ABSTRACT: Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening falciparum malaria in African children. Despite this clear protection, the molecular mechanisms by which HbAS confers these protective phenotypes remain incompletely understood. As a forward genetic screen for aberrant parasite transcriptional responses associated with parasite neutralization in HbAS red blood cells (RBCs), we performed comparative transcriptomic analyses of Plasmodium falciparum in normal (HbAA) and HbAS erythrocytes during both in vitro cultivation of reference parasite strains and naturally occurring P. falciparum infections in Malian children with HbAA or HbAS. During in vitro cultivation, parasites matured normally in HbAS RBCs, and the temporal expression was largely unperturbed of the highly ordered transcriptional program that underlies the parasite's maturation throughout the intraerythrocytic development cycle (IDC). However, differential expression analysis identified hundreds of transcripts aberrantly expressed in HbAS, largely occurring late in the IDC. Surprisingly, transcripts encoding members of the Maurer's clefts were overexpressed in HbAS despite impaired parasite protein export in these RBCs, while parasites in HbAS RBCs underexpressed transcripts associated with the endoplasmic reticulum and those encoding serine repeat antigen proteases that promote parasite egress. Analyses of P. falciparum transcriptomes from 32 children with uncomplicated malaria identified stage-specific differential expression: among infections composed of ring-stage parasites, only cyclophilin 19B was underexpressed in children with HbAS, while trophozoite-stage infections identified a range of differentially expressed transcripts, including downregulation in HbAS of several transcripts associated with severe malaria in collateral studies. Collectively, our comparative transcriptomic screen in vitro and in vivo indicates that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Because HbAS consistently protects from severe P. falciparum, modulation of these responses may offer avenues by which to neutralize P. falciparum parasites. IMPORTANCE Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening malaria, yet the molecular mechanisms that underlie HbAS protection from severe malaria remain incompletely understood. Here, we used transcriptome sequencing (RNA-seq) to measure the impact of HbAS on the blood-stage transcriptome of Plasmodium falciparum in in vitro time series experiments and in vivo samples from natural infections. Our in vitro time series data reveal that, during its blood stage, P. falciparum's gene expression in HbAS is impacted primarily through alterations in the abundance of gene products as opposed to variations in the timing of gene expression. Collectively, our in vitro and in vivo data indicate that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Due to the persistent association of HbAS and protection from severe disease, these processes that are modified in HbAS may offer strategies to neutralize P. falciparum.
