Kota Arun Kumar

Mitochondrial anomalies are associated with the induction of intrinsic cell death proteins-Bcl2, Bax, cytochrome-c and p53 in mice brain during experimental fatal murine cerebral malaria

The levels of apoptosis associated proteins Bcl(2), 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 Bcl(2), 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 Bcl(2), 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.

Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites

ABSTRACT Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity. IMPORTANCE Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite’s successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite’s autophagic pathway may represent a novel approach to control malarial infections. Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite’s successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite’s autophagic pathway may represent a novel approach to control malarial infections.

Gene disruption reveals a dispensable role for Plasmepsin VII in the Plasmodium berghei life cycle

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.

Experimental Selenium Toxicity in Guinea Pigs: Biochemical Studies

Biochemical studies were conducted in experimentally induced selenium toxicity in recently weaned guinea pigs. A significant drop in blood glucose level, in comparison to controls, was observed in animals fed selenium-enriched barley (organic form) as well as those fed ordinary barley mixed with sodium selenite (inorganic form). Estimation of total serum proteins also revealed a significant drop in both these groups. SGOT (EC 2.6.1.1) activity was comparatively lower but no significant alteration was noticed in SGPT (EC 2.6.1.2). The erythrocytic glutathione peroxidase activity was significantly increased in inorganic selenosis followed by that in organic one, in comparison to controls. All these alterations were of mild degree in guinea pigs which were given sodium arsenite (10 ppm) along with sodium selenite (30 ppm) in the feed.

A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

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.

Plasmodium berghei plasmepsin VIII is essential for sporozoite gliding motility.

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 PbPM 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.

Genetic ablation of plasmoDJ1, a multi-activity enzyme, attenuates parasite virulence and reduces oocyst production

Malaria parasites must respond to stresses and environmental signals to perpetuate efficiently during their multistage development in diverse environments. To gain insights into the parasites stress response mechanisms, we investigated a conserved Plasmodium protein, which we 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 berghei ANKA attenuated virulence and reduced oocyst production, suggesting a major role for plasmoDJ1 in parasite development, which probably depends on its multiple activities.

Modulation of host cell SUMOylation facilitates efficient development of Plasmodium berghei and Toxoplasma gondii

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.

The shikimate pathway enzyme that generates chorismate is not required for the development of Plasmodium berghei in the mammalian host nor the mosquito vector

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.

CaM kinase II-alpha activity, levels and Ca/calmodulin dependent phosphorylation of substrate proteins in mice brain during fatal murine cerebral malaria

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, Ca(2+)/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.