Stochastic expression of invasion genes in Plasmodium falciparum schizonts

Abstract

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms. Here, we investigate transcriptional heterogeneity and stochastic gene expression in Plasmodium falciparum by performing the quasilinear multiple annealing and looping based amplification cycles (MALBAC) based amplification and single cell RNA sequencing of blood stage schizonts. Our data reveals significant transcriptional variations in the schizont stage with a distinct group of highly variable invasion gene transcripts being identified. Moreover, the data reflects several diversification processes including putative developmental “checkpoint”; transcriptomically distinct parasite sub-populations and transcriptional switches in variable gene families (var, rifin, phist). Most of these features of transcriptional variability are preserved in isogenic parasite cell populations (albeit with a lesser amplitude) suggesting a role of epigenetic factors in cell-to-cell transcriptional variations in human malaria parasites. Lastly, we apply quantitative RT-PCR and RNA-FISH approach and confirm stochastic expression of key invasion genes, such as, msp1, msp3, msp7, eba181 and ama1 which represent prime candidates for invasion-blocking vaccines.

Highly Variable Expression of Merozoite Surface Protein MSPDBL2 in Diverse Plasmodium falciparum Clinical Isolates and Transcriptome Scans for Correlating Genes

“…Other genes shown previously to be variably expressed in schizont stages of clinical isolates, including different members of the msp3-like gene family (6), and members of the eba and Rh gene families (31,32), were not among those having a highly significant positive or negative correlation with MSPDBL2 expression here. This finding is consistent with previous results indicating that transcripts of mspdbl2 in clinical isolates assayed by RT-qPCR were not associated with variation in relative transcript levels of other members of the msp3-like family ( 6) and independent regulation of expression of the other merozoite antigen genes (31,33,34).…”

The artemisinin-induced dormant stages ofPlasmodium falciparumexhibit hallmarks of cellular senescence and drug resilience

“…The main motivation was to evaluate if these pyknotic cell-like forms represent the genuine dormant stage(s) of P. falciparum and if so, what is their biochemical and cellular physiology. For this, we used the above-mentioned FACS-based cell isolation strategy coupled with “few-cells” RNA sequencing that allows morphology/stage-associated transcriptomic analysis of a defined number of P. falciparum cells (including single cell) 29 . Briefly, 500-parasite cells were captured from the pyknotic/ring stage FACS gate each day and subjected to SMARTseq2-based RNA sequencing for whole transcriptome profiling 30 (for more details see Material and Methods ).…”

The artemisinin-induced dormant stages of Plasmodium falciparum exhibit hallmarks of cellular senescence and drug resilience

“…The main motivation was to evaluate if these pyknotic cell-like forms represent the genuine dormant stage(s) of P. falciparum and if so, what is their biochemical and cellular physiology. For this, we used the above-mentioned FACS-based cell isolation strategy coupled with "few-cells" RNA sequencing that allows morphology/stage-associated transcriptomic analysis of a de ned number of P. falciparum cells (including single cell) 29 . Brie y, 500parasite cells were captured from the pyknotic/ring stage FACS gate each day and subjected to SMARTseq2-based RNA sequencing for whole transcriptome pro ling 30 (for more details see Material and Methods).…”