Beneficiary 1 (IP) conducted 3 parallel experimental evolution experiments (EE1, EE2, EE3) using different splenic amastigote isolates as starting material to derive three independent promastigote populations. Applying comparative genomic analysis, we assessed population heterogeneity before and after adaptation and mapped distinct adaptive trajectories in over 50 clones derived from non-adapted (passage p2), short-term adapted (p20) and long-term adapted cultures (>p150). We assessed the role of translational control and co-translational mRNA degradation across the three EEs in convergent fitness gain observed between genetically distinct populations and identified signals of genomic adaptation in parasite exposed to different forms of environmental stress. We screened for fitness factors relevant for infection by conducting an in vivo screen in hamsters inoculated with hyper-evolvable parasite strains that were transfected with a cosmid library covering the entire Leishmania genome. Finally, we have identified the nuclear fusion protein GEX-1 as a first putative evolvability gene and established corresponding knock-out and add-back strains for future experimental evolution studies.
Beneficiary 2 (BIU) : The cycling of Leishmania between its hosts involve changes in temperature, pH and food resources. We obtained evidence that the ribosome plays a major role in this adaptation via changing rRNA modification. Single KO of a snoRNA by CRISPR-KO guiding a developmentally regulated pseudouridine generated specialized ribosomes. Based on CryoEM the structure of these ribosomes was changed affecting the translation of hundreds of mRNAs. We revealed two novel functions of snoRNAs (i) acting as chaperones during rRNA formation and (ii) serving as anti-sense regulators alike microRNAs that do not exist in these parasites. Such novel activities were not reported yet. We obtained promastigotes adapted to the cues that induce transformation between stages and found by DNA and RNA-seq that the parasites used for adaptation: (i) changing chromosomal ploidy, (ii) changes in gene copy number variation, (iii) activation of distinct polycistrons and (iv) activation of post-transcriptional regulation on mRNAs (v) changing rRNA modification, suggesting a complex regulated network specific for each adaptation.
Beneficiary 3: We analyzed large-scale nascent transcription, RNAseq, and genome sequencing data across several Trypanosomatid species to explore how these organisms control gene expression regulation. We found that these organisms adjust transcription at the level of gene clusters (known as polycistronic transcription units or PTUs), not individual genes. We showed that this tuning is active, common, and distinct from known post-transcriptional mechanisms of gene expression regulation. Further, we discovered a consistent drop in transcription output along the length of these clusters. Finally, we traced how the unusual polycistronic genome organization originated in the course of evolution, as Trypanosomatids diverged from free-living ancestors that lacked PTUs. Our work reveals hidden complexity in how these parasites regulate genes and adapt to their environments.
