AbstractLeprosy is a chronic infection where the skin and peripheral nervous system is invaded byMycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet forM.leprae.Mce1A protein (442 aa) is coded by mce1A (1326 bp) ofM.leprae. The mce1A homolog inMycobacterium tuberculosisis known to be associated withM.tuberculosisepithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that mce1A ofM.lepraeis also associated with epithelial cell entry. This study is aimed at identifying particular sequences within mce1A associated withM.lepraeepithelial cell entry.Recombinant proteins having N-terminus and C-terminus truncations of the mce1A region ofM.lepraewere created inEschericia 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 ofE.coli.Entry of theseE.coliinto monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. OnlyE.coliharboring 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.RecombinantE.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 forM.lepraeinvasion into nasal epithelial cells.Author SummaryMce1A protein is encoded by the mce1A region of mce1 locus ofM.tuberculosisandM.leprae, and is involved in the bacteria’s invasion into epithelial cells. The present study revealed that the active sequence ofM.lepraeinvolved in the invasion into nasal mucosa epithelial cells is present in the 316-531 bp region of mce1A.The most important region of mce1A protein involved in the invasion ofM.tuberculosisinto human epithelial cells is called the InvIII region, which is located between amino acids at position 130 to 152. The InvIII region ofM.tuberculosiscorresponds to InvXb ofM.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 betweenM.lepraeandM.tuberculosis. Suppression test results also indicated that the most important region of mce1A protein ofM.lepraeinvolved in the invasion into human epithelial cells is different from thatM.tuberculosis. WhileM.tuberculosishas 3,959 protein-encoding genes and only 6 pseudogenes,M.lepraehas only 1,604 protein-encoding genes but has 1,116 pseudogenes indicating that inM.leprae, far more proteins are inactivated as compared toM.tuberculosis. The present study also revealed that, as inM.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 ofM.lepraeinto nasal mucosa epithelial cells.
Transcriptome Analysis Unveils the Effects of Proline on Gene Expression in the Yeast Komagataella phaffii
