The RNA helicase DDX1 modulates intraerythrocytic development and gene expression in Plasmodium falciparum

Abstract

RNA helicases participate in multiple aspects of RNA metabolism, but the functions of most helicases in malaria parasites remain poorly understood. PfDDX1 is a conserved DEAD-box RNA helicase whose role during the asexual blood-stage development of Plasmodium falciparum has not been defined. Endogenous PfDDX1 fusion-protein lines were generated using CRISPR/Cas9-mediated genome editing. PfDDX1 expression and subcellular distribution were examined by Western blotting and immunofluorescence assays. A PfDDX1-HA-ddFKBP conditional knockdown line was used to assess parasite proliferation, intraerythrocytic developmental cycle (IDC) progression, merozoite production, and erythrocyte invasion. Comparative RNA sequencing was performed using morphologically stage-matched ring- and schizont-stage parasites. RNA immunoprecipitation sequencing (RIP-seq) was used to identify PfDDX1-associated RNAs. PfDDX1 contains conserved helicase-associated domains and showed a predominantly nuclear or perinuclear distribution during asexual blood-stage development. PfDDX1 was detected at the ring, trophozoite, and schizont stages, with the highest protein abundance in trophozoites. Conditional PfDDX1 knockdown was associated with earlier progression through the IDC and increased merozoite production per schizont beginning in the first cycle. Parasitemia increased cumulatively over successive cycles and became significantly higher in the fourth cycle, whereas merozoite invasion efficiency was unchanged. RNA-seq revealed stage-dependent changes in transcript abundance, with relatively limited changes at the ring stage and a broader upregulated response at the schizont stage. Schizont-stage upregulated genes were enriched for functions associated with merozoite development and invasion-related cellular structures, including rhoptries, micronemes, the inner membrane complex, and the cell surface. RIP-seq identified stage-associated PfDDX1-enriched RNAs, and integration with RNA-seq revealed a subset of PfDDX1-associated transcripts whose abundance increased following knockdown. PfDDX1 knockdown is associated with altered intraerythrocytic development, increased merozoite production, and stage-dependent changes in transcript abundance in P. falciparum . Although several invasion-associated transcripts were upregulated, erythrocyte invasion efficiency was not detectably altered. These findings support a role for PfDDX1 in RNA-associated regulation during asexual blood-stage development, while the precise transcriptional or post-transcriptional mechanisms require further investigation.