The unique tropism of Mycobacterium leprae to the nasal epithelial cells can be explained by the mammalian cell entry protein 1A

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. Mce1A 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. Recombinant proteins having N-terminus and C-terminus truncations of the Mce1A region of M. leprae were created in Escherichia coli. Entry activity of latex beads, coated with these truncated proteins (r-lep37 kDa and r-lep27 kDa), 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. Recombinant 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.

麻风病（Leprosy）是一类慢性感染性疾病，麻风分枝杆菌（Mycobacterium leprae）可侵袭宿主皮肤与外周神经系统。由于尚未建立麻风分枝杆菌的体外培养方法，其感染机制仍有部分未明。麻风分枝杆菌的mce1A基因（1326 碱基对（base pair））编码Mce1A蛋白（442个氨基酸（amino acid））。已知结核分枝杆菌（Mycobacterium tuberculosis）中的Mce1A同源蛋白与结核分枝杆菌上皮细胞入侵、巨噬细胞内存活及增殖密切相关。已有重组蛋白相关研究证实，麻风分枝杆菌的Mce1A蛋白同样参与上皮细胞入侵过程。本研究旨在定位麻风分枝杆菌Mce1A蛋白中与上皮细胞入侵相关的特定序列区段。我们在大肠杆菌（Escherichia coli）中构建了携带麻风分枝杆菌Mce1A蛋白N端与C端截短片段的重组蛋白。将这些截短蛋白（分别为r-lep37 kDa与r-lep27 kDa）包被乳胶微球后，通过电子显微镜观察其入侵HeLa细胞的活性。实验结果显示，即便分别从N端截去315 bp（对应105个氨基酸）、从C端截去922 bp（对应308个氨基酸），Mce1A蛋白仍保留入侵活性。我们将该保留活性的316–921 bp区段划分为三个亚区域：316–531 bp（命名为InvX）、532–753 bp（InvY）以及754–921 bp（InvZ）。将每个亚区域克隆至AIDA载体，并在大肠杆菌表面进行表达。通过电子显微镜观察这些重组大肠杆菌对单层培养的HeLa细胞与RPMI2650细胞的入侵能力。仅携带InvX亚区域的重组大肠杆菌可介导细胞入侵。我们进一步将InvX划分为四个结构域：InvXa至InvXd，分别对应氨基酸序列1–24、25–46、47–57及58–72位残基。将分别表达InvXa至InvXd的重组大肠杆菌与对应结构域的抗体共孵育后，添加至单层培养的RPMI细胞中。通过菌落计数法评估这些抗体对细胞入侵过程的阻断效果。实验结果表明，针对InvXa、InvXb与InvXd的抗体可显著抑制细胞入侵活性。上述结果提示，Mce1A蛋白的这三个InvX结构域对于麻风分枝杆菌入侵鼻腔上皮细胞至关重要。