Nutritional stress and intracellular traffic in Tripanosomatids

GUILLERMO D. ALONSO; TAMARA STERNLIEB; ALEJANDRA C. SCHOIJET

Santa Fe

Congreso; XXVIII Reunión Anual de la Sociedad Argentina de Protozoología y Enfermedades Parasitarias.; 2016

Sociedad Argentina de Protozoología y CURSO - SIMPOSIO Internacional de Biología Celular y Molecular de la Enfermedad de Chagas

Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. This is highly conserved across a taxonomically diverse range of eukaryotes including parasitic protists belonging to the family Trypanosomatidae; however, few proteins implicated in this process have been characterized so far in these parasites. Moreover, it has been shown that autophagy is involved in Trypanosoma cruzidifferentiation and thus might have a role in pathogenicity. The phosphatidylinositol 3-kinase (PI3K) Vps34 is a protein that plays an important role in many processes such as vesicular traffic, endocytosis and autophagy. This protein is found in yeast and mammalian forming a complex with the kinase Vps15. This kinase is postulated to be necessary for Vps34 activity, but the role of this protein is not well elucidated. Previously in our laboratory, we have identified the first PI3K in T. cruzi, named TcVps34, which plays a role in vital processes for the parasite survival such as osmoregulation, vesicular acidification, and vesicular traffic. In this work we report the cloning and characterization of both TcVps15 in T. cruzi and TbVps15 in T. brucei. We demonstrated on one side that TcVps15 is involved in autophagy in T. cruziby co-localization studies with the autophagosome marker TcAtg8 and by using the autofluorescence drug monodansylcadaverine (MDC). In addition, we found that TcVps15-K219D (a catalytic domain mutant version of TcVps15) -overexpressing parasites, showed reduced protein kinase activity when compared with the overexpression of the wild type protein, are less efficient modulating the lipid kinase activity of TcVps34 and displayed lower autophagy levels than those observed when wild type TcVps15 is overexpressed. Regarding TbVps15, the orthologue in T. brucei, we demonstrated that the knockdown of this gene in procyclic parasites produces a severe growth defect, with a block of cytokinesis, accompanied by a variety of morphological abnormalities. Moreover, cells induced with tetracycline also showed a significant reduction of transport of Concanavalin A to the lysosome. Overall, our results reveal for the first time a role of TcVps15 in autophagy in T. cruziwhereas initially we demonstrated that TbVps15 has a role in intracellular traffic in T. brucei.