Cloning, expression and immunological reactivity of two mammalian cell entry proteins encoded by the mce1 operon of Mycobacterium tuberculosis.

Abstract

The DNA segments corresponding to two members of the mammalian cell entry operon 1 (mce1) encoding Mce1A and Mce1E proteins were amplified from Mycobacterium tuberculosis genomic DNA by polymerase chain reaction, cloned and subcloned into pGEM-T and pGEX-4T-3 vectors, respectively, and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) of Schistosoma japonicum as the fusion partner. The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels at the expected molecular mass of 68 kDa and 64 kDa for GST-Mce1A and GST-Mce1E, respectively. The identity of each fusion protein was confirmed by reactivity with anti-GST antibodies in Western immunoblots. The fusion proteins were purified to near homogeneity by affinity chromatography, and purified Mce1A and Mce1E, free of the fusion partner, were recovered following specific proteolytic cleavage of the GST portion by thrombin protease. Purified Mce1E appeared as a single band of 38 kDa, whereas purified Mce1A tended to exist in degraded as well as aggregated forms of different sizes. The fusion proteins, free GST and monomeric Mce1A and Mce1E reacted in Western immunoblots with antibodies in pools of human sera from six to 11 tuberculosis patients. Similar analysis showed the presence of antibodies to GST and Mce1A, in pools of human sera from M. bovis BCG-vaccinated healthy subjects. When pure Mce1E was blotted against individual sera, antibodies in 4/10 sera from tuberculosis patients reacted, whereas no reaction was seen with 10 individual sera from M. bovis BCG-vaccinated healthy subjects. However, when the same sera were tested for reactivity to the purified preparation of Mce1A, 8/10 sera from both tuberculosis patients and M. bovis BCG-vaccinated healthy subjects showed positive reactivity. These findings demonstrate that both Mce1A and Mce1E are expressed and immunogenic during natural infection with M. tuberculosis.