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

The DNA segments corresponding to two members of the mammalian cell entry o peron 1 (mce1) encoding Mce1A and Mce1E proteins were amplified from Mycoba cterium tuberculosis genomic DNA by polymerase chain reaction, cloned and s ubcloned 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 appe ared 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 id entity of each fusion protein was confirmed by reactivity with anti-GST ant ibodies in Western immunoblots. The fusion proteins were purified to near h omogeneity by affinity chromatography, and purified Mce1A and Mce1E, free o f the fusion partner, were recovered following specific proteolytic cleavag e of the GST portion by thrombin protease. Purified Mce1E appeared as a sin gle band of 38 kDa, whereas purified Mce1A tended to exist in degraded as w ell as aggregated forms of different sizes. The fusion proteins, free GST a nd monomeric Mce1A and Mce1E reacted in Western immunoblots with antibodies in pools of human sera from six to 11 tuberculosis patients. Similar analy sis showed the presence of antibodies to GST and Mce1A, in pools of human s era from M. bovis BCG-vaccinated healthy subjects. When pure Mce1E was blot ted against individual sera, antibodies in 4/10 sera from tuberculosis pati ents reacted, whereas no reaction was seen with 10 individual sera from M. bovis BCG-vaccinated healthy subjects. However, when the same sera were tes ted for reactivity to the purified preparation of Mce1A, 8/10 sera from bot h 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 .