Calmodulin-like activity in mycobacteria.

Calmodulin-like activity has been reported for the first time in mycobacterial species, namely Mycobacterium tuberculosis BCG and M. smegmatis ATCC 14468. The activity was mainly located in the soluble fraction of the mycobacterial cells, Radioimmunoassay revealed maximum levels of calmodulin in young growing cells (early logarithmic phase of growth). Calmodulin-dependent phosphodiesterase activation assay revealed low activity (22%) of partially purified calmodulin either due to insufficient amount of calmodulin to activate phosphodiesterase or due to the presence of some factors interfering with the assay. Calmodulin antagonists, viz. trifluoperazine and phenothiazine, significantly inhibited the 32Pi incorporation into mycobacterial phospholipids. Similar inhibition was observed when EGTA (which removes calcium) was added to the medium. Significant inhibition of 32Pi incorporation in the presence of calmodulin antagonists suggested the involvement of calmodulin in mycobacterial phospholipid metabolism.

Serodiagnosis of tuberculosis using purified mycobacterial proteins.

With a view to detect specific M. tuberculosis infection, mycobacterial proteins were purified initially by ammonium sulphate precipitation followed by ion-exchange chromatography. Out of the three fractions, namely P1, P2 and P3 precipitated with increasing concentrations (0-25%, 26-65% and 66-100% respectively) of ammonium sulphate, P2 fraction was found to be more immunoreactive. P2 fraction proteins were further fractionated into five fractions by salt gradient using DEAE-cellulose DE-52 ion-exchange chromatography. Immunoreactivity against tuberculous patients sera of all the fractions was assessed using ELISA test. The last fraction (DE-V) eluted with high salt concentration was found to have a more specific immunoreactive set of proteins within the range of 55 kD to 67 kD molecular weight. Multiple non-specific proteins were distributed in all the other fractions. ELISA test using P2 fraction proteins against tuberculous patients sera showed significantly higher (p less than 0.01) titre even in the absence of any other bacteriological evidence. DE-V fraction of P2 proteins was found to have a significantly high specificity for detecting M. tuberculosis infection in clinically confirmed and suspected tuberculous patients indicating its application in the diagnosis of the disease.