microbiology, Vol 142, 1375-1383, Copyright © 1996 by Society for General Microbiology
A major T-cell-inducing cytosolic 23 kDa protein antigen of the vaccine candidate Mycobacterium habana is superoxide dismutase
D Bisht, J Mehrotra, MS Dhindsa, NB Singh and S Sinha
Division of Membrane Biology, Central Drug Research Institute, Lucknow, India.
This study describes the purification and immunochemical characterization
of a major 23 kDa cytosolic protein antigen of the vaccine candidate
Mycobacterium habana (TMC 5135). The 23 kDa protein alone was salted out
from the cytosol at an ammonium sulfate saturation of 80-95%. It
represented about 1.5% of the total cytosolic protein, appeared
glycosylated by staining with periodic acid/Schiff's reagent, and showed a
pl of approximately 5.3. Its native molecular mass was determined as
approximately 48 kDa, suggesting a homodimeric configuration.
Immunoblotting with the WHO-IMMLEP/IMMTUB mAbs mc5041 and IT61 and activity
staining after native PAGE established its identity as a mycobacterial
superoxide dismutase (SOD) of the Fe/Mn type. The sequence of the 18
N-terminal amino acids, which also contained the binding site for mc5041,
showed a close resemblance, not only with the reported deduced sequences of
Mycobacterium leprae and Mycobacterium tuberculosis Fe/MnSODs, but also
with human MnSOD. In order to study its immunopathological relevance, the
protein was subjected to in vivo and in vitro assays for T cell activation.
It induced, in a dose-related manner, skin delayed hypersensitivity in
guinea-pigs and lymphocyte proliferation in BALB/c mice primed with M.
habana. Most significantly, it also induced lymphocyte proliferative
responses, in a manner analogous to M. Ieprae, in human subjects comprising
tuberculoid leprosy patients and healthy contacts.
