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Mammalian Cell‐Entry Proteins Encoded by the mce3 Operon of Mycobacterium tuberculosis are Expressed During Natural Infection in Humans

The mammalian cell‐entry (mce)3 operon is one of four homologous mce operons on Mycobacterium tuberculosis genome that encodes six putative invasin/ adhesin‐like proteins (Mce3A–F) possibly involved in the entry and survival of this bacterium inside macrophages. To study the in vivo expression of the mce3 operon‐encoded proteins during natural human infection, the genes encoding Mce3A–F were cloned and expressed in Escherichia coli as fusion proteins with glutathione‐S‐transferase (GST) at the N‐terminal and a ×6 histidine (His) tag at the C‐terminal end. The recombinant proteins appeared as major cellular proteins in SDS‐PAGE gels and reacted with anti‐GST and antipenta‐His antibodies at the expected molecular mass of 72, 61, 78, 80, 66 and 78 kDa for GST‐Mce3A, GST‐Mce3B, GST‐Mce3C, GST‐Mce3D, GST‐Mce3E and GST‐Mce3F, respectively. In Western immunoblots, all the six fusion proteins, particularly GST‐Mce3A, GST‐Mce3C, GST‐Mce3D and GST‐Mce3E, reacted with antibodies in combined human serum from 11 tuberculosis (TB) patients. Pure Mce3A, Mce3D and Mce3E could be isolated by specific proteolytic cleavage by thrombin protease of the respective purified fusion protein followed by preparative SDS‐PAGE. The pure Mce3A, Mce3D and Mce3E reacted to various extents with antibodies in serum samples from TB patients. The Mce3E reacted with 51 of 55 (93%) and all the three proteins reacted with 34 of 55 (62%) serum samples. The Mce3A, Mce3D and Mce3E proteins also reacted, albeit at lower frequency, with one of 23 (4%) serum sample obtained from M. bovis bacillus Calmette–Guérin‐vaccinated healthy subjects and four of 18 (22%) serum samples from long‐term contacts of TB patients showing reactivity with all the three Mce3 proteins. The data show that Mce3A, Mce3D and Mce3E encoded by mce3 operon of M. tuberculosis are expressed and elicit antibody responses in humans during natural infection with this pathogen.