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 genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa 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 native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective 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. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis

Purification and characterization of phospholipase C of Salmonella gallinarum.

Phospholipase C was isolated from an outbreak strain of Salmonella gallinarum with ciprofloxacin extraction, dialysis, gel filtration, ion exchange chromatography and chromatofocussing. Purified phospholipase C (mol wt. 65 KDa; isoelectric point, pI 3.5) was resistant to pasteurization, stomach enzyme (pepsin), bacterial protease and lipase but lost its activity on trypsin and chymotrypsin treatment. It was sensitive to pH > or = 8.0. It was haemolytic, embryotoxic, enterohaemorrhagic, lethal to birds, cytotoxic to Vero and MDBK cells, dermonecrotoxic in rabbit and antigenically active protein. Antisera raised against purified phospholipase C neutralized its all biological activities and agglutinated the producer Salmonella strains. Serologically it was proved similar to phospholipase C of Klebsiella pneumoniae and S. weltevreden. Fluorescent antibody technique (FAT) was standardized to detect phospholipase producer strains.

Distribution of phospholipase C isozymes in normal human lung tissue and their immunohistochemical localization

Phospholipase C(PLC) plays a central role in signal transduction and it is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC identified and cloned. However, there are no report of PLC distribution in human lung tissue or their significances in pulmonary diseases. Presence of various PLC isozymes in normal human lung tissue was studied from surgical specimens. PLC isozymes in tissue extracts of the lung were partially purified by successive chromatographic steps on heparin-sepharose CL-6B conventional and TSKgel heparin-5PW HPLC columns and their activities were assayed. PLC activity peaks identified in the chromatography were immunoblotted with specific antibodies against ten known mammalian PLC isozymes(PLC-beta 1-4, -gamma 1-2, and -delta 1-4). In addition, immunohistochemical staining of the lung tissue was performed to determine subcellular and histological localization of PLC isozymes. The results indicate that normal human lungs contain beta 1, beta 3, gamma 1, and delta 1, isozymes of PLC. The order of amount present in the lung tissue was PLC-delta 1 > gamma 1 >beta 1 >> beta 3, in descending order. On immunohistochemistry, PLC-gamma 1 was most widely distributed and was present in bronchiolar epithelium, in type I and type II pneumocytes as well as in fibroblasts of the interstitial tissue. PLC-delta 1 was present in the cytoplasm of the bronchiolar epithelium whereas PLC-beta 1 was localized to the apical membranous portion of the same epithelium. PLC-beta 3 was seen in the nucleus of the respiratory and alveolar lining epithelium as well as in the nucleus of lung fibroblasts.