Our Research
We study gene regulation at the post-transcriptional level in protozoa parasites.
Ribosome specialization in protozoa parasites
Ribosomes in general are viewed as constitutive macromolecular machines where protein synthesis takes place, however, this view has been recently challenged supporting the hypothesis of ribosome specialization and opening a completely new field of research. Recent studies have demonstrated that ribosomes are heterogenous in their nature and can provide another layer of gene expression control by regulating translation. Heterogeneities in ribosomal RNA and ribosomal proteins that compose them favor the selective translation of different sub-pools of mRNAs and functional specialization. While ribosome specialization is an exciting direction to discover another layer of gene expression regulation at the ribosomes, however, we are still at infancy to understand the functional specialization of ribosomes. This project investigates the fundamental concept of ribosome specialization in protozoa using Leishmania as a model organism. Leishmania is a great model to study ribosome specialization since it has a limited use of transcriptional control of gene expression comparing to other eukaryotic organisms. We hypothesize that ribosomes undergo a change in its composition during transformation of promastigotes (insect vector form) to amastigotes (mammalian form) and specialized ribosome components are essential to support selective translation of transcripts during differentiation. To test this hypothesis, we use an integrative strategy using polysome profiling followed by deep RNA-sequencing, proteomic analysis of ribosomes and CRISPR-Cas9 technology. We separate actively translated polysomes in sucrose gradients and analyze changes in protein composition of monosomes, light and heavy polysomes during stress/differentiation by proteomic approach, and identify transcripts engaged in translation using advances of next generation sequencing (NGS). We generate knock-out for specialized ribosome components using CRISPR-Cas9 gene editing and examine their role in survival in mammalian host using macrophage and mouse models for infection experiments.
Drug resistance in Leishmania
The second project is focused on the molecular mechanisms of drug resistance in Leishmania parasites. Specifically, we explore the role of translational reprogramming in the development of drug resistance. Current studies on drug resistance in Leishmania are mostly limited to the research at genomic level. However, changes at genomic level cannot explain all possible mechanisms of resistance and treatment failures without genetic-based resistance are very widespread. Our data demonstrate that Leishmania parasites undergo a dramatic reprogramming of mRNA translation during development of drug resistance. As a consequence, it leads not only to changes in proteome but also to lipidome and metabolome remodeling and contribute to drug resistance. We propose a novel model (Figure 1) that establishes translational control as a major driver of antimony drug resistance in Leishmania. In future directions gene editing knock-out screen will be performed to validate targets involved in drug resistance.
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Figure 1: Model of the translational regulation as a central driver of Leishmania’s antimony-resistant phenotypes.
A) Translational remodeling orchestrates a pre-emptive adaptation to drug challenges. B) Drug-resistant parasites activate a highly selective translation in response to the drug exposure.
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Our methods
We study the gene regulation through translatomic, proteomic, lipidomic and metabolomic approaches, complemented with basic molecular techniques. Our main research methods include: polysome profiling to study translatome and proteome changes and CRISPR-Cas9 gene editing to generate gene knock-outs and examine the gene function.
