bioRxiv2018
Leprosy is a chronic infection where the skin and peripheral nervous system is invaded by Mycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet for M.leprae.\n\nMce1A protein (442 aa) is coded by mce1A (1326 bp) of M.leprae. The mce1A homolog in Mycobacterium tuberculosis is known to be associated with M.tuberculosis epithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that mce1A of M.leprae is also associated with epithelial cell entry. This study is aimed at identifying particular sequences within mce1A associated with M.leprae epithelial cell entry.\n\nRecombinant proteins having N-terminus and C-terminus truncations of the mce1A region of M.leprae were created in Eschericia coli. Entry activity of latex beads, coated with these truncated proteins (r-lep37kDa and r-lep27kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316 - 921 bp region was divided into three sub-regions: 316 - 531 bp (InvX), 532 - 753 bp (InvY), and 754 - 921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface of E.coli. Entry of these E.coli into monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. Only E.coli harboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa - InvXd, containing sequences 1 - 24 aa, 25 - 46 aa, 47 - 57 aa, and 58 - 72 aa, respectively.\n\nRecombinant E.coli, expressing each of InvXa - InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of mce1A are important for M.leprae invasion into nasal epithelial cells.\n\nAuthor SummaryMce1A protein is encoded by the mce1A region of mce1 locus of M.tuberculosis and M.leprae, and is involved in the bacterias invasion into epithelial cells. The present study revealed that the active sequence of M.leprae involved in the invasion into nasal mucosa epithelial cells is present in the 316-531 bp region of mce1A.\n\nThe most important region of mce1A protein involved in the invasion of M.tuberculosis into human epithelial cells is called the InvIII region, which is located between amino acids at position 130 to 152. The InvIII region of M.tuberculosis corresponds to InvXb of M.leprae. The sequences of these regions are identical between amino acids at positions 10 to position 22 as counted from the N terminus, except that amino acids at positions 1 to 3, 5, 8, 9, 13 are different between M.leprae and M.tuberculosis. Suppression test results also indicated that the most important region of mce1A protein of M.leprae involved in the invasion into human epithelial cells is different from that M.tuberculosis. While M.tuberculosis has 3,959 protein-encoding genes and only 6 pseudogenes, M.leprae has only 1,604 protein-encoding genes but has 1,116 pseudogenes indicating that in M.leprae, far more proteins are inactivated as compared to M.tuberculosis. The present study also revealed that, as in M.tuberculosis, the mce1A protein is expressed on the surface of bacteria as a native protein. In light of these data, the mce1A protein is considered to be one of the most important proteins involved in the invasion of M.leprae into nasal mucosa epithelial cells.