Haemolysin from Mycobacterium avium complex isolates from AIDS patients.

Cell-bound haemolytic activity was observed in isolates of Mycobacterium avium complex (MAC) from AIDS patients. M. avium type strains showed negligible activity. None of the culture supernates exhibited any haemolytic activity. Zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulphonate (CHAPS) was used to extract haemolysin from ethanol-treated M. avium complex strain 101 (MAC101) cells. Haemolysin was isolated from CHAPS extract (CE) by metal affinity chromatography and identified as a 32-kDa protein by polyclonal antibodies raised against M. tuberculosis haemolysin. Treatment of CE with trypsin resulted in reduction of haemolytic activity, whereas heating at 100 degrees C for 10 min did not affect its activity. A similar 32-kDa haemolysin was extracted from cells of M. avium K128 which was isolated from a monkey infected with simian immunodeficiency virus (SIV). The haemolysin produced by M. avium strains isolated from AIDS patients may be associated with the pathogenesis of M. avium infection.

Isolation of a contact-dependent haemolysin from Mycobacterium tuberculosis.

Contact-dependent haemolytic activity was observed with cells of Mycobacterium tuberculosis H(37)Rv and M. tuberculosis H(37)Ra, 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 H(37)Rv 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 M(r) of >200000 by gel-filtration on Sephadex G-200 and a subunit M(r) of 66000 by SDS-PAGE. It was sensitive to trypsin but stable when heated at 60 degrees 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.

Isolation of a 33-kDa protein antigen from delipidified Mycobacterium tuberculosis H37Rv.

A 33-kDa protein (TB33) was isolated from a delipidated cell sonicate (CS) of Mycobacterium tuberculosis H37Rv (grown in Middlebrook 7H9 broth supplemented with glucose) using immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (Ni-NTA) column. TB33 could not be isolated from the culture filtrate (CF) of M. tuberculosis H37Rv using Ni-NTA. TB33 was recognized by monoclonal antibodies (mAb) known to react with proteins of M. tuberculosis with a molecular mass of 33/34 kDa; namely, mAb F126-5, F67-1 and F126-2. The N-terminal amino acid sequence of TB33 was found to be Xaa-Xaa-Thr-Pro-Ala-Asp-Val-Ser/Cys-Asn-Val-Ala-Ile and thus, shows identity with the N-terminal of antigen 84 of M. tuberculosis except for two mismatches. Antibodies to TB33 could be raised in mice by administering four injections of TB33 (40 micrograms total protein). Sera from tuberculosis patients reacted with TB33, while those from normal healthy individuals did not.

Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate.

The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti-M. leprae hyperimmune serum coupled to CNBr-Sepharose 4B. A high molecular weight (M(r)) M. leprae protein (MLP) with a subunit M(r) of 22,000 was isolated. MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M. leprae. The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M. paratuberculosis. It showed 44% homology with N-terminal end of E. coli bacterioferritin. In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera. The role of bacterioferritin in M. leprae and the importance of MLP as an immunogen has been discussed.

Isolation of a 43 kDa protein from Mycobacterium tuberculosis H37Rv and its identification as a pyridine nucleotide transhydrogenase.

A 43 kDa protein (TB43) was isolated from the cell sonicate (CS) of Mycobacterium tuberculosis H37Rv with immobilized metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid column. Two-dimensional electrophoresis of the IMAC fraction showed a major spot with an M(r) of 43,000 and a pI of approximately 6.0. The N-terminal amino acid sequence of TB43 was met-arg-val-gly-ile-pro-asn-glu-thr-lys-asn-asn-glu-phe-arg-val-ala- ile-thr-pro-ala. It showed 86% homology with the N-terminal end of the alanine dehydrogenase of Myco. tuberculosis and 65% homology with the N-terminal end of the alpha-subunit of the Escherichia coli pyridine nucleotide transhydrogenase (Tsh). TB43 did not show any alanine dehydrogenase activity and did not react with monoclonal antibody (MAb) HBT10, which is known to recognize the 40 kDa alanine dehydrogenase of Myco. tuberculosis. It was also not recognized by MAb F29-29 which is known to react with a 43 kDa protein of Myco. tuberculosis complex. This protein exhibited strong Tsh activity. A similar 43 kDa protein showing Tsh activity was also isolated by IMAC from Myco. bovis CS. However, the pI of the protein was approximately 7.0. A similar protein could not be isolated from the CS or culture filtrate of Myco. bovis BCG and Myco. tuberculosis H37Ra. TB43 is a cell-associated pyridine nucleotide transhydrogenase and is distinct from the 40/44 kDa secreted alanine dehydrogenase of Myco. tuberculosis.

Skin reactivity and fibronectin-binding property of TB66 (66-kDa protein of Mycobacterium tuberculosis).

A 66-kDa protein (TB66) was purified from culture filtrate (CF) and cell sonicate (CS) of Mycobacterium tuberculosis H37Rv by immobilised metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid (NTA) column. TB66 was found to be a fibronectin-binding protein as determined by ELISA and could be purified by affinity chromatography with fibronectin-Sepharose. A similar 66-kDa protein could be isolated also from M. bovis, M. bovis BCG, M. africanum and M. tuberculosis H37Ra by IMAC, but not from any other mycobacteria. The NH2-terminal amino-acid sequence of TB66 from H37Rv and M. bovis was identical and showed 85% homology with the N-terminal sequence of bovine serum albumin (BSA). A monoclonal antibody (MAb) OD4AG3 recognised a heat-stable and trypsin-sensitive epitope near the C-terminal end of TB66. This MAb also recognised the 66-kDa protein isolated from the other members of the M. tuberculosis complex. In tests of immunogenicity, TB66 elicited a delayed type hypersensitivity reaction in guinea-pigs immunised with either TB66 or with M. tuberculosis H37Rv. TB66 also elicited an antibody response in immunised guinea-pigs and stimulated murine macrophages to produce tumour necrosis factor.

Purification and partial characterisation of a novel 66-kDa seroreactive protein of Mycobacterium tuberculosis H37Rv.

A seroreactive protein (TB66) was purified from culture filtrate (CF) and cell sonicate (CS) of Mycobacterium tuberculosis H37Rv by immobilised metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid (NTA) column. The TB66 preparations obtained by IMAC contained predominantly a 66-kDa protein with a pI of c. 5.5 as determined by two-dimensional electrophoresis. TB66 was detected in the CF as early as 1 week of growth of H37Rv. The NH2-terminal amino-acid sequence showed 85% homology with the N-terminal sequence of bovine serum albumin (BSA) and 80% homology with human serum albumin. Amino-acid analysis indicated a difference in the amino-acid content of TB66 when compared to BSA, with an abundance of acidic amino acids. A monoclonal antibody (MAb) OD4AG3, raised in this laboratory against an M. avium complex (MAC 101) sonicate cross-reacting with H37Rv sonicate, recognised a heat-stable and trypsin-sensitive epitope of this protein. TB66 was also recognised by MAbs IT1 and IT20 which also react with the 14-kDa antigen of the M. tuberculosis complex. Antibodies against TB66 were present in the sera of 62 of 64 patients with tuberculosis; sera from normal healthy individuals showed no significant reactivity. TB66 appears to be a predominant secretory protein of M. tuberculosis and could play an important role in the pathogenesis of this organism.

Superoxide dismutase activity of Mycobacterium tuberculosis isolated from tuberculosis patients and the immunoreactivity of superoxide dismutase from M. tuberculosis H37Rv.

OBJECTIVE: To determine the superoxide dismutase (SOD) activity from clinical isolates of Mycobacterium tuberculosis and to study the seroreactivity of SOD from M. tuberculosis H37Rv. DESIGN: Crude cell extracts of 16 strains of M. tuberculosis isolated from tuberculosis (TB) patients were assayed for SOD activity. SOD from H37Rv was partially purified and characterized, and the seroreactivity was studied by ELISA using sera from 36 active pulmonary TB and 31 leprosy patients. RESULTS: SOD activity was detected in all the 16 strains of M. tuberculosis and also in the medium of logarithmic and stationary cultures of H37Rv. SOD activity from H37Rv extract was not affected by 1 mM KCN or by 5 mM H2O2 and was only 20% inhibited by 10 mM NaN3, suggesting that it is a Mn-containing enzyme. SOD was partially purified from H37Rv extract by gel filtration chromatography as a tetramer of molecular weight (MW) of 80,000 and a subunit MW of approximately 23,000. A delayed type hypersensitivity was elicited by SOD in guinea pigs sensitized with H37Rv or M. leprae sonicate. ELISA using SOD as antigen indicated 100% positivity with TB sera, while 84% positivity was observed with leprosy sera. Western blotting with pooled TB and leprosy sera indicated the presence of antibodies to the 23 kD SOD protein. CONCLUSION: Our data indicate that M. tuberculosis strains are rich in SOD, and the secretion of SOD may play a valuable role in the pathogenesis of M. tuberculosis.