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Plasmodium sporozoites are extracellular forms introduced during mosquito bite that selectively invade mammalian hepatocytes. Sporozoites are delimited by a cell membrane that is linked to the underlying acto‐myosin molecular motor. While membrane proteins with roles in motility and invasion have been well studied, very little is known about proteins that maintain the sporozoite shape. We demonstrate that in Plasmodium berghei (Pb) a conserved hypothetical gene, PBANKA_1422900 specifies sporozoite structural integrity maintenance protein (SIMP) required for maintaining the sporozoite shape and motility. Sporozoites lacking SIMP exhibited loss of regular shape, extensive membrane blebbing at multiple foci, and membrane detachment. The mutant sporozoites failed to infect hepatocytes, though the altered shape did not affect the organization of cytoskeleton or inner membrane complex (IMC). Interestingly, the components of IMC failed to extend under the membrane blebs likely suggesting that SIMP may assist in anchoring the membrane to IMC. Endogenous C‐terminal HA tagging localized SIMP to membrane and revealed the C‐terminus of the protein to be extracellular. Since SIMP is highly conserved among Plasmodium species, these findings have important implications for utilizing it as a novel sporozoite‐specific vaccine candidate. [ABSTRACT FROM AUTHOR]

Summary: The interplay between ATP generating and utilizing pathways in a cell is responsible for maintaining cellular ATP/energy homeostasis that is reflected by Adenylate Energy Charge (AEC) ratio. Adenylate kinase (AK), that catalyzes inter‐conversion of ADP, ATP and AMP, plays a major role in maintaining AEC and is regulated by cellular AMP levels. Hence, the enzymes AMP deaminase (AMPD) and nucleotidases, which catabolize AMP, indirectly regulate AK activity and in‐turn affect AEC. Here, we present the first report on AMPD from Plasmodium, the causative agent of malaria. The recombinant enzyme expressed in Saccharomyces cerevisiae was studied using functional complementation assay and residues vital for enzyme activity have been identified. Similarities and differences between Plasmodium falciparum AMPD (PfAMPD) and its homologs from yeast, Arabidopsis and humans are also discussed. The AMPD gene was deleted in the murine malaria parasite P. berghei and was found to be dispensable during all stages of the parasite life cycle. However, when episomal expression was attempted, viable parasites were not obtained, suggesting that perturbing AMP homeostasis by over‐expressing AMPD might be lethal. As AMPD is known to be allosterically modulated by ATP, GTP and phosphate, allosteric activators of PfAMPD could be developed as anti‐parasitic agents. [ABSTRACT FROM AUTHOR]

SUMOylation is a reversible post translational modification of proteins that regulates protein stabilization, nucleocytoplasmic transport, and protein-protein interactions. Several viruses and bacteria modulate host SUMOylation machinery for efficient infection. Plasmodium sporozoites are infective forms of malaria parasite that invade mammalian hepatocytes and transforms into exoerythrocytic forms (EEFs). Here, we show that during EEF development, the distribution of SUMOylated proteins in host cell nuclei was significantly reduced and expression of the SUMOylation enzymes was downregulated. Plasmodium EEFs destabilized the host cytoplasmic protein SMAD4 by inhibiting its SUMOylation. SUMO1 overexpression was detrimental to EEF growth, and insufficiency of the only conjugating enzyme Ubc9/E2 promoted EEF growth. The expression of genes involved in suppression of host cell defense pathways during infection was reversed during SUMO1 overexpression, as revealed by transcriptomic analysis. The inhibition of host cell SUMOylation was also observed during Toxoplasma infection. We provide a hitherto unknown mechanism of regulating host gene expression by Apicomplexan parasites through altering host SUMOylation. [ABSTRACT FROM AUTHOR]

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]

Immunization with radiation attenuated Plasmodium sporozoites (RAS) elicits sterile protective immunity against sporozoite challenge in murine models and in humans. Similarly to RAS, the genetically attenuated sporozoites (GAPs) named uis3(-), uis4(-) and P36p(-) have arrested growth during the liver stage development, and generate a powerful protective immune response in mice. We compared the protective mechanisms in P. yoelii RAS, uis3(-) and uis4(-) in BALB/c mice. In RAS and GAPs, sterile immunity is only achieved after one or more booster injections. There were no differences in the immune responses to the circumsporozoite protein (CSP) generated by RAS and GAPs. To evaluate the role of non-CSP T-cell antigens we immunized antibody deficient, CSP-transgenic BALB/c mice, that are T cell tolerant to CSP, with P. yoelii RAS or with uis3(-) or uis4(-) GAPs, and challenged them with wild type sporozoites. In every instance the parasite liver stage burden was approximately 3 logs higher in antibody deficient CSP transgenic mice as compared to antibody deficient mice alone. We conclude that CSP is a powerful protective antigen in both RAS and GAPs viz., uis3(-) and uis4(-) and that the protective mechanisms are similar independently of the method of sporozoite attenuation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A fundamental problem in systems biology and whole genome sequence analysis is how to infer functions for the many uncharacterized proteins that are identified, whether they are conserved across organisms of different phyla or are phylum-specific. This problem is especially acute in pathogens, such as malaria parasites, where genetic and biochemical investigations are likely to be more difficult. Here we perform comparative expression analysis on Plasmodium parasite life cycle data derived from P. falciparum blood, sporozoite, zygote and ookinete stages, and P. yoelii mosquito oocyst and salivary gland sporozoites, blood and liver stages and show that type II fatty acid biosynthesis genes are upregulated in liver and insect stages relative to asexual blood stages. We also show that some universally uncharacterized genes with orthologs in Plasmodium species, Saccharomyces cerevisiae and humans show coordinated transcription patterns in large collections of human and yeast expression data and that the function of the uncharacterized genes can sometimes be predicted based on the expression patterns across these diverse organisms. We also use a comprehensive and unbiased literature mining method to predict which uncharacterized parasite-specific genes are likely to have roles in processes such as gliding motility, host-cell interactions, sporozoite stage, or rhoptry function. These analyses, together with proteinprotein interaction data, provide probabilistic models that predict the function of 926 uncharacterized malaria genes and also suggest that malaria parasites may provide a simple model system for the study of some human processes. These data also provide a foundation for further studies of transcriptional regulation in malaria parasites. [ABSTRACT FROM AUTHOR]

Abstract: Malaria sporozoites migrate through several cells prior to a productive invasion that involves the formation of a parasitophorous vacuole (PV) where sporozoites undergo transformation into Exo-erythorcytic forms (EEFs). The precise mechanism leading to sporozoite activation for invasion is unknown, but prior traversal of host cells is required. During cell migration sporozoites are exposed to large shifts in K+ concentration. We report here that incubation of sporozoites to the intracellular K+ concentration enhances 8–10 times the infectivity of Plasmodium berghei and 4–5 times the infectivity of Plasmodium yoelli sporozoites for a hepatocyte cell line, while simultaneously decreasing cell passage activity. The K+ enhancing effect was time and concentration dependent, and was significantly decreased by K+ channel inhibitors. Potassium-treated P. berghei sporozoites also showed enhanced numbers of EEFs in non-permissive cell lines. Treated sporozoites had reduced infectivity for mice, but infectivity was enhanced upon Kupffer cell depletion. Transcriptional analysis of K+ treated and control sporozoites revealed a high degree of correlation in their levels of gene expression, indicating that the observed phenotypic changes are not due to radical changes in gene transcription. Only seven genes were upregulated by more than two-fold in K+ treated sporozoites. The highest level was noted in PP2C, a phosphatase known to dephosphorylate the AKT potassium channel in plants. [Copyright &y& Elsevier]

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]

The circumsporozoite (CS) protein is the major surface protein of Plasmodium sporozoites. Antibodies to the immunodominant repeat domain of CS immobilize sporozoites and prevent infection of hepatocytes. Plasmodium falciparum vaccines containing CS repeats are undergoing human trials in endemic areas, and proof of efficacy has been obtained. The correlates of protection are under investigation. Levels of anti-repeat antibodies in the serum of the human volunteers have been measured mostly by enzyme-linked immunosorbent assay (ELISA) and IFA. Assays that measure the effect of the serum antibodies on parasite infectivity (serum neutralization assays SNAs) are not usually performed because they require a susceptible host and P. falciparum sporozoites are highly infectious only to humans. To overcome this limitation, we developed a new assay named transgenic sporozoite neutralization assay (TSNA) that uses as neutralization target, a transgenic rodent malaria parasite Plasmodium berghei that bears the P. falciparum CS repeats [CS(Pf)]. Following incubation with human serum, CS(Pf) infectivity of HepG2 cells is evaluated by real-time PCR. We have compared ELISA titers and TSNAs in a limited number of sera from humans immunized with (T1B)4 MAP, a peptide vaccine containing P. falciparum CS repeats. A comparison between the two assays did not reach significance (p=0.175) when analyzed by non-parametric Spearman correlation method. Ongoing human trials of CS-based vaccines should provide an opportunity to determine whether TSNAs will provide better correlates of protective immunity than ELISA assays. [Copyright &y& Elsevier]

The levels of nuclear transcription factor-kappa B (NF-κB) subunits p65 and p50 and its associated kinase, inhibitory kappa B kinase (IKK) alpha and beta were monitored in cytosolic and nuclear fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Since protein kinase C (PKC) and protein tyrosine kinases (PTK) are known to collaborately regulate the NF-κB activation, we also studied the activity of these two kinases in cytosol and membrane fraction. In parallel, the levels of two PKC isoforms (alpha and delta) and tyrosine phosphorylated proteins were monitored to correlate the observed changes in the activity. Our results underscore the involvement of IKK-beta as an essential mediator of NF-κB activation as evinced by the nuclear translocation of p65 and p50 during CM pathology. Additional findings confirm altered activity and levels of PKC and enhanced activation of PTK and tyrosine phosphorylation of proteins during CM pathology. These signaling intricacies involving an interplay between rel family (NF-κB) of transcription factors, PKC and PTK may serve as an important cue in understanding the possible continuation of the post receptor signaling events associated with tumor necrosis factor-alpha induction during FMCM pathology. [Copyright &y& Elsevier]

The activity and levels of CaM kinase II-alpha was investigated in the cytosolic and membrane fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). In parallel, Ca2+/Calmodulin dependent phosphorylation of target substrate proteins was studied using syntide-2 as substrate. Pathology of FMCM resulted in decreased CaM kinase-II activity in both cortex and cerebellum though western analysis revealed no appreciable changes in the levels of CaM kinase-II alpha in cytosol and membrane fractions from control and cerebral malaria infected brain. Given the abundant expression of Cam kinase-II in neuronal tissue, its significance in neurotransmitter release and synthesis and signal transduction during apoptosis, decreased levels of enzyme activity and altered phosphorylation of substrate proteins by CaM kinase II may serve as important cues in understanding the CaM kinase signal transduction events central to neurological disorders during FMCM. [Copyright &y& Elsevier]

The levels of apoptosis associated proteins Bcl2, Bax, cytochrome-c and p53 was investigated in mice cerebral cortex and cerebellum, using an experimental model of fatal murine cerebral malaria (FMCM). Owing to the activation of events central to mitochondrial dysfunctions, we monitored the structural integrity of mitochondria in cerebral malaria (CM) infected brain tissue by transmission EM (TEM) studies. Western blot analysis revealed the induction of Bcl2, Bax, cytochrome-c and p53 in both cortex and cerebellum. The TEM studies revealed extensive vacuolation and swelling of mitochondria in infected brain suggestive of a late stage of degeneration. Our results underscore the activation of an intrinsic cell death pathway as evinced by the induction of mitochondria associated apoptotic proteins Bcl2, Bax and cytochrome-c and further envisages the induction of p53 as a possible continuation of the post receptor signaling events associated with tumor necrosis factor induction following inflammatory responses during CM. These findings may be crucial to mitochondrial dysfunctions underlying the pathology of FMCM. [Copyright &y& Elsevier]

A fundamental problem in systems biology and whole genome sequence analysis is how to infer functions for the many uncharacterized proteins that are identified, whether they are conserved across organisms of different phyla or are phylum-specific. This problem is especially acute in pathogens, such as malaria parasites, where genetic and biochemical investigations are likely to be more difficult. Here we perform comparative expression analysis on Plasmodium parasite life cycle data derived from P. falciparum blood, sporozoite, zygote and ookinete stages, and P. yoelii mosquito oocyst and salivary gland sporozoites, blood and liver stages and show that type II fatty acid biosynthesis genes are upregulated in liver and insect stages relative to asexual blood stages. We also show that some universally uncharacterized genes with orthologs in Plasmodium species, Saccharomyces cerevisiae and humans show coordinated transcription patterns in large collections of human and yeast expression data and that the function of the uncharacterized genes can sometimes be predicted based on the expression patterns across these diverse organisms. We also use a comprehensive and unbiased literature mining method to predict which uncharacterized parasite-specific genes are likely to have roles in processes such as gliding motility, host-cell interactions, sporozoite stage, or rhoptry function. These analyses, together with protein-protein interaction data, provide probabilistic models that predict the function of 926 uncharacterized malaria genes and also suggest that malaria parasites may provide a simple model system for the study of some human processes. These data also provide a foundation for further studies of transcriptional regulation in malaria parasites.

Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus. [ABSTRACT FROM AUTHOR]

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. [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]

Plasmepsins (PM), aspartic proteases of Plasmodium, comprises a family of ten proteins that perform critical functions in Plasmodium life cycle. Except VII and VIII, functions of the remaining plasmepsin members have been well characterized. Here, we have generated a mutant parasite lacking PM VII in Plasmodium berghei using reverse genetics approach. Systematic comparison of growth kinetics and infection in both mosquito and vertebrate host revealed that PM VII depleted mutants exhibited no defects in development and progressed normally throughout the parasite life cycle. These studies suggest a dispensable role for PM VII in Plasmodium berghei life cycle. [ABSTRACT FROM AUTHOR]

• PbS10 and PbS23/SSP3 are sporozoite specific genes. • PbS10 gene disruption leads to an early arrest in the liver stage development. • PbS23/SSP3 gene deletion leads to an arrest in the late liver stage development. 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. [ABSTRACT FROM AUTHOR]