Dataset Information

Overexpression of the HECT ubiquitin ligase PfUT prolongs the intraerythrocytic cycle and reduces invasion efficiency of Plasmodium falciparum.

ABSTRACT: The glms ribozyme system has been used as an amenable tool to conditionally control expression of genes of interest. It is generally assumed that insertion of the ribozyme sequence does not affect expression of the targeted gene in the absence of the inducer glucosamine-6-phosphate, although experimental support for this assumption is scarce. Here, we report the unexpected finding that integration of the glms ribozyme sequence in the 3' untranslated region of a gene encoding a HECT E3 ubiquitin ligase, termed Plasmodium falciparum ubiquitin transferase (PfUT), increased steady state RNA and protein levels 2.5-fold in the human malaria parasite P. falciparum. Overexpression of pfut resulted in an S/M phase-associated lengthening of the parasite's intraerythrocytic developmental cycle and a reduced merozoite invasion efficiency. The addition of glucosamine partially restored the wild type phenotype. Our study suggests a role of PfUT in controlling cell cycle progression and merozoite invasion. Our study further raises awareness regarding unexpected effects on gene expression when inserting the glms ribozyme sequence into a gene locus.

SUBMITTER: Jankowska-Dollken M

PROVIDER: S-EPMC6893019 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Overexpression of the HECT ubiquitin ligase PfUT prolongs the intraerythrocytic cycle and reduces invasion efficiency of Plasmodium falciparum.

Jankowska-Döllken Monika M Sanchez Cecilia P CP Cyrklaff Marek M Lanzer Michael M

Scientific reports 20191204 1

The glms ribozyme system has been used as an amenable tool to conditionally control expression of genes of interest. It is generally assumed that insertion of the ribozyme sequence does not affect expression of the targeted gene in the absence of the inducer glucosamine-6-phosphate, although experimental support for this assumption is scarce. Here, we report the unexpected finding that integration of the glms ribozyme sequence in the 3' untranslated region of a gene encoding a HECT E3 ubiquitin ...[more]

PMID: 31797898

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Project description:The sphingolipid pool is key regulator of vital cellular functions in Plasmodium falciparum a causative agent for deadly malaria. Erythrocytes, the host for asexual stage of Plasmodium, are major reservoir for Sphingosine-1-phosphate (S1P). Erythrocyte possesses Sphingosine kinase (SphK) that catalyzed its biosynthesis from sphingosine (Sph). Since, Plasmodium lacks SphK homologous protein it can be envisaged that it co-opts sphingolipids from both intraerythrocytic as well as extracellular pools for its growth and development. Herein, by sphingosine-NBD probing, we report that infected erythrocytes imports Sph from extracellular pool, which is converted to S1P and thereby taken by P. falciparum. Next, by targeting of the SphK through specific inhibitor N,N-Dimethylsphingosine DMS, we show a reduction in erythrocyte endogenous S1P pool and SphK-phosphorylation that led to inhibition in growth and development of ring stage P. falciparum. Owing to the role of S1P in erythrocyte glycolysis we analyzed uptake of NBD-Glucose and production of lactate in DMS treated and untreated plasmodium. DMS treatment led to decreased glycolysis in Plasmodium. Interestingly the host free Plasmodium did not show any effect on glycolysis with DMS treatment indicating its host-mediated effect. Further to understand the in-vivo anti-plasmodial effects of exogenous and endogenous erythrocyte S1P level, Sphingosine-1-phosphate lyase (S1PL) inhibitor (THI), S1P and SphK-1 inhibitor (DMS), were used in Plasmodium berghei ANKA (PbA) mice model. DMS treatment led to reduction of endogenous S1P conferred significant decrease in parasite load, whereas the plasma level S1P modulated by (THI) and exogenous S1P have no effect on growth of Plasmodium. This suggested erythrocyte endogenous S1P pool is important for Plasmodium growth whereas the plasma level S1P has no effect. Altogether, this study provides insight on cellular processes regulated by S1P in P. falciparum and highlights the novel mechanistically distinct molecular target i.e. SphK-1.

Project description:Plasmodium falciparum is the causative agent of the most burdensome form of human malaria, affecting 200-300 million individuals per year worldwide. The recently sequenced genome of P. falciparum revealed over 5,400 genes, of which 60% encode proteins of unknown function. Insights into the biochemical function and regulation of these genes will provide the foundation for future drug and vaccine development efforts toward eradication of this disease. By analyzing the complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of the HB3 strain of P. falciparum, we demonstrate that at least 60% of the genome is transcriptionally active during this stage. Our data demonstrate that this parasite has evolved an extremely specialized mode of transcriptional regulation that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium-specific functionalities, such as genes involved in erythrocyte invasion. The data reveal that genes contiguous along the chromosomes are rarely coregulated, while transcription from the plastid genome is highly coregulated and likely polycistronic. Comparative genomic hybridization between HB3 and the reference genome strain (3D7) was used to distinguish between genes not expressed during the IDC and genes not detected because of possible sequence variations. Genomic differences between these strains were found almost exclusively in the highly antigenic subtelomeric regions of chromosomes. The simple cascade of gene regulation that directs the asexual development of P. falciparum is unprecedented in eukaryotic biology. The transcriptome of the IDC resembles a "just-in-time" manufacturing process whereby induction of any given gene occurs once per cycle and only at a time when it is required. These data provide to our knowledge the first comprehensive view of the timing of transcription throughout the intraerythrocytic development of P. falciparum and provide a resource for the identification of new chemotherapeutic and vaccine candidates.

Dataset Information

Overexpression of the HECT ubiquitin ligase PfUT prolongs the intraerythrocytic cycle and reduces invasion efficiency of Plasmodium falciparum.

Publications

Overexpression of the HECT ubiquitin ligase PfUT prolongs the intraerythrocytic cycle and reduces invasion efficiency of Plasmodium falciparum.

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