Isolation of a contact-dependent haemolysin from Mycobacterium tuberculosis

Abstract

Contact-dependent haemolytic activity was observed with cells of Mycobacterium tuberculosis H37Rv and M. tuberculosis H37Ra, but not with those of M. bovis, M. bovis BCG and M. africanum. Culture filtrates of all these strains did not exhibit any haemolytic activity. M. tuberculosis H37Rv was subsequently used for the isolation of haemolysin. Haemolytic activity was retained in the cell debris even after sonication of the cells and treatment with Tween 80 and lysozyme. Solubilisation of haemolysin was possible only after the cell debris was washed with ethanol 70% and then treated with Tween 80 0.1%. The haemolysin thus obtained showed a micellar Mr of >200000 by gelfiltration on Sephadex G-200 and a subunit Mr of 66000 by SDS-PAGE. It was sensitive to trypsin but stable when heated at 60°C for 10 min. Polyclonal serum raised in rabbits against the haemolysin neutralised the haemolytic activity. The N-terminal amino-acid sequence of the 66-kDa subunit of haemolysin showed identity with TB66, the 66-kDa secretory protein of M. tuberculosis, and 30% homology with the haemolysin A precursor of Vibrio cholerae. Phosphatidylglycerol inhibited lysis of sheep erythrocytes by the haemolysin and is probably the receptor for the haemolysin. Haemolysin not only lysed erythrocytes, but was also cytotoxic to human lung cells. It appears that, among the members of the M. tuberculosis complex, the cell-bound contact-dependent haemolysin/cytolysin is restricted to M. tuberculosis and it may be associated with the pathogenesis of M. tuberculosis.

Necrosis of Lung Epithelial Cells during Infection withMycobacterium tuberculosisIs Preceded by Cell Permeation

“…As with other bacteria that induce host cell necrosis, such as Legionella pneumophila (22,55), it is likely that numerous complex events contribute to the M. tuberculosis necrotic phenotype of epithelial cells, including invasion, intracellular growth, differential gene expression, and permeation of the membrane. M. tuberculosis infection of host cells could contribute to this necrosis through secretion of pore-forming molecules, such as previously described hemolysins, or other putative cytotoxic molecules including broad-spectrum esterases or phospholipases (4,11,15,19,21,26,40,52).…”

Characterization of a haemolytic phospholipase A2 activity in clinical isolates of Campylobacter concisus

“…Sonication of cells did not release the haemolysin from cell debris and treatment with lysozyme and detergent was needed for effective haemolysin extraction, indicating the presence of a membrane-bound haemolysin (Deshpande et al, 1997). Pronase-treated cell suspensions, CHEs and HEs showed negligible haemolytic activity in comparison with control untreated samples, when tested using the haemolysin assay, indicating that the haemolysin was proteinaceous in nature.…”

Mycobacterium tuberculosis Can Utilize Heme as an Iron Source

“…Contact-dependent hemolytic activity has been reported in Mycobacterium avium (24) and M. tuberculosis (10). However, the molecular determinants of the M. tuberculosis hemolytic activity are not entirely clear.…”