Cell signaling inTrypanosoma cruzi: Cloning and expressing a Class I PI-3 kinase (TcPI3K)

GIMENEZ A; SCHOIJET A.; TORRES H.; FLAWIÁ M.; MACHADO E.; ALONSO G.

Tucumán, Argentina

Congreso; XLV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

The life cycle of Trypanosoma cruzi, protozoan parasite agent of Chagas’ disease, comprises three different morphological and functional forms: amastigotes, epimastigotes and trypomastigotes. Under stress conditions, intermediate forms between epi- and trypomastigotes are also observed. Previously, we have found that epimastigotes possess PI3K activity which is a component in the signaling pathway activated by hyperosmotic stress. In adition, this activity increases in the intermediate forms respect to epimastigotes. The aim of this work was to define if class I PI3K is functional in this parasite, through cloning and expressing a putative T. cruzi class I PI3K (TcPI3K) in BL 21 E. coli cells. Primer sequences were designed from Tc00.1047053510167.10 gene and PCR product was sequenced and compared with the original sequence found at GeneDB. We used pET22 b(+) expression vector and protein induction with IPTG was performed at 25ºC to obtain enough soluble protein for its purification. Southern and Northern blot analysis showed that TcPI3K is a single copy gene and its mRNA is expressed in epimastigote forms. Moreover, recombinant TcPI3K recovered from soluble fraction was assayed in order to determinate kinase activity, confirming a functional PI3K. Our results suggest for the first time, the presence of class I PI3K signaling pathway system in trypanosomas.