Project description:Background: Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, the quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the precise correlations between transcript and protein abundance remain under-scrutinized. Results: Here, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34-46 hours post-invasion) based on 2D-gel electrophoresis of protein samples labeled with DIGE fluorescent dyes. For this purpose we analyzed parasite samples taken at four-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins including actin-I, enolase, eIF4A, eIF5A, and several heat shock proteins are represented by more than one isoform, presumably due to post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal significant post-transcriptional gene expression regulation. Conclusions: Together, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra-erythrocytic development of P. falciparum. Additional comment: In essence, the microarray data generated in this study is a repeat of the transcriptome analysis described in Bozdech et al., PLoS Biol 2003, 1(1):E5. Differences include (1) an improved set of oligonucleotides [see Hu et al., BMC Bioinformatics 2007, 8:350] used in the study associated with this GEO entry, and (2) the sampling at only four timepoints during the intra-erythrocytic development cycle (schizont stage) to match and complement the samples processed in the concomitant proteomics analysis. Four timepoint samples were harvested from a tightly synchronized 6 liter biofermenter culture of P. falciparum during schizont stage (at 34, 38, 42, and 46 hours post-invasion) and compared against a 3D7 RNA reference pool.

Project description:Abstract of associated publication: Background: During the latter half of the natural 48-hour intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge. Methods: We developed a custom nCounter® gene expression platform, and used this platform to measure malaria parasite gene expression profiles in vitro and in vivo. We also used imputation to generate global transcriptional profiles, and assessed differential gene expression between parasites growing in vitro and those recovered from malaria-infected patient tissues collected at autopsy. Results: We demonstrate, for the first time, global transcriptional expression profiles from in vivo malaria parasites sequestered in human tissues. We found that parasite physiology can be correlated with in vitro data from an existing life cycle data set, and that parasites in sequestered tissues show an expected schizont-like transcriptional profile, which is conserved across tissues from the same patient. Imputation based on 60 landmark genes generated global transcriptional profiles that were highly correlated with genome-wide expression patterns from the same sample measured by microarray. Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections. Conclusions: Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.

Project description:Background: Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, the quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the precise correlations between transcript and protein abundance remain under-scrutinized. Results: Here, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34-46 hours post-invasion) based on 2D-gel electrophoresis of protein samples labeled with DIGE fluorescent dyes. For this purpose we analyzed parasite samples taken at four-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins including actin-I, enolase, eIF4A, eIF5A, and several heat shock proteins are represented by more than one isoform, presumably due to post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal significant post-transcriptional gene expression regulation. Conclusions: Together, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra-erythrocytic development of P. falciparum. Additional comment: In essence, the microarray data generated in this study is a repeat of the transcriptome analysis described in Bozdech et al., PLoS Biol 2003, 1(1):E5. Differences include (1) an improved set of oligonucleotides [see Hu et al., BMC Bioinformatics 2007, 8:350] used in the study associated with this GEO entry, and (2) the sampling at only four timepoints during the intra-erythrocytic development cycle (schizont stage) to match and complement the samples processed in the concomitant proteomics analysis.

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Plasmodium falciparum

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