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Plasmodium sporozoites are infective forms of the parasite to mammalian hepatocytes. Sporozoite surface or secreted proteins likely play an important role in recognition, invasion and successful establishment of hepatocyte infection. By approaches of reverse genetics, we report the functional analysis of two Plasmodium berghei (Pb) sporozoite specific genes- PbS10 and PbS23/SSP3 that encode for proteins with a putative signal peptide. The expression of both genes was high in oocyst and salivary gland sporozoite stages as compared to other life cycle stages and PbS23/SSP3 protein was detected in salivary gland sporozoites. Both mutants were indistinguishable to wild-type parasites with regard to asexual growth in RBC, ability to complete sexual reproduction and form sporozoites in vector host. While the sporozoite stage of both mutants were able to glide and invade hepatocytes normally in vitro and in vivo, PbS10 mutants suffered growth attenuation at an early stage while PbS23/SSP3 mutants manifested defect during late exo-erythrocytic form maturation. Interestingly, both mutants gave rare breakthrough infections, suggesting that while both were critical for liver stage development, their depletion did not completely abrogate blood stage infection. These findings have important implications for weakening sporozoites by multiple gene attenuation towards the generation of a safe whole organism vaccine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In Plasmodium, the shikimate pathway is a potential target for malaria chemotherapy owing to its absence in the mammalian host. Chorismate, the end product of this pathway, serves as a precursor for aromatic amino acids, Para-aminobenzoic acid and ubiquinone, and is synthesised by Chorismate synthase (CS). Therefore, it follows that the Cs locus may be refractory to genetic manipulation. By utilising a conditional mutagenesis system of yeast Flp/FRT, we demonstrate an unexpectedly dispensable role of CS in Plasmodium. Our studies reiterate the need to establish an obligate reliance on Plasmodium metabolic enzymes through genetic approaches before their selection as drug targets., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.

The irradiated-sporozoite vaccine elicits sterile immunity against Plasmodium parasites in experimental rodent hosts and human volunteers. Based on rodent malaria models, it has been proposed that CD8+ T cells are the key protective effector mechanism required in sporozoite-induced immunity. To investigate the role of class II-restricted immunity in protective immunity, we immunized beta2-microglobulin knockout (beta2M-/-) mice with irradiated Plasmodium yoelii or P. berghei sporozoites. Sterile immunity was obtained in the CD8+-T-cell-deficient mice immunized with either P. berghei or P. yoelii sporozoites. beta2M-/- mice with the BALB/c (H-2d) genetic background as well as those with the C57BL (H-2b) genetic background were protected. Effector mechanisms included CD4+ T cells, mediated in part through the production of gamma interferon, and neutralizing antibodies that targeted the extracellular sporozoites. We conclude that in the absence of class I-restricted CD8+ T cells, sporozoite-induced protective immunity can be effectively mediated by class II-restricted immune effector mechanisms. These results support efforts to develop subunit vaccines that effectively elicit high levels of antibody and CD4+ T cells to target Plasmodium pre-erythrocytic stages.

Background: Emerging resistance of the malaria parasite Plasmodium to current therapies underscores the critical importance of exploring novel strategies for disease eradication. Plasmodium species are obligate intracellular protozoan parasites. They rely on an unusual form of substrate-dependent motility for their migration on and across host-cell membranes and for host cell invasion. This peculiar motility mechanism is driven by the 'glideosome', an actin-myosin associated, macromolecular complex anchored to the inner membrane complex of the parasite. Myosin A, actin, aldolase, and thrombospondin-related anonymous protein (TRAP) constitute the molecular core of the glideosome in the sporozoite, the mosquito stage that brings the infection into mammals. Methods: Virtual library screening of a large compound library against the PfAldolase-TRAP complex was used to identify candidate compounds that stabilize and prevent the disassembly of the glideosome. The mechanism of these compounds was confirmed by biochemical, biophysical and parasitological methods. Results: A novel inhibitory effect on the parasite was achieved by stabilizing a protein-protein interaction within the glideosome components. Compound 24 disrupts the gliding and invasive capabilities of Plasmodium parasites in in vitro parasite assays. A high-resolution, ternary X-ray crystal structure of PfAldolase-TRAP in complex with compound 24 confirms the mode of interaction and serves as a platform for future ligand optimization. Conclusion: This proof-of-concept study presents a novel approach to anti-malarial drug discovery and design. By strengthening a protein-protein interaction within the parasite, an avenue towards inhibiting a previously "undruggable" target is revealed and the motility motor responsible for successful invasion of host cells is rendered inactive. This study provides new insights into the malaria parasite cell invasion machinery and convincingly demonstrates that liver cell invasion is dramatically reduced by 95% in the presence of the small molecule stabilizer compound 24. [ABSTRACT FROM AUTHOR]

Malaria parasites must respond to stresses and environmental signals to perpetuate efficiently during their multistage development in diverse environments. To gain insights into the parasite's stress response mechanisms, we investigated a conserved Plasmodium protein, whichwe have named plasmoDJ1 on the basis of the presence of a putative cysteine protease motif of the DJ-1/PfpI superfamily, for its activities, potential to respond to stresses and role in parasite development. PlasmoDJ1 is expressed in all intraerythrocytic stages and ookinetes. Its expression was increased 7-9-fold upon heat shock and oxidative stress due to H2O2 and artemisinin; its expression in a stress-sensitive Escherichia coli mutant conferred tolerance against oxidative stress, indicating that plasmoDJ1 has the potential to sense and/or protect from stresses. Recombinant plasmoDJ1 efficiently neutralized H2O2, facilitated renaturation of denatured citrate synthase and showed protease activity, indicating that plasmoDJ1 is a multi-activity protein. Mutation of the catalytic cysteine residue, but not other residues, reduced H2O2-neutralization activity by ~90% and significantly decreased chaperone and protease activities, indicating that these activities are intrinsic to plasmoDJ1. The plasmoDJ1 gene knockout in Plasmodium bergheiANKAattenuated virulence and reduced oocyst production, suggesting a major role for plasmoDJ1 in parasite development, which probably depends on its multiple activities. [ABSTRACT FROM AUTHOR]

Abstract: Plasmodium berghei ANKA (Pb ANKA) is a lethal strain of malaria that causes experimental cerebral malaria (ECM) in rodent models. Pathology of the disease is associated with the sequestration of the infected rbc (irbc) in the micro vessels of brain. In the present study, we analyzed the nature of the glycoprotein modification occurring in irbc membrane during erythrocytic stages of Pb ANKA infection. Titration of naturally occurring glycoproteins with concanavalin A (Con A) and wheat germ agglutinin (WGA) lectins revealed an enhanced lectin binding ability for the irbc membrane preparations. Partial characterization of the Con A specific determinants (α-d-methyl mannoside specificity) by lectin affinity chromatography followed by 2D electrophoresis and WGA specific determinants (sialic acid specificity) by Western analysis revealed the association of novel lectin specific determinants in irbc membrane. To correlate the biochemical changes with the morphological changes, SEM of irbc, and TEM of sequestered irbc were performed. These ultra structural studies revealed variable and irregular surface protrusions and deep surface indentations on irbc. These observations implicate that altered glycoprotein profiles may lead to cytoarchitectural changes in irbc membrane and such changes may be essential to establish contact with the host endothelial cells. These observations may be central to the microvascular sequestration and pathology of ECM. [Copyright &y& Elsevier]

[Display omitted] • Plasmodium DDI1 protein is expressed in all major life cycle stages and is crucial for parasite development. • Infection of mice with DDI1 knock-down parasites confers immunity to subsequent infection. • Plasmodium DDI1 is associated with chromatin and DNA-protein crosslinks. • DDI1 knock-down parasites show enhanced susceptibility to HIV protease inhibitor lopinavir and antimalarial artemisinin. • Plasmodium DDI1 could be a therapeutic target for malaria control. DNA damage inducible 1 protein (DDI1) is involved in a variety of cellular processes including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of a RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating the potential of DDI1 knock-down parasites as a whole organism vaccine. Plasmodium falciparum DDI1 (Pf DDI1) is associated with chromatin and DNA-protein crosslinks. Pf DDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA-damaging chemicals, indicating a role of Pf DDI1 in removal of DNA-protein crosslinks. Knock-down of Pf DDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control. [ABSTRACT FROM AUTHOR]

Plasmodium aspartic proteases, termed plasmepsins (PMs) play many critical roles such as haemoglobin degradation, cleavage of PEXEL proteins and sporozoite development in the parasite life cycle. Most of the plasmepsins are well characterized, however the role of PM VIII in Plasmodium remains unknown. Here, we elucidate the functions of PM VIII (PBANKA_132910) in the rodent malaria parasite Plasmodium berghei ( Pb ). By targeted gene deletion, we show that Pb PM VIII is critical for sporozoite egress from an oocyst and gliding motility, which is a prerequisite for the invasion of salivary glands and subsequent transmission to the vertebrate host. [ABSTRACT FROM AUTHOR]