Leishmania are parasitic protozoans that are responsible for the disease leishmaniasis. Leishmaniasis is endemic in some 88 countries worldwide and treatment options are limited and expensive where available. It has been reported that Leishmania secrete acid phosphatases (SAP 1 and SAP 2) that may play a role during host infection. Our previous studies using supernatant from L. tarentolae (13 day) cultures had detectable SAP activity that was substantially inhibited by decavanadate. Thus, SAP 1 and SAP 2 are of interest as potential drug targets. However, studies using these enzymes are limited by amounts of enzyme available. Thus, we have employed recombinant technology.The 1263 and 675 nucleotide genes for Leishmania major SAP 1 and SAP 2, respectively, were amplified from genomic DNA using PCR. After ligation into the pLEXSY Con 2 expression vector (Jena Bioscience) containing NTC for selection, the genes were transfected into competent Leishmania cells by electroporation (450V). The successfully transfected cells, containing either SAP 1 or SAP 2 gene with a 6X his-tag and antibiotic resistance to NTC, were grown either in liquid culture containing NTC, penicillin, and streptomycin or on BHI agar plates with the same antibiotics. Cultures with cells containing the genes were centrifuged at 1250 xg and supernatants were tested for the SAP 1 or SAP 2 enzyme via the ILAP� plate (SIGMA) which selectively binds the 6X his-tagged proteins. Enzyme activity was monitored (A405 nm) using para-nitrophenylphosphate as substrate in 0.5M acetate buffer at pH 4.8. Preliminary results indicate that the SAP1 and SAP 2 genes were successfully transfected into L. tarentolae, and that enzyme activity was higher than from control cells. Future work includes enzyme inhibition tests using select vanadium complexes with the recombinant SAP 1 and SAP 2 enzymes, and determining Km and Vmax Values.

Monitoring activity of recombinant Leishmania secreted acid phosphatases

246th National Meeting (2013)

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