Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous ge nes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginos a phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the na tive proteins in M. tuberculosis H37Rv has not been demonstrated. The objec tive of the present study was to demonstrate that native PLC-a is expressed in M, tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E, coil were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tub erculosis extracts. No bands were visible in M, tuberculosis culture supern atants or extracts from M. avium, M, bovis and M. smegmatis. A 550-bp DNA f ragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of beta -galactosidase activity. beta -Galactosidase activity was detected in M. smegmatis transf ormed with recombinant pJEM12 grown in vitro and inside macrophages. The pu tative promoter was active both in vitro and in vivo, suggesting that expre ssion is constitutive. In conclusion, expression of non-secreted native PLC -a was demonstrated in M, tuberculosis.