Molecular characterisation of a novel lipoglycoprotein from Mycobacterium tuberculosis and Mycobacterium bovis
In Great Britain a recent independent scientific review into bovine
tuberculosis concluded that the best prospect for control of TB in the
national herd is to develop a vaccine. The secreted antigens of the
Mycobacterium tuberculosis complex have received much interest as
possible vaccine candidates due to their ability to confer protection
against tuberculosis in small animal models. The recent discovery that
some of these mycobacterial antigens are glycosylated, a modification
ubiquitous in eukaryotic proteins, may provide some novel insights into
the properties of these antigens.
The antigen MPB70 is the major secreted protein of Mycobacterium
bovis, the causative agent of tuberculosis in cattle. It has previously
been reported that this antigen, encoded for by the gene mpb70, is
present in at least two forms, a 22 kDa unglycosylated form and a 25
kDa glycosylated form. A clone was isolated from an M. tuberculosis
H37Rv mycobacterial shuttle cosmid library which expressed both the
22 and 25 kDa antigens.
Genetic analysis of this cosmid revealed that the two antigens were
encoded by separate genes. The gene encoding the 25 kDa antigen
(MPT83), subsequently designated mpt83, is situated 2.4 kb upstream of
mpt70 and transcribed in the same direction. Using a mycobacterial
expression system and alkaline phosphate (PhoA) fusions, it was shown
that MPT83 but not MPT70 is glycosylated by Mycobacterium smegmatis.
Moreover, neither fusion protein was glycosylated in E. cW
demonstrating that glycosylation of MPT83 was specific to the
mycobacterial species. The use of site directed mutagenesis in
conjunction with the PhoA reporter system identified both 0 and N-linked
glycosylation sites within MPT83.
Preliminary investigations into the role of glycosylation in the
alteration of immune recognition showed a possible influence of
cilycosylation on a T cell epitope of MPT83.