Identification, cloning and expression of a novel Phospholipase A from Leishmania braziliensis.

BELAUNZARÁN, ML; VELANDIA, A; LAMMEL, EM; GIMÉNEZ, G; BOTT, EMANUEL; BARBIERI, MA; ISOLA, ELD

Puerto Madryn, Chubut

Otro; XLVI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010

Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

We have reported in Trypanosoma cruzi infective stages the presence of Phospholipase A1 (PLA1), a membrane and secreted activity that modified Vero cells lipid profile and activated PKC, suggesting its involvement in the signaling events of parasite invasion (Belaunzarán et al, 2007). To extend these studies to Leishmania braziliensis, we here describe the cloning and expression of PLA from this parasite. We identify in the genome database TriTrypDb (http://tritrypdb.org/tritrypdb/) 2 putative genes with lipase motif GXSXG: LbrM33_V2.1730 and LbrM31_V2.2750. As the first codifies for a putative precursor, LbrM31_V2.2750 was chosen. Primers were designed and PCR was done using genomic DNA of the reference clone MHOM/BR/75M2904. A unique band (1129 bp) was obtained and cloned into pGEMT vector, sequence was corroborated and then cloned into pET30a and pGEX-6p1 plasmids. Expression was assayed at 21-37ºC and 0.05-1mM IPTG. The best yields were obtained with pET30a in BL21C43(DE3)pLysS cells at 1mM IPTG, 37°C, 3 hs. Protein identity was confirmed by Western blot with anti-T. cruzi and anti-T. brucei PLA1 antibodies. Enzyme activity was assay with 14C Phosphatidylcholine confirming that the expressed gene has PLA activity. Further studies will allow us to determine the role of PLA in L. braziliensis biology as well as its potential pathogenic effects. Supported by FONCYT, CONICET and NIH.