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Single Cell RNA Sequencing Reveals Transcriptional Heterogeneity and Stochastic Gene Expression 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 such as yeast and bacteria. Here, we investigated transcriptional heterogeneity and stochastic gene expression in P. falciparum by performing single cell RNA sequencing on blood stage schizonts. Our data reveals significant transcriptional variations in the schizonts stage that appear to reflect several diversification processes including putative developmental “checkpoint”; transcriptomically distinct parasite cells sub-populations; transcriptional switches in variable gene families and a distinct group of highly variable transcripts. Most of these features of transcriptional variability were 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. Finally, to validate our findings, we applied quantitative qRT-PCR and RNA-FISH approach and confirmed stochastic expression of several genes such as msp1, msp3, msp7, rhopH2 and eba181, some of which represent key candidates for invasion blocking vaccines

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE164459 | GEO | 2021/01/09

REPOSITORIES: GEO

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Project description:Plasmodium falciparum merozoite surface protein MSPDBL2 is a highly polymorphic target of naturally acquired immune responses encoded by a single copy gene under strong balancing selection in natural populations. The MSPDBL2 protein is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene expression in culture is increased in response to overexpression of the gametocyte development inducer GDV1 in an engineered parasite clone, so there is a need to characterise its natural expression variation. In this study, MSPDBL2 in mature schizonts was analyzed in the first ex vivo culture cycle of 102 clinical isolates from four endemic populations in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as eleven isolates had more than 3% schizonts positive and one had 73% positive. The expression frequencies were similar across the four endemic populations, and there was no significant difference between the clinical isolates overall and frequencies of positive schizonts previously seen in cultured P. falciparum laboratory lines. To explore whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBP2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of many genes previously implicated as likely to be involved in gametocytogenesis, but not including other genes including the gametocytogenesis master regulator ap2g. Although MSPDBL2 expression occurs in a highly variable proportion of schizonts in clinical isolates, and correlation with expression of some gametocytogenesis-related genes is consistent with regulation by GDV1, it is not apparently a direct marker of sexual commitment and its function in the parasite remains to be determined.

Project description:During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes M-bM-^@M-^S sexual stage precursor cells M-bM-^@M-^S likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the blood stream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and ~30 hours post invasion and mature gametocytes after around 7 days post invasion. We used 164/TdTom, a transgenic parasite line expressing a red fluorescent protein reporter under a gametocyte-specific promoter to generate schizont samples. Schizonts were subsequently isolated from both the fluorescent and non-fluorescent population by FACS and prepared for microarray analysis. Two biological replicates were produced for both the fluorescent and the non-fluorescent samples.

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Single Cell RNA Sequencing Reveals Transcriptional Heterogeneity and Stochastic Gene Expression in Plasmodium falciparum Schizonts

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