Fig 1.
Production of sporozoites and in vitro liver culture infection.
Rhesus macaques (1) are infected with blood stage parasites (A) of P. cynomolgi or P. knowlesi. Blood is drawn to feed mosquitoes and harvest the sporozoites (B) used in the in vitro infection of cultured rhesus hepatocytes (2). Upon infection of primary hepatocytes, sporozoites give rise to developing liver stages and hypnozoites in P. cynomolgi and developing liver stages only in P. knowlesi. Primary hepatocytes used in in vitro cultures are freshly harvested from an independent rhesus macaque each time (three biological replicates).
Fig 2.
Experimental set-up for metabolomics.
Samples for metabolomics were obtained from P. cynomolgi or P. knowlesi infected and uninfected in vitro liver cultures. Parasites were cultures in three conditions: standard (no drug treatment), Atovaquone (ATQ) and PI4K-treated. The treatment with Atovaquone started at d0 after sporozoite infection, while the treatment with PI4K started at day 1. Cultures were treated for 5 consecutive days. The rationale for using two drugs is briefly presented. The sampling strategy is shown in parallel with the culture read-out at the time of sampling. The downstream workup of the samples is shown prior to the metabolomics.
Table 1.
Total metabolites detected by category in the three P. cynomolgi-infected biological replicated and in the single P. knowlesi-infected replicate.
Fig 3.
(A) Venn diagram of significantly up- or down-regulated metabolites. A Venn diagram of the number of significantly up- or down-regulated metabolites detected in the different biological replicates is presented. A comparison between significantly up- or down-regulated metabolites in P. cynomolgi samples and in P. knowlesi samples is also shown. (B) Venn diagram of the selected potentially hypnozoite-infection specific metabolites. A Venn diagram of the selected potentially hypnozoite-infection specific metabolites is presented showing in which biological replicates they were found. Some selected metabolites (*), while being found only in one biological replicate have been detected multiple times and were found to be always statistically significant or highly statistically significant at late timepoints indicative of the enrichment with hypnozoites in the cultures.
Table 2.
Metabolites likely to be hypnozite infection specific.
Table 3.
Detection of potentially hypnozoite-associated candidate metabolites across replicates.
Table 4.
Predicted structures for seven candidate metabolites initially lacking identification.