• Host and Parasite Transcriptomic Changes upon Successive Plasmodium falciparum Infections in Early Childhood

      Bradwell, Katie R; Coulibaly, Drissa; Koné, Abdoulaye K; Laurens, Matthew B; Dembélé, Ahmadou; Tolo, Youssouf; Traoré, Karim; Niangaly, Amadou; Berry, Andrea A; Kouriba, Bourema; et al. (American Society for Microbiology, 2020-08)
      Children are highly susceptible to clinical malaria, and in regions where malaria is endemic, their immune systems must face successive encounters with Plasmodium falciparum parasites before they develop immunity, first against severe disease and later against uncomplicated malaria. Understanding cellular and molecular interactions between host and parasites during an infection could provide insights into the processes underlying this gradual acquisition of immunity, as well as to how parasites adapt to infect hosts that are successively more malaria experienced. Here, we describe methods to analyze the host and parasite gene expression profiles generated simultaneously from blood samples collected from five consecutive symptomatic P. falciparum infections in three Malian children. We show that the data generated enable statistical assessment of the proportions of (i) each white blood cell subset and (ii) the parasite developmental stages, as well as investigations of host-parasite gene coexpression. We also use the sequences generated to analyze allelic variations in transcribed regions and determine the complexity of each infection. While limited by the modest sample size, our analyses suggest that host gene expression profiles primarily clustered by individual, while the parasite gene expression profiles seemed to differentiate early from late infections. Overall, this study provides a solid framework to examine the mechanisms underlying acquisition of immunity to malaria infections using whole-blood transcriptome sequencing (RNA-seq).IMPORTANCE We show that dual RNA-seq from patient blood samples allows characterization of host/parasite interactions during malaria infections and can provide a solid framework to study the acquisition of antimalarial immunity, as well as the adaptations of P. falciparum to malaria-experienced hosts.
    • Successful Profiling of Plasmodium falciparum Gene Expression in Clinical Samples via a Custom Capture Array

      Stucke, Emily M; Dara, Antoine; Dwivedi, Ankit; Hodges, Theresa K; Ott, Sandra; Coulibaly, Drissa; Koné, Abdoulaye K; Traoré, Karim; Guindo, Bouréima; Tangara, Bourama M; et al. (American Society for Microbiology, 2021-11-30)
      var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche's SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var-like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum, this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.