaceous pores, which may contribute to acquisition of host nutrients and disposal of parasite waste products. Members of the early transcribed membrane protein (ETRAMP) family, single-pass transmembrane proteins conserved among Plasmodium spp., which are highly expressed and developmentally regulated in both blood and liver stage parasites (23, 24), could be candidates for mediating uptake of host resources. Such a role in lipid uptake has indeed been proposed for the P. berghei ETRAMP UIS3 on the basis of its interaction with host-cell L-FABP (liver fatty acid binding protein) (25).Although Plasmodium parasites must use host resources to support their own growth in both mammalian stages, the single cycle replicative output of the liver stage parasite is vastly greater than that of the blood stage, which may reflect a similarly increased need for host resources. In this respect, the hepatocyte constitutes far superior “raw material” compared with the RBC; hepatocytes are not only metabolically active, but also highly versatile cells, which are capable of altering uptake, storage, production, and degradation of a wide array of macromolecules in response to cellular and organismal requirements. The presence of a growing Plasmodium parasite is sensed by the host hepatocyte, which responds with activation of cellular stress responses and altered metabolism (26, 27). The mammalian target of rapamycin (mTOR) kinase integrates signals from amino acids, stress, oxygen, energy, and growth factors and responds by altering cellular protein and lipid synthesis, as well as autophagy (28). As such, we sought to determine how inhibition of host mTOR signaling would affect Plasmodium liver stage development. Here we show that torins, a single structural class of mTOR inhibitors, are highly potent antiplasmodial compounds targeting both mammalian stages in vitro and in vivo. Independent of host-cell mTOR, torins impair trafficking of Plasmodium liver stage PVM-resident proteins, revealing the fast turnover of these proteins at the liver stage PVM, and provoke elimination of liver stage parasites.