Single-cell RNA profiling of Plasmodium vivax-infected hepatocytes reveals parasite- and host- specific transcriptomic signatures and therapeutic targets

Abstract

The resilience of Plasmodium vivax, the most widely distributed malaria-causing parasite in humans worldwide, is attributed to its ability to produce dormant liver forms known as hypnozoites, which can activate weeks, months, or even years after an initial mosquito bite. The factors underlying hypnozoite formation and activation are poorly understood, as is the parasite's influence on the host hepatocyte. Here, we shed light on transcriptome-wide signatures of both the parasite and the infected host cell by sequencing over 1,000 P. vivax liver forms at single-cell resolution. We distinguish between replicating schizonts and hypnozoites at the transcriptional level, identifying key differences in transcripts encoding for RNA-binding proteins associated with cell fate. In infected hepatocytes, we show that genes associated with energy metabolism and antioxidant stress response were upregulated, and those involved in the host immune response downregulated, suggesting both schizonts and hypnozoites alter the host intracellular environment. The transcriptional markers in schizonts, hypnozoites, and infected hepatocytes revealed here pinpoint potential factors underlying dormancy and can inform drug targets against P. vivax liver-stage infection.

Plasmodium berghei-Mediated NRF2 Activation in Infected Hepatocytes Enhances Parasite Survival

“…Another recent transcriptome analysis on cells infected with Plasmodium vivax, a human infecting Plasmodium species, found enrichment of transcripts governed by NRF2 upon infection of primary human hepatocytes. Upregulation of genes controlled by NRF2 was observed in all infected cells, suggesting a broad importance of the transcription factor for parasite persistence and survival [14]. These transcriptome studies strongly support our findings that NRF2 is constitutively activated during Plasmodium infection of hepatocytes.…”

“…Primary mouse hepatocytes were infected with PbmCherry parasites (red). 6 hpi, cells were fixed and stained with either anti-UIS4 (magenta; upper panel) or anti-LC3 antibodies (magenta; middle panel) to visualize 14 Cellular Microbiology the PVM. p62 was stained using an anti-p62 antibody or an antibody specifically recognizing p62 phosphorylated at serine 351.…”

“…Importantly, several NRF2 target genes have recently been shown by single cell mRNA seq analysis to be upregulated upon P. berghei infection of hepatocytes [13]. Another transcriptome study in P. vivax-infected hepatocytes also identified NRF2 regulated expression as a pathway supporting parasite persistence suggesting an important and conserved function of this host cell transcription factor [14].…”

Systems biology of malaria explored with nonhuman primates

“…This type of research comes with many challenges, given the need to distinguish quiescent infected cells that have low metabolic activity from developing or newly activated forms [ 217 , 218 ]. Today, such culture systems, single-cell -omic technologies [ 219 ], and cellular imaging advances [ 220 , 221 ] are paving the way for using these model systems to understand P. vivax sporozoites [ 222 ] and host–parasite interactions in infected hepatocytes, including dormant and activated hypnozoites [ 223 ].…”

“…These feats have been groundbreaking, but—if P. vivax clinical cases continue to decline, as hoped—logistical challenges will be compounded that include having sufficient availability of patient infected-blood donors, mosquito insectary operations, and experts for establishing, infecting, and analyzing the data coming from hepatocyte cultures. Meanwhile, there is much yet to be learned about the biology of hypnozoites to effectively identify targets for drug intervention, and multi-omic approaches that can distinguish host and parasite targets will be essential [ 218 , 223 ]. With such knowledge and systems biological analyses, vaccination to eliminate hypnozoites, or at a minimum delay or reduce the number of relapses [ 225 ], may one day also become a reality, in addition to targeting the universal parasite developmental life cycle forms or stages known across all species as liver-stage forms (LSFs, or exoerythrocytic stages) and blood-stage forms (BSFs, or erythrocytic stages) (Fig.…”