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Transcriptome Analysis Reveal Molecular Targets of Erythrocyte Invasion Phenotype Diversity in Natural Plasmodium falciparum Isolates from Cameroon.

ABSTRACT: Further understanding of the molecular mediators of alternative RBC invasion phenotypes in endemic malaria parasites will support malaria blood stage vaccine or drug development. This study investigated the prevalence of SA-dependent and SA-independent RBC invasion pathways in endemic P. falciparum parasites from Cameroon and compared the schizont stage transcriptomes in these two groups with the goal of uncovering the wider repertoire of transcriptional variation associated with the use of alternative RBC invasion pathway phenotypes. Two-colour flow cytometry-based invasion-inhibition assay against RBCs treated with neuraminidase, trypsin and chymotrypsin and deep RNA sequencing of schizont stages harvested in the first ex-vivo replication cycle in culture were employed in this investigation. RBC invasion phenotypes were determined for 63 isolates from asymptomatic children with uncomplicated malaria. Approximately 80% of the isolates invaded neuraminidase-treated but not chymotrypsin-treated RBCs, representing sialic acid (SA)-independent pathways of RBC invasion. The schizont transcriptome profiles of 16 isolates with invasion phenotypes revealed a total of 5136 gene transcripts, with 85% of isolates predicted at schizont stages. Two distinct transcriptome profile clusters belonging to SA-dependent and SA-independent parasites were obtained by data reduction with principal component analysis. Differential analysis of gene expression between the two clusters implicated in addition to the well characterized adhesins, the up-regulation of genes encoding proteins mediating merozoite organelle discharges and several conserved, virulent, merozoite associated and exported proteins. The latter majority of which have been shown to have structural and physiological relevance to RBC surface remodelling and immune evasion in malaria and thus, have potential as anti-invasion targets.

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE264687 | GEO | 2025/01/22

REPOSITORIES: GEO

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Project description:Ubiquitylation is a common post translational modification of eukaryotic proteins. Protein ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite in the asexual blood stage cycle of the human malaria parasite, Plasmodium falciparum (Pf). Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified. 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor, MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We constructed a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite’s elaboration of a new food vacuole.

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Transcriptome Analysis Reveal Molecular Targets of Erythrocyte Invasion Phenotype Diversity in Natural Plasmodium falciparum Isolates from Cameroon.

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