Discussion

We applied systems-wide analyses at genomic, transcriptomic, proteomic and metabolomic levels to compare bona fide tissue-purified amastigotes with culture-derived virulent promastigotes shortly after in vitro conversion with the aim to reveal L. donovani stage-specific expression profiles and gain new insight into post-transcriptional and -translational regulatory mechanisms. RNAseq and Gene Set Enrichment Analyses  identified significant increased transcript abundance for 387 genes in amastigotes with significant enrichment in RNA binding and RNA processing functions, and for 991 genes in promastigotes showing enrichment in proteolysis and glycolysis activities. Using label-free quantitative proteomics, a total of 3805 proteins were identified. Significant increases in stage-specific abundance were observed in splenic amastigotes and promastigotes for 561 and 1252 proteins, respectively. The comparison of transcript and protein abundance allowed us to distinguish three regulatory groups, including (i) genes with a stage-specific increase in both transcript and protein abundance, suggesting regulation by RNA stability, (ii) genes with unchanged or reduced RNA abundance but increased protein abundance in one of the stages, suggesting regulation on the level of translational efficiency or protein stability, and (iii) genes with stage-specific increase in transcript abundance but no change or reduction in protein abundance, suggesting regulation by protein degradation. This first systems-wide comparison of L. donovani amastigote and promastigote under physiological conditions shed new light on stage-specific mechanisms regulating Leishmania gene expression at transcript and protein levels.