The transcriptome of dormant and developing liver stages of P. cynomolgi
Background. Plasmodium vivax liver stage hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The simian parasite Plasmodium cynomolgi is closely related to P. vivax and one of only five malaria species that form hypnozoites, making it an excellent model for relapsing malaria. We have developed an in vitro P. cynomolgi liver stage culture platform as well as transgenic P. cynomolgi expressing GFP and mCherry for drug screening and biological studies on hypnozoites. Materials and Methods. Primary rhesus hepatocytes were infected in vitro with transgenic fluorescent P. cynomolgi parasites. Using fluorescence-activated cell sorting (FACS), we separated hypnozoite- and schizont- infected hepatocytes 6-7 days post infection. Amplified cDNA libraries from hypnozoites and developing liver stages were prepared and used for RNA-sequencing. Validation was performed by immunofluorescence (IFA) using antibodies generated against recombinant proteins from differentially expressed genes. In addition, sensitive fluorescent in situ hybridization was developed for P. cynomolgi liver stages and used for validation. Results. About 80% of the parasite’s predicted genes are expressed in liver schizonts. Many pathways previously known to be biologically important in other non-relapsing Plasmodium species are also robustly expressed in P. cynomolgi developing liver schizonts. In contrast, gene expression levels in hypnozoites are globally reduced and only a limited number of pathways are detected in the hypnozoite transcriptome. Interestingly, a few genes were upregulated in hypnozoites. Antibodies against the gene with the highest differential expression between hypnozoites and schizonts detected the protein in blood stage parasites but not in hypnozoites. We found that alternatively spliced transcripts were present in the hypnozoite population. Molecular analyses of other differentially expressed genes are being performed. Conclusion. This data set provides a wealth of information on gene expression in both liver schizonts and hypnozoites, that can be exploited for vaccine and drug development, as well as for further biological studies on hypnozoites.