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Pfcerli2, a duplicated gene in the malaria parasite Plasmodium falciparum essential for invasion of erythrocytes as revealed by phylogenetic and cell biological analysis.

ABSTRACT: Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1, a related cytosolically exposed rhoptry bulb protein. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade after formation of the tight junction. PfCERLI2 knockdown was associated with inhibition of rhoptry antigen processing and a significant elongation of the rhoptries, suggesting that the inability of merozoites to invade is caused by aberrant rhoptry function due to PfCERLI2 deficiency. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Plasmodium Falciparum Homo Sapiens (human)

TISSUE(S): Blood Cell, Blood

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: Ghizal Siddiqui

LAB HEAD: Danny Wilson

PROVIDER: PXD028937 | Pride | 2021-11-15

REPOSITORIES: Pride

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Project description:Background: Considerable work has been carried out to understand the biology of the intermediate stages, the tachyzoite and bradyzoite, of Toxoplasma gondii in large part due to the accessible culturing methods for these stages. However, culturing methods for stages beyond the bradyzoite, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. We begin to unravel the molecular aspects of the merozoite stage focusing on gene expression. Results: To initiate this, we harvested merozoite parasites and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by combining it with a previously published study that generated array data for the oocyst, tachyzoite, and bradyzoite stages (Fritz HM et al. PLoS One, 2012). Principal component analysis highlights the unique profile of the merozoite samples, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen p30 (SAG1) and many dense granule proteins do not label merozoites, and our microarray data confirms that these genes are not expressed at this stage. Also, the expression for many rhoptry and microneme proteins is drastically reduced while the expression for many surface antigens is increased at the merozoite stage. Gene Ontology and KEGG analysis reveals that genes involved in transcription/translation and many metabolic pathways are upregulated at the merozoite stage, highlighting unique growth requirements of this stage. We also show that an upstream promoter region of a merozoite specific gene is sufficient to control stage specific expression at the merozoite stage. Conclusion: The merozoite represents the first developmental stage within the gut of the definitive host. Determining the correct conditions that coax the parasite into the merozoite stage in vitro may allow the parasite to complete sexual development. The data presented here describe the global gene expression profile of merozoite stage and the creation of transgenic parasite strains that will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate coccidian development. The ToxoGeneChip microarray was used to measure both tachyzoite and merozoite mRNA expression in the type II TgNmBr1 strain.

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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Pfcerli2, a duplicated gene in the malaria parasite Plasmodium falciparum essential for invasion of erythrocytes as revealed by phylogenetic and cell biological analysis.

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