ISOLATION OF A CONTACT-DEPENDENT HEMOLYSIN FROM MYCOBACTERIUM-TUBERCULOSIS

Contact-dependent haemolytic activity was observed with cells of Mycob acterium tuberculosis H(37)RV 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. tubercu losis H(37)Rv was subsequently used for the isolation of haemolysin. H aemolytic activity was retained in the cell debris even after sonicati on of the cells and treatment with Tween 80 and lysozyme. Solubilisati on of haemolysin was possible only after the cell debris was washed wi th ethanol 70% and then treated with Tween 80 0.1%, The haemolysin thu s obtained showed a micellar M(r) of >200 000 by gel-filtration on Sep hadex G-200 and a subunit M(r) of 66 000 by SDS-PAGE, It was sensitive to trypsin but stable when heated at 60 degrees C for 10 min, Polyclo nal serum raised in rabbits against the haemolysin neutralised the hae molytic activity, The N-terminal amino-acid sequence of the 66-kDa sub unit of haemolysin showed identity with TB66, the 66-kDa secretory pro tein of M. tuberculosis, and 30% homology with the haemolysin A precur sor of Vibrio cholerae. Phosphatidylglycerol inhibited lysis of sheep erythrocytes by the haemolysin and is probably the receptor for the ha emolysin. Haemolysin not only lysed erythrocytes, but was also cytotox ic to human lung cells, It appears that, among the members of the M. t uberculosis complex, the cell-bound contact-dependent haemolysin/cytol ysin is restricted to M. tuberculosis and it may be associated with th e pathogenesis of M. tuberculosis.