Investigation of Stage-Specific Distinction in the Leishmania mexicana Translational Machinery

Leishmaniasis is a parasitic infection found in tropical, subtropical and southern European regions. The Leishmania life cycle is digenic and transitions between the sandfly and human hosts, which have distinct environmental conditions. Near-constitutive transcription in kinetoplasts renders gene regulation overwhelmingly post-transcriptional, placing heightened regulatory emphasis on RNA binding protein function. Modulation of translation plays a major role in parasite survival and adaptation. Our knowledge of the molecular mechanisms of translation in Leishmania remains limited, and it is unknown to what extent the composition of the translational machinery varies throughout the parasite life cycle Therefore, to expand this knowledge, enrichment strategies for Leishmania ribosomes were evaluated in order to generate samples suitable for mass spectrometry analysis of stage-specific ribosome-associated proteins. Immunoprecipitation was employed using antibodies against ribosomal protein P0 (also known as uL10) to capture the ribosomes on beads, which were subsequently analysed by mass spectrometry. Polysome profiling was also performed to identify the ideal conditions for RNA digestion to ensure the enrichment of ribosomes, to isolate both translating and non-translating ribosomes from L. mexicana promastigote stages and specifically exclude any RNA binding proteins (RBPs) interacting with an mRNA mid-translation. Further optimisation is required to realise this strategy. Bioinformatic analyses of mass spectrometry data have enabled comparisons of the translational machinery between the procyclic (PCF) and the metacyclic promastigotes (META). This has highlighted differentially abundant proteins involved in specific processes, which are key to cell regulation, translation and differentiation. It also illustrates how post-translational modifications (PTM) promote parasitic adaptation; specifically, protein kinase activity and phosphoregulation in META differentiation and metacyclogenesis. Key findings from our mass spectrometry include the enrichment of CRK9, RDK2 kinases and serine/threonine phosphatases in META.