Leishmaniasis is a tropical disease caused by the Leishmania parasite. It has two stages: promastigote and amastigote, the latter being the cause of disease in the mammalian host. In Leishmania, a state of quiescence has been described in the amastigote. Quiescence is clinically relevant because it allows pathogens to persist in the host and reactivate disease years after clinical cure. It is important to understand the mechanisms that trigger quiescence to propose strategies for the control of the disease. Leishmania gene expression is noteworthy because genes are constitutively expressed with only 0.2% to 5% of different mRNA sequences between stages. Therefore, the regulation of gene expression must occur mainly at the post-transcriptional level. We hypothesize that ribosomes may play a discriminatory role to translate specific mRNAs. Recent studies suggest that the heterogeneity of ribosomal components, i.e. rRNA and ribosomal proteins, could play a role in the regulation of gene expression by selective association with different mRNA subgroups and/or performing extra-ribosomal functions. In yeast, the differential expression of paralogous ribosomal proteins was associated with the transition from a proliferation stage to quiescence. Therefore, we propose that the ribosomal fraction of quiescent amastigotes must present significant proteomic changes.
To obtain a population of mainly quiescent cells, amastigotes were cultured in conditions of nutrient-poor medium and hypoxia. Using flow cytometry, the quiescence state was monitored by analyzing a biosensor consisting of the insertion of GFP in the 18S ribosomal DNA locus. The decrease in 18S ribosomal RNA is a feature of quiescence found in other organisms and previously reported in Leishmania. We showed that the conditions of nutrients limitation and hypoxia allow us to obtain a quiescent population with 90.13% of cells that are rGFP negative. This group was compared with stationary promastigotes and the previous model of axenic amastigotes, which presents a heterogeneous population with 47.15% of rGFP negative.
The ribosomal fraction from the high percentage of quiescent amastigotes was compared with stationary promastigotes and analyzed by two-dimensional gels electrophoresis, using silver stain. Comparison of two dimensional gel electrophoresis patterns using Melanie 2D gel analysis software showed that 16 out of 220 corresponding individual spots presented a differential pattern. By comparing the isoelectric points and molecular weights of each spot with Uniprot database of ribosomal proteins of Leishmania mexicana. We were able to propose that two of them may be ribosomal proteins: RPL7/RPL12-like, that was underexpressed, and RPS4, that was overexpressed. Interestingly, the ribosomal protein RPL7/RPL12 decrease was reported in bacteria to be associated with low efficiency in protein production, a characteristic of the quiescent state. From the results mentioned above, we conclude that it is important to identify the translational machinery components or other proteins present in the ribosomal fraction by mass spectrometry to look for differences that explains the phenotype of Leishmania quiescent amastigotes.
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