The six mammalian cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during in vitro growth of Mycobocterium tuberculosis

The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. The mammalian cell entry (mce)3 operon is one of four homologous mce operons that encodes six putative invasin-like exported proteins (Mce3A-F), possibly involved in entry and survival of M. tuberculosis inside macrophages. We have recently shown that Mce3A, Mce3D and Mce3E are expressed and elicit antibody responses in a majority of human subjects during natural infection with M. tuberculosis. In this study, we demonstrate the expression of Mce3A-F proteins and their mRNA during in vitro growth of M. tuberculosis. To demonstrate the expression of mce3A-F proteins, the antibodies were raised in rabbits against three pure proteins (Mce3A, Mce3D and Mce3E), and their specificity was checked by immunoblotting with recombinant Mce1A-F proteins encoded by mce1 operon. The antibodies were also generated against all the six Mce3 proteins, which were expressed and purified as fusion proteins with glutathione S-transferase (GST) as the fusion partner (GST-Mce3A-F). The antibodies reacted, in each case, with a protein of expected molecular mass (Mr) for the corresponding Mce3 protein in the cell wall fraction but not in the soluble fraction of in vitro-grown M. tuberculosis cells. The presence of mRNA for mce3A-F genes was also shown by using mce3A-F gene-specific primers, and total RNA isolated from in vitro-grown M. tuberculosis cells by reverse transcription-polymerase chain reaction (RT-PCR). Pretreatment of the RNA preparation with RNase A abolished amplification in RT-PCR confirming that mce3A-F mRNA rather than genomic DNA was being amplified. The data show that Mce3A-F encoded by the mce3 operon are expressed during in vitro growth of M. tuberculosis.