Cloning and Expression of Recombinant Phospholipase A1 from Trypanosoma cruzi.

BELAUNZARÁN M.L; GIMÉNEZ, G; MARTI, R; LAMMEL, E. M; WILKOWSKY S. E; ISOLA, E.L.D

Huerta Grande, Córdoba, Argentina

Congreso; XXI Reunion Anual de la Sociedad Argentina de Protozoología; 2006

Sociedad Argentina de Protozoología

Previous results from our laboratory demonstrated in Trypanosoma cruzi the presence of Phospholipase A1 (Tc-PlaseA1) mainly as a membrane-bound activity and also secreted by the infective stages. After stimulation with purified Tc-PlaseA1, the host cell (Vero cells) lipid profile was modified generating second lipid messengers. Concomitantly host cell Protein Kinase C activation was observed, indicating that Tc-PlaseA1 could play a critical role on parasite-host cell interaction (Biocell, 2004; Acta Bioquímica Clínica Latinoamericana, 2005). We here report the identification of putative genes encoding Plase A1 from the Trypanosoma cruzi Genome Project. Primers were designed considering T. brucei PlaseA1 sequence and PCRs were performed. A unique band of 1050 bp was obtained, cloned into a TOPO-TA plasmid (Invitrogen) and expressed in BL21 Escherichia coli using a pet 28 (Novagen) expression plasmid. Polyclonal antibodies were generated in Balb C mice in order to obtain antisera against recombinat T. brucei PlaseA1 (kindly provided by Dr. Smith, Dundee University, UK) and native Tc-PlaseA1 (amastigote purified). The recombinant Tc-PlaseA1 was recognized by these antibodies in Western blot analyses confirming that the cloned enzyme was a PlaseA1. A unique band was identified in lysates from the different T. cruzi stages of around 40 kDa, in accordance with the MW previously determined for epimastigote PlaseA1 (Wainszelbaum et al, 2001). The identification and the recombinant production of Tc-PlaseA1 will allow us to determine its role in the early events of parasite invasion and in consecuence, its involvement in the pathogenesis of Chagas disease.   Supported by UBA and Agencia Nacional de Promoción Científica y Tecnológica.