Characterization of a 34-kilodalton protein of Mycobacterium leprae that is isologous to the immunodominant 34-kilodalton antigen of Mycobacterium paratuberculosis

Abstract

During DNA sequence analysis of cosmid L373 from the Mycobacterium leprae genome, an open reading frame of 1.4 kb encoding a protein with some homology to the immunodominant 34-kDa protein of Mycobacterium paratuberculosis, but lacking significant serological activity, was detected. The DNA sequence predicted a signal peptide with a modified lipoprotein consensus sequence, but the protein proved to be devoid of lipid attachment.

FIG. 1

Comparison of the deduced amino acid sequences of the M. leprae (LEPRAE) and M. paratuberculosis (PARATU) 34-kDa proteins. The global comparison shows 62% identity between proteins of M. leprae and M. paratuberculosis. The comparison also shows a higher level of homology (73% identity) at the N terminus (170 aa), which comprises the four predicted transmembrane regions, than at the C terminus (46% identity; the last 142 aa) (1, 2). The configuration of the proposed N terminus is based on the presence of positively charged amino acids in the N terminus and hydrophobic amino acids along the signal sequence, in addition to a pseudolipoprotein consensus sequence. The signal sequence is indicated by the upper line with a small arrow for the cleavage site of the signal peptidase. The oligopeptides that were synthesized and used for the serum blocking experiments are marked P-1, P-2, and P-3. Dashes show identity between the two sequences. The arrow above M. leprae aa 201 indicates the first amino acid of the C-terminal protein expressed in E. coli. Gray shading represents conserved substitution; black shading represents identical amino acids; no shading represents different sequences.

FIG. 2

Comparison of oligopeptides (P1 to P3) in terms of the inhibition of the seroreactivity of the recombinant 34-kDa antigen against sera from leprosy and tuberculosis patients. Lep-1, leprosy patient 1; Lep-2, leprosy patient 2; TB, tuberculosis patient 1; Serum ID, inhibition by the P-3 peptide.

FIG. 3

Expression and localization of the M. leprae 34-kDa protein with and without the signal peptide. The gene was amplified by PCR, cloned into pMV261, and transformed into M. smegmatis mc²155. Cells were grown and fractionated into cell wall, membrane, and cytosol fractions as described in the text. The different fractions were analyzed by Western blotting of SDS–15% PAGE gel using sera from a mouse immunized with the C-terminal portion of the 34-kDa protein expressed in E. coli. Lanes 1, 2, and 3 are the cell wall, membrane, and cytosolic fractions, respectively, of M. smegmatis cells expressing the 34-kDa protein with the signal peptide. Lanes 4, 5, and 6 indicate the cell wall, membrane, and cytosolic fractions, respectively, of M. smegmatis cells expressing the 34-kDa protein without the signal. Lanes 7, 8, and 9 are the cell wall, membrane, and cytosolic fractions, respectively, of M. smegmatis containing vector pMV261 as the control. Lanes 10, 11, and 12 show M. leprae cell wall, membrane, and cytosolic fractions, respectively.