Genome-wide analysis of strand-specific transcription and DNA methylation in Plasmodium falciparum severe malaria.

INTRODUCTION

Gene expression and regulation are central to the survival and adaptation of Plasmodium falciparum. However, the interplay between transcriptional activity, Natural Antisense Transcripts (NATs), and DNA methylation in clinical isolates associated with different malaria manifestations remains poorly understood.

METHODS

Here, we present an integrated, strand-specific transcriptome and methylome analysis of P. falciparum derived from patients with uncomplicated malaria (UNC), hepatic dysfunction (HD), and cerebral malaria (CM), using custom, strand-aware microarrays, and directional short-read RNA sequencing. We also performed long-read Nanopore sequencing to simultaneously identify 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) modifications in a subset of these isolates.

RESULTS

We identified widespread NATs expressed in distinct disease manifestations. Transcriptional regulation in P. falciparum appears to require an expression flux in which coordinated S-AS expression occurs in the nucleus, whereas mitochondrial genes exhibit a high functional demand with limited antisense control. NATs complementary to rRNA species reveal a potential regulatory role for rRNA stability in parasites. We report novel associations between mitochondrial respiration and disease severity. Despite globally low methylation levels, intragenic methylation is consistently associated with transcription, with the enrichment of differentially methylated events within exons.

DISCUSSION: Together, we provide the first integrated view of DNA methylation and global transcription in clinical isolates of P. falciparum, elucidating a multilayered regulatory mechanism of parasite adaptation in the host, paving the way for disease-specific, informed antimalarial approaches.