RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

Abstract

The apicomplexan parasites Plasmodium spp. are the causative agents of malaria, a disease that poses a significant global health burden. Plasmodium spp. initiate infection of the human host by transforming and replicating within hepatocytes. This liver stage (LS) is poorly understood compared to other Plasmodium life stages, which has hindered our ability to target these parasites for disease prevention. We conducted an extensive transcriptome sequencing (RNA-Seq) analysis throughout the Plasmodium berghei LS, covering as early as 2 h postinfection (hpi) and extending to 48 hpi. Our data revealed that hundreds of genes are differentially expressed at 2 hpi and that multiple genes shown to be important for later infection are upregulated as early as 12 hpi. Using hierarchical clustering along with coexpression analysis, we identified clusters functionally enriched for important liver-stage processes such as interactions with the host cell and redox homeostasis. Furthermore, some of these clusters were highly correlated to the expression of ApiAP2 transcription factors, while showing enrichment of mostly uncharacterized DNA binding motifs. This finding indicates potential LS targets for these transcription factors, while also hinting at alternative uncharacterized DNA binding motifs and transcription factors during this stage. Our work presents a window into the previously undescribed transcriptome of Plasmodium upon host hepatocyte infection to enable a comprehensive view of the parasite’s LS. These findings also provide a blueprint for future studies that extend hypotheses concerning LS gene function in P. berghei to human-infective Plasmodium parasites. IMPORTANCE The LS of Plasmodium infection is an asymptomatic yet necessary stage for producing blood-infective parasites, the causative agents of malaria. Blocking the liver stage of the life cycle can prevent clinical malaria, but relatively less is known about the parasite’s biology at this stage. Using the rodent model P. berghei, we investigated whole-transcriptome changes occurring as early as 2 hpi of hepatocytes. The transcriptional profiles of early time points (2, 4, 12, and 18 hpi) have not been accessible before due to the technical challenges associated with liver-stage infections. Our data now provide insights into these early parasite fluxes that may facilitate establishment of infection, transformation, and replication in the liver.

Prion‐like proteins: from computational approaches to proteome‐wide analysis

“…Between the prion-like constituent functional domains, the most enriched ones were those related to nucleotide-binding, including the ubiquitous RRM, but also domains not detected in other organisms such as the ApiAP2 domain. Indeed, [118], playing crucial roles in gametocyte maturation [119], organ infection [120] and the development of host immunity [121]. Thus, the analysis uncovered the association of putative PrLDs with essential functions connected with Plasmodium life cycle regulation and its adaptation to the host.…”

“…Indeed, 50% of the ApiAP2 protein family was shown to bear a PrLD. These proteins are the central transcriptional regulators in Plasmodium parasites and the other Apicomplexa [117], playing crucial roles in gametocyte maturation [118], organ infection [119], and the development of host immunity [120]. Thus, the analysis uncovered the association of putative PrLDs with essential functions connected with Plasmodium life cycle regulation and its adaptation to the host.…”

The PfAP2‐G2 transcription factor is a critical regulator of gametocyte maturation

“…In another study, disruption of pbap2‐g2 also caused premature expression of liver stage and sporozoite stage genes during asexual development in red blood cells (Modrzynska et al., 2017). Moreover, a P. berghei liver‐stage (LS) transcriptome of P. berghei reported strong upregulation of pbap2‐g2 in early LS that is negatively correlated with the expression of liver stage‐specific transcripts (Toro‐Moreno et al., 2020). Together, these findings suggest that AP2‐G2 acts as a repressor in both the asexual and sexual stages and during early liver‐stage infection in P. berghei .…”

Analysis of Pir Gene Expression Across the Plasmodium Life Cycle

“…The 6h Liver samples from [34] were removed due to low read counts, also removed by the authors in the original publication. Additionally, a 2h Liver sample (SRR11142819) from [35], and two schizont samples (SRR3437888 and SRR3437912) from [36] were removed because hierarchical clustering showed that they were dissimilar to the other samples.…”