Comparative Study of the Growth and Survival of Recombinant Mycobacterium smegmatis Expressing Mce4A and Mce4E from Mycobacterium bovis

Cholesterol is important for the growth and persistence of Mycobacterium tuberculosis in macrophages. The mce4 locus, which is conserved in both M. tuberculosis and Mycobacterium bovis, is thought to be responsible for cholesterol transport into the bacteria. However, the exact roles of specific genes within the sophisticated mce4 system remain poorly understood. In this study, Mce4A and Mce4E of M. bovis, two proteins that are encoded by the mce4 locus, were expressed in Mycobacterium smegmatis. The recombinant strain expressing the Mce4E protein (M. smeg::E) performed better than that expressing the Mce4A protein (M. smeg::A) in a minimal medium with and without glycerol or cholesterol, which may be the reason why M. smeg::E showed better survival in ANA-1 macrophages than did M. smeg::A. Cytokine expression profiles were similar in macrophages infected with either recombinant strain. We also investigated the role of CD36 in recognizing Mce4A and Mce4E proteins. However, CD36 did not appear to be specific for these proteins and showed little impact on the ultimate clearance of the recombinant strains. Reduced interleukin-1 beta, inducible nitric oxide synthase, and tumor necrosis factor-alpha mRNA expression at 6 h postinfection in macrophages infected with M. smeg::E was observed using a CD36-specific monocular antibody to block the receptor, whereas no obvious changes in the expression of these cytokines were observed in cells infected with M. smeg::A with or without exposure to the CD36 antibody. Conclusively, the different performances of the recombinant strains suggest that the Mce4A and Mce4E proteins enhance mycobacterial adaptation to the harsh environment within macrophages after phagocytosis.