Genetic and transcriptional analysis of phosphoinositide-specific phospholipase C in Plasmodium

Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, which has been shown to be important in various processes of the malaria parasite Plasmodium. PI-PLC is generally implicated in calcium liberation from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate, and is itself dependent on calcium for its activation. Here we describe the plc genes from Plasmodium species. The encoded proteins contain all domains typically found in PI-PLCs of the δ class but are almost twice as long as their orthologues in mammals. Transcriptional analysis by qRT-PCR of plc during the erythrocytic cycle of P. falciparum revealed steady expression levels that increased at the late schizont stages. Genetic analysis in the P. berghei model revealed that the plc locus was targetable but that plc gene knock-outs could not be obtained, thereby strongly indicating that the gene is essential during blood stage development. Alternatively, we attempted to modify plc expression through a promoter exchange approach but found the gene to be refractory to over-expression indicating that plc expression levels might additionally be tightly controlled

Phosphatidylinositol 3-Monophosphate Is Involved in Toxoplasma Apicoplast Biogenesis

Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs

Toxoplasmosis: Current and Emerging Parasite Druggable Targets

Toxoplasmosis is a prevalent disease affecting a wide range of hosts including approximately one-third of the human population. It is caused by the sporozoan parasite Toxoplasma gondii (T. gondii), which instigates a range of symptoms, manifesting as acute and chronic forms and varying from ocular to deleterious congenital or neuro-toxoplasmosis. Toxoplasmosis may cause serious health problems in fetuses, newborns, and immunocompromised patients. Recently, associations between toxoplasmosis and various neuropathies and different types of cancer were documented. In the veterinary sector, toxoplasmosis results in recurring abortions, leading to significant economic losses. Treatment of toxoplasmosis remains intricate and encompasses general antiparasitic and antibacterial drugs. The efficacy of these drugs is hindered by intolerance, side effects, and emergence of parasite resistance. Furthermore, all currently used drugs in the clinic target acute toxoplasmosis, with no or little effect on the chronic form. In this review, we will provide a comprehensive overview on the currently used and emergent drugs and their respective parasitic targets to combat toxoplasmosis. We will also abridge the repurposing of certain drugs, their targets, and highlight future druggable targets to enhance the therapeutic efficacy against toxoplasmosis, hence lessening its burden and potentially alleviating the complications of its associated diseases

A Key Role for Plasmodium Subtilisin-like SUB1 Protease in Egress of Malaria Parasites from Host Hepatocytes

International audienceIn their mammalian host, Plasmodium parasites have two obligatory intracellular development phases, first in hepatocytes and subsequently in erythrocytes. Both involve an orchestrated process of invasion into and egress from host cells. The Plasmodium SUB1 protease plays a dual role at the blood stage by enabling egress of the progeny merozoites from the infected erythrocyte and priming merozoites for subsequent erythrocyte invasion. Here, using conditional mutagenesis in P. berghei, we show that SUB1 plays an essential role at the hepatic stage. Stage-specific sub1 invalidation during prehepatocytic development showed that SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and to egress from hepatocytes. Furthermore, mechanically released parasites were not adequately primed and failed to establish a blood stage infection in vivo. The critical involvement of SUB1 in both pre-erythrocytic and erythrocytic developmental phases qualifies SUB1 as an attractive multistage target for prophylactic and therapeutic anti-Plasmodium intervention strategies

Cumulation and injury determination of the European Community in anti-dumping cases

Available from British Library Document Supply Centre-DSC:3487.2848(97/17) / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase

International audienceThe Plasmodium subtilase SUB1 plays a pivotal role during the egress of malaria parasites from host hepatocytes and erythrocytes. Here we report the crystal structure of full-length SUB1 from the human-infecting parasite Plasmodium vivax, revealing a bacterial-like catalytic domain in complex with a Plasmodium-specific prodomain. The latter displays a novel architecture with an amino-terminal insertion that functions as a 'belt', embracing the catalytic domain to further stabilize the quaternary structure of the pre-protease, and undergoes calcium-dependent autoprocessing during subsequent activation. Although dispensable for recombinant enzymatic activity, the SUB1 'belt' could not be deleted in Plasmodium berghei, suggesting an essential role of this domain for parasite development in vivo. The SUB1 structure not only provides a valuable platform to develop new anti-malarial candidates against this promising drug target, but also defines the Plasmodium-specific 'belt' domain as a key calcium-dependent regulator of SUB1 during parasite egress from host cells

FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei

International audienc