Post-transcriptional regulation of Leishmania fitness gain

Background

Parasites of the genus Leishmania cause a spectrum of diseases known collectively as leishmaniases. These parasites cause over 12 million infections worldwide, making it one of the five most important parasitic diseases on the planet. One curious aspect of the molecular biology of Leishmania parasites is their plastic or unstable genome. These parasites can vary the copy number of individual genes and even chromosomes to adapt to different conditions. Some of these changes are linked to the ability to develop drug resistance or different tissue tropism in individual parasites. However, how do Leishmania parasites deal with such an unstable genome and the deleterious effects of gene dosage, because of the variations in the number of genes and chromosomes?

In this preprint, Piel and colleagues study the mechanisms of evolution of the L. donovani parasites to better understand how these parasites adapt. To induce experimental evolution, L. donovani parasites were grown in in vitro cell culture conditions for 190 generations (approximately 20 weeks in culture), where the medium is rich in nutrients and there is no immune system to evade. After adaptation, the authors compared these new culture-adapted parasites with parasites that were kept in culture for only 20 generations. This allowed them to observe how the parasites modified the expression of their genes after so many generations and if there were any costs to this experimental evolution. They looked at the number of chromosomes, the levels of mRNA and the amount of proteins to discern the different layers of regulation during adaptation and evolution.

 

Key findings

• L. donovani parasites that were adapted long term to in vitro culture show less ability to infect macrophages and to differentiate into the next stage of their life cycle. Conversely, parasites that are not adapted to in vitro culture are better at infecting macrophages and differentiating. This means that accelerated growth in vitro was gained with attenuation of virulence in vivo.
• Comparing the karyotypes of these two parasite strains, the authors observed aneuploidy, which is the presence of an abnormal number of chromosomes. In fact, L. donovani adaptation to cell culture was accompanied by triploidy of several chromosomes.
• When looking at the transcriptomes, the authors observed that culture-adapted parasites express more genes related to ribosome biogenesis and assembly, translation and non-coding RNA processing. However, this variation was not fully explained by the variation in the number of chromosomes, suggesting that there is another mechanism of gene regulation besides copy number variation.
• Also, the authors performed proteomics and observed that presence of some proteins did not correlate with either the gene copy number or the abundance of mRNA transcripts. This suggests that there is a new layer of regulation after mRNA post-transcriptional regulation.
• Parasite adaptation to cell culture also increased the levels of some small nucleolar RNAs. These non-coding RNAs have the ability to modify rRNAs and thus regulate translation. In fact, higher levels of rRNA pseudouridylation were observed in culture-adapted strains, suggesting a translational control mechanism during adaptation.

 

Importance

Leishmania parasites have two life cycle phases , one extracellular phase in the midgut of sand flies and another intracellular phase inside macrophages of mammals. As such, the adaptation to different environments is crucial for transmission and success of the infection. To better survive, parasites need to express the proper genes and proteins for the host and the tissue they are colonizing.

Importantly, Leishmania parasites cannot control protein levels by transcriptional control of their genes, as most eukaryotes. These organisms cannot simply turn on one gene at a time because their gene expression is more similar to bacterial operons, expressing multiple protein coding genes at the same time in long polycistronic units. These polycistronic transcripts are trans-spliced and polyadenylated to generate one mature mRNA transcript per coding gene. As such, in Leishmania, mRNA abundance is regulated by gene copy number (more gene copies, more expression) or post-transcriptionally (each mature mRNA is individually regulated).

This study on experimental evolution by Piel and colleagues, adds another layer of regulation, as small nucleolar RNAs change ribosomes by pseudouridylation, thus creating ribosomes that have a higher preference for certain mRNAs. In fact, rRNA pseudouridylation affects ribosomal-mRNA binding and translational fidelity in human and yeast cells. This way Leishmania parasites might be able to select which mRNAs are behind translated into proteins, avoiding the expression of deleterious proteins after chromosome duplication. It will be interesting to see how mRNAs are being selected for translation, and if messenger RNA modifications work in tandem with ribosomal RNA modifications to create this subset of translatable transcripts.

Genomic instability can be thought as a necessary part for adaptation and evolution for some organisms. In fact, cancer cells take advantage of their genomic instability as a driver for advantageous mutations and chromosomal rearrangements. Genomic instability therefore creates a genetically heterogenous population that has better chances of survival given a new stimulus (such as antibiotics, drugs, the immune system). This new manuscript explores an innovative way through which eukaryotic cells deal with genomic instability by modifying ribosomes, the components of the cell that are responsible for protein synthesis. This discovery might be useful to understand how other organisms deal with their own genomic instability and provide therapeutic options to deregulate it.

Tags: karyotype, leishmania, ncrna, proteomics, snorna, transcriptomics

doi: https://doi.org/10.1242/prelights.27991

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