Ectopic overexpression of <i>Plasmodium falciparum</i> DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development

Alba domain-containing proteins are ubiquitously found in archaea and eukaryotes. By binding to either DNA, RNA, or DNA:RNA hybrids, these proteins function in genome stabilization, chromatin organization, gene regulation, and/or translational modulation. In the malaria parasite <i>Plasmodium falciparum</i>, six Alba domain proteins PfAlba1-6 have been described, of which PfAlba1 has emerged as a "master regulator" of translation during parasite intra-erythrocytic development (IED). Given that a tight control of gene expression is especially important during IED, when malaria pathogenesis manifests, in this study, we focus on three other <i>P. falciparum</i> Albas, PfAlba2-4. Because genetic manipulation of the genomic loci of PfAlba2-4 was unsuccessful, we overexpressed each of these proteins from an episome under a strong constitutive promoter. We observed that PfAlba2 or PfAlba3 overexpression strongly reduced parasite growth and impacted IED stage transitions. In contrast, elevated levels of PfAlba4 were well-tolerated by the parasite. In keeping with this, differential gene expression analysis using RNA-seq of PfAlba2 or PfAlba3 overexpressing strains revealed a significant misregulation of mRNAs encoding virulence factors, such as those related to erythrocyte invasion; a general repression of <i>var</i> gene expression was also apparent. PfAlba4 overexpression, on the other hand, did not significantly perturb the steady-state transcriptome of IED stages and appeared to enhance <i>var</i> mRNA levels. Moreover, distinct sets of genes were targeted by each PfAlba for regulation. Taken together, this study highlights the nonredundant roles of PfAlba proteins in the <i>P. falciparum</i> IED, emphasizing their importance in subtelomeric chromatin biology and RNA regulation.IMPORTANCEThe malaria parasite <i>Plasmodium falciparum</i> tightly controls the expression of its genes at the epigenetic, transcriptional, post-transcriptional, and translational levels to synthesize essential proteins, including virulence factors, in a timely and spatially coordinated manner. A family of six proteins implicated in this process is called PfAlba, characterized by the presence of the DNA-, RNA- or DNA:RNA hybrid-binding Alba domain. To better understand the cellular pathways regulated by this protein family, we overexpressed three PfAlbas during <i>P. falciparum</i> intra-erythrocytic growth and found that high levels of PfAlba2 and PfAlba3 were detrimental to parasite development. This was accompanied by significant changes in the parasite's transcriptome, either with regards to mRNA steady-state levels or expression timing. PfAlba4 overexpression, on the other hand, was well-tolerated by the parasite. Overall, our results delineate specific pathways targeted by individual PfAlbas for regulation and link PfAlba2/PfAlba3 to mutually exclusive expression of the virulence-promoting surface antigen PfEMP1.