Trypanosoma cruzi: cloning, expression and partial characterization of recombinant phospholipase A1

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

Noordwijkerhout, Holanda.

Congreso; FEBS Special Meeting. New concepts in lipidology; 2006

FEBS

Trypanosoma cruzi, the etiological agent of Chagas disease, is still a public health problem that affects 16 to 18 million people in the Americas (World Health Organization, 2002). This parasite has three main stages throughout its life cycle: the non infective epimastigote (found in the gut of the insect vector), the infective trypomastigote (able to invade nucleated cells) and the amastigote (intracellular replicating form), both found in the mammalian host. Previous results from our laboratory demonstrated the presence of Phospholipase A1 (Plase A1) in T. cruzi, mainly detected as a membrane-bound activity in the infective stages. As this enzyme was also secreted by these stages, the host cell lipid profile was analyzed after interaction with purified T. cruzi Plase A1 and significant changes were observed, with generation of second lipid messengers. Concomitantly host cell Protein Kinase C activation was observed. These results indicated that T. cruzi Plase A1 could play a critical role on parasite-host cell interaction (Biocell, 28: 3, 2004; Acta Bioquímica Clínica Latinoamericana, Spl. 3, 2005). We here report the identification of putative genes encoding T. cruzi Plase A1 from the Trypanosoma cruzi Genome Project. In order to clone and expressed these genes, primers were designed taking in consideration T. brucei Plase A1 published sequence and PCR was performed. The unique band of aproximatelly 1050 bp obtained was cloned into a TOPO-TA plasmid (Invitrogen) and expressed in BL21 Escherichia coli using a pet 28 (Novagen) expression plasmid. To confirm if the recombinant enzyme obtained was a Plase A1, polyclonal antibodies against both recombinat T. brucei Plase A1 (kindly provided by Dr. T. Smith, Dundee University, UK) and native T. cruzi Plase A1 (purified from amastigotes) were generated in Balb C mice. The antisera obtained were tested by Western blotting, using whole lysates from the different T. cruzi stages as antigens and a unique band of around 40 kDa was visualized, in accordance with the MW previously determined for epimastigote Plase A1 (Wainszelbaum et al, 2001). The recombinant T. cruzi Plase A1 was recognized by these antibodies in Western blot analyses. The identification and the recombinant obtaintion of this enzyme 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.