Characterization of P. vivax blood stage transcriptomes from field isolates reveals similarities among infections and complex gene isoforms

Kim, Adam; Popovici, Jean; Vantaux, Amélie; Samreth, Reingsey; Bin, Sophalai; Kim, Saorin; Roesch, Camille; Liang, Li; Davies, Huw; Felgner, Philip L.; Herrera, Sócrates; Arévalo‐Herrera, Myriam; Ménard, Didier; Serre, David

doi:10.1038/s41598-017-07275-9

Cited by 32 publications

(61 citation statements)

References 40 publications

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“…We used a more stringent threshold to avoid merging adjacent UTRs on the same strand; the threshold used to define a block of continuous transcription was iteratively increased, in increments of 5 reads. The mean length of 5' UTRs was 1,007 nt (median length 815 nt), and was 818 nt for 3' UTRs (median was 685 nt), which is longer than those described in previous studies in P. vivax 14,15 . These are also longer than the 5' and 3' UTR lengths recently described in P. falciparum 23 (577 nt and 453 nt, respectively).…”

Section: The Architecture Of the Plasmodium Vivax Schizont Transcriptcontrasting

confidence: 55%

“…In the first P. vivax RNA-seq study 14 , the median length of 5' UTRs was 295 nt (n=3,633), and the median length of the 3' UTRs was 203 nt (n=3,967). In a more recent P. vivax RNA-seq study 15 , the median lengths of the was 754 nt for 5' UTRs and 785 nt for the 3' UTRs (n =3,230); the lack of a difference in relative lengths of the 5' and 3' UTRs is likely to be a feature of the computational approach used in that work, which did not consider each UTR independently. The improved annotation of UTRs in this data was enabled by a more even coverage of AT-rich regions of the genome compared to other studies, in tandem with a computational approach developed specifically for Plasmodium genomes.…”

Section: Discussionmentioning

confidence: 95%

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Analysis ofPlasmodium vivaxschizont transcriptomes from field isolates reveals heterogeneity of expression of genes involved in host-parasite interactions

Siegel

Chappell

Hostetler

et al. 2020

Preprint

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Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol "DAFT-seq" to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5' and 3' untranslated regions (UTRs), some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets. Results Preparation of purified late-stage schizont transcriptomesFour blood samples from Cambodian patients were selected to undergo short-term ex-vivo culture. After the majority of parasites had matured, as judged by microscopy, late-stage schizonts were purified using Percoll gradients. Four RNA-seq libraries were generated using a modified version of the DAFT-seq protocol, which was optimised for highly AT-rich Plasmodium parasites 23 , and sequenced using the Illumina platform to generate 55-63 million reads per patient sample. In all cases >85% of reads mapped to the P. vivax PvP01 reference genome (Table S1). This data was used to improve the gene models of 352 genes in the PvP01 genome, including identifying 20 novel gene transcripts (Table S2). Comparison of the expression values of these RNA-seq libraries to blood stage microarray time course from a P. vivax dataset (containing three patient isolates) 12 (Fig. S1) and a more densely sampled P. falciparum dataset 24 (Fig. S2) confirmed that the samples were late-stage schizonts (as they correlated most strongly to these time points in the prior datasets) and that the transcriptomes were highly similar to each other (as the correlation of the normalised expression values of the RNA-seq libraries was close to 1) (Fig. S3). More apparent heterogeneity was found between the patient isolates using the PVP01 genome than using only the gene IDs present in the Sal1 genome (Fig. S4), consistent with most of the heterogeneity within the patient isolate transcriptomes being present in multigene families that are difficult to assemble and annotate, and are thus under-represented in the Sal1 genome.

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Section: The Architecture Of the Plasmodium Vivax Schizont Transcriptcontrasting

confidence: 55%

Section: Discussionmentioning

confidence: 95%

Analysis ofPlasmodium vivaxschizont transcriptomes from field isolates reveals heterogeneity of expression of genes involved in host-parasite interactions

Siegel

Chappell

Hostetler

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…We extracted RNA from all blood samples stored in Trizol using the Zymo Direct-zol kit with an in-column DNAse step and eluted RNA into 20 µL of water. We then prepared Illumina stranded libraries after ribosomal RNA and globin mRNA reduction [7] and sequenced them on a HiSeq 2500 to generate a total of 1.2 billion paired-end reads of 50 bp ( Supplemental Table S1 ).…”

Section: Methodsmentioning

confidence: 99%

“…Here, we expand on previous analyses demonstrating that RNA-seq data could be generated directly from P. vivax -infected patients [7] and characterize the transcriptomes of 26 clinical P. vivax isolates obtained from Cambodian patients enrolled in a chloroquine efficacy study ([8], Popovici et al , under review). First, we describe variations in parasite gene expression among infected patients and identify novel potential gametocyte markers.…”

Section: Introductionmentioning

confidence: 90%

In vivogene expression analyses provide unique insights onP. vivaxgametocytogenesis and chloroquine response

Kim

Popovici

Ménard

et al. 2018

Preprint

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13Studies of gene expression have provided insights on the regulation of Plasmodium parasites. 14 However, few studies have targeted P. vivax, the cause of one third of all human malaria cases 15 outside Africa, due to the lack of in vitro culture system and the difficulties associated with 16 studying clinical samples. Here, we describe robust RNA-seq profiles of P. vivax parasites 17 generated directly from infected patient blood. Gene expression deconvolution analysis reveals 18 that most parasite mRNAs derive from trophozoites and that the asynchronicity of P. vivax 19 infections is unlikely to confound gene expression studies. We also show that gametocyte genes 20 form two clusters of co-regulated genes, possibly indicating the independent regulation of male 21 and female gametocytogeneses. Finally, despite a large effect on parasitemia, we find that 22 the drug on P. falciparum. Overall, our study exemplifies the biological insights that can be gained 35 from applying modern genomic tools to study this difficult human pathogen. 36 37

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“…The systems analysis of P. vivax sporozoites that reside in the mosquito salivary glands and are poised for transmission and liver infection offer a key opportunity to gain insight into P. vivax infection. Plasmodium vivax sporozoites have been explored previously by microarray [24] and most recently, in a single RNA-seq replicate [25] and a study on sporozoite activation {Roth, 2018 #66}. Epigenetic regulation in sporozoites has only been explored in P. falciparum [26, 27].…”

Section: Introductionmentioning

confidence: 99%

Transcriptome and histone epigenome ofPlasmodium vivaxsalivary-gland sporozoites point to tight regulatory control and potential mechanisms for liver-stage differentiation

Müller

et al. 2017

Preprint

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1 salivary-gland sporozoites point to tight regulatory control 2 and potential mechanisms for liver-stage differentiation. ABSTRACT 2 8Plasmodium vivax is the key obstacle to malaria elimination in Asia and Latin America, 2 9 largely attributed to its ability to form resilient hypnozoites (sleeper-cells) in the host liver 3 0 that escape treatment and cause relapsing infections. The decision to form hypnozoites is 3 1 made early in the liver infection and may already be set in sporozoites prior to invasion. To 3 2 better understand these early stages of infection, we undertook a comprehensive 3 3 transcriptomic and histone epigenetic characterization of P. vivax sporozoites. The salivary-3 4 gland sporozoite transcriptome is heavily composed of transcripts associated with functions 3 5 needed for early infection of the vertebrate host and development within hepatocytes. 3 6Through comparisons to recently published proteome data for the P. vivax sporozoite, our 3 7 study finds that although highly transcribed, these transcripts are not detectable as proteins 3 8 and may be regulated through translational repression; a finding we test for a small subset of 3 9 transcripts and proteins through immunofluorescent microscopy of sporozoites and liver 4 0 stages in humanized mice. We identify differential transcription between the sporozoite and 4 1 published transcriptomes of asexual blood-stages and mixed versus hypnozoite-enriched liver 4 2 stages. These comparisons point to multiple layers of transcriptional, post-transcriptional and 4 3 post-translational control that appear active in sporozoites and to a lesser extent hypnozoites, 4 4 but largely absent in replicating liver schizonts or mixed blood-stages. Common transcripts 4 5 up-regulated in sporozoites and hypnozoites compared to mixed (i.e., schizont) liver-stages 4 6identify genes linked to dormancy/persistence in bacteria, amoebae and plants. We also 4 7 characterise histone epigenetic modifications in the P. vivax sporozoite and explore their role 4 8 in regulating transcription. Collectively, these data support the hypothesis that the sporozoite 4 9 as a tightly programmed stage primed to infect the human host and identifies potential 5 0 mechanisms for hypnozoite-formation that may be further explored in liver stage models. 5 1 5 2

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Characterization of P. vivax blood stage transcriptomes from field isolates reveals similarities among infections and complex gene isoforms

Cited by 32 publications

References 40 publications

Analysis ofPlasmodium vivaxschizont transcriptomes from field isolates reveals heterogeneity of expression of genes involved in host-parasite interactions

Analysis ofPlasmodium vivaxschizont transcriptomes from field isolates reveals heterogeneity of expression of genes involved in host-parasite interactions

In vivogene expression analyses provide unique insights onP. vivaxgametocytogenesis and chloroquine response

Transcriptome and histone epigenome ofPlasmodium vivaxsalivary-gland sporozoites point to tight regulatory control and potential mechanisms for liver-stage differentiation

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