Role of Calcium Signaling in the Transcriptional Regulation of the Apicoplast Genome of Plasmodium falciparum

Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses of Plasmodium falciparum to two calcium ionophores (A23187 and ionomycin) that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up- or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure of P. falciparum to both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand) that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1) in P. falciparum cells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that the P. falciparum apicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1

Calcium ionophores induce transcriptional changes in erythrocytic stages of human malaria parasite plasmodium falciparum.

178 p.Calcium is a universal second messenger molecule that plays a significant role in several biological processes. Presence of calcium sensors (calmodulins) and calcium dependent protein kinases in Plasmodium species suggest an important role of calcium dependent signaling pathways in the regulation of cellular processes in the malaria parasites. In general, changes in the intracellular calcium ion levels are interpreted as a signal that mediates biological processes varying from transcription to secretion. It was shown that in Plasmodium falciparum the intracellular calcium concentration rises as a result of exposure of the parasite cells to calcium ionophores (2-3). In this study, parasite response to calcium ionophores (ionomycin and A23187) and calcium channel blockers (thapsigargin and verapamil) was analyzed using DNA microarray. Global changes in various mRNA abundance levels have been analyzed and several pathways were found to be affected in the calcium ionophore treated parasites. Calcium channel blockers did not show any major effect. Interestingly, the treatment with calcium ionophores resulted in a significant down regulation of essentially all the protein-coding genes of the apicoplast genome. The effect of ionomycin was particularly commendable as it was more specific when compared to the wide range changes caused by A23187. Chelation of extracellular Ca2+ using EGTA still produced the same effect suggesting that external Ca2+ influx is not responsible for the apicoplast gene down regulation. Underlining the involvement of intracellular Ca2+ in this process, a nuclear encoded apicoplast targeted protein with an EF-hand domain was identified and localized to the apicoplast. Reduced sensitivity towards ionomycin was observed in the parasites expressing this protein suggesting its possible involvement in the phenomenon. Apicoplast enriched fractions isolated from the ionomycin treated cells was subjected to proteomic analysis using 2-D DIGE and a few non-apicoplast proteins were found to be down-regulated whose significance in the context of ionomycin treatment needs further exploration.​Doctor of Philosophy (SBS

Role of Calcium Signaling in the Transcriptional Regulation of the Apicoplast Genome of Plasmodium falciparum

Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses of Plasmodium falciparum to two calcium ionophores (A23187 and ionomycin) that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up-or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure of P. falciparum to both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand) that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1) in P. falciparum cells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that the P. falciparum apicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1

Role of calcium signaling in the transcriptional regulation of the apicoplast genome of Plasmodium falciparum

Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses of Plasmodium falciparum to two calcium ionophores (A23187 and ionomycin) that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up- or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure of P. falciparum to both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand) that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1) in P. falciparum cells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that the P. falciparum apicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1.Published versio

Optimized AAV rh.10 Vectors That Partially Evade Neutralizing Antibodies during Hepatic Gene Transfer

Of the 12 common serotypes used for gene delivery applications, Adeno-associated virus (AAV)rh.10 serotype has shown sustained hepatic transduction and has the lowest seropositivity in humans. We have evaluated if further modifications to AAVrh.10 at its phosphodegron like regions or predicted immunogenic epitopes could improve its hepatic gene transfer and immune evasion potential. Mutant AAVrh.10 vectors were generated by site directed mutagenesis of the predicted targets. These mutant vectors were first tested for their transduction efficiency in HeLa and HEK293T cells. The optimal vector was further evaluated for their cellular uptake, entry, and intracellular trafficking by quantitative PCR and time-lapse confocal microscopy. To evaluate their potential during hepatic gene therapy, C57BL/6 mice were administered with wild-type or optimal mutant AAVrh.10 and the luciferase transgene expression was documented by serial bioluminescence imaging at 14, 30, 45, and 72 days post-gene transfer. Their hepatic transduction was further verified by a quantitative PCR analysis of AAV copy number in the liver tissue. The optimal AAVrh.10 vector was further evaluated for their immune escape potential, in animals pre-immunized with human intravenous immunoglobulin. Our results demonstrate that a modified AAVrh.10 S671A vector had enhanced cellular entry (3.6 fold), migrate rapidly to the perinuclear region (1 vs. >2 h for wild type vectors) in vitro, which further translates to modest increase in hepatic gene transfer efficiency in vivo. More importantly, the mutant AAVrh.10 vector was able to partially evade neutralizing antibodies (~27–64 fold) in pre-immunized animals. The development of an AAV vector system that can escape the circulating neutralizing antibodies in the host will substantially widen the scope of gene therapy applications in humans

Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum

Functions have yet to be defined for the majority of genes of Plasmodium falciparum, the agent responsible for the most serious form of human malaria. Here we report changes in P. falciparum gene expression induced by 20 compounds that inhibit growth of the schizont stage of the intraerythrocytic development cycle. In contrast with previous studies, which reported only minimal changes in response to chemically induced perturbations of P. falciparum growth, we find that ~59% of its coding genes display over three-fold changes in expression in response to at least one of the chemicals we tested. We use this compendium for guilt-by-association prediction of protein function using an interaction network constructed from gene co-expression, sequence homology, domain-domain and yeast two-hybrid data. The subcellular localizations of 31 of 42 proteins linked with merozoite invasion is consistent with their role in this process, a key target for malaria control. Our network may facilitate identification of novel antimalarial drugs and vaccines.<br /