EIF2αphosphorylation is regulated in intracellular amastigotes for the generation of infectiveTrypanosoma cruzitrypomastigote forms

Abstract

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNA met to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2α T169A or with an endogenous CRISPR/Cas9-generated eIF2α T169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2α T169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.

Transcriptomic analysis of N-terminal mutated Trypanosoma cruzi UBP1 knockdown underlines the importance of this RNA-binding protein in parasite development

“…An important finding from the first RNA-Seq transcriptome and translatome of this parasite was that a key factor in the control of the gene expression profiles during T. cruzi differentiation is translation regulation [5]. The molecular mechanisms that could explain this regulation have been previously described by us and other authors [8,19,20].…”

Transcriptomic analysis of N-terminal mutatedTrypanosoma cruziUBP1 knockdown underlines the importance of this RNA-binding protein in parasite development

“…The knockout of TcK2 protein kinase, which phosphorylates T. cruzi eIF2α, showed lower release of cell-derived trypomastigotes than the control [47]. Moreover, the overexpression of eIF2α in amastigotes increases translation levels and decreases differentiation to infective trypomastigotes [20]. In the same line, overexpression of UBP1 in a replicative stage decreases the abundance of numerous transcripts coding for ribosomal proteins, probably withdrawing translation global levels and triggering an infective-type expression profile.…”

“…These results highlight a substantial overlap between UBP1-OE and epimastigote stationary phase DEGs, supporting the notion that UBP1-overexpressing parasites exhibit characteristics of a transitional parasite form between epimastigotes and metacyclic trypomastigotes in the insect host. The transition from replicative to infective-type cell also requires a decrease in the rate of translation, and we currently know that this step is regulated by the expression and phosphorylation levels of the initiation factor eIF2α [20]. The knockout of TcK2 protein kinase, which phosphorylates T. cruzi eIF2α, showed lower release of cell-derived trypomastigotes than the control [47].…”

Trypanosoma cruziVDU deubiquitinase mediates surface protein trafficking and infectivity

“…Metacyclic trypomastigotes were purified from the culture supernatant by ion-exchange chromatography using 2-(diethylamino) ethyl ether cellulose (DEAE-cellulose) columns [98]. T. cruzi tissue-culture derived trypomastigotes (TcT) were obtained from supernatants of LLC-MK2 cells (Rhesus monkey kidney epithelial cells, ATCC CCL-7), or U-2OS cells (Human osteosarcoma cells, Banco de Células do Rio de Janeiro) maintained as described [99]. The cells were infected with metacyclic-trypomastigotes, or with trypomastigotes released from infected cells.…”

“…The parasites were incubated for 24 h with the cells, the wells were PBS washed and incubated with fresh medium for further 48 h hours. The number of intracellular amastigotes were quantified as above as described previously [99]. In parallel, TcT egress was determined by counting the number of parasites released in the culture supernatant.…”