Department of Microbiology, College of Science, Oregon State University, Corvallis, OR, United States.
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, United States.
Front Cell Infect Microbiol. 2018 Jun 27;8:217. doi: 10.3389/fcimb.2018.00217. eCollection 2018.
subspecies (MAP) is the causative agent of Johne's disease, chronic and ultimately fatal enteritis that affects ruminant populations worldwide. One mode of MAP transmission is oral when young animals ingest bacteria from the collostrum and milk of infected dams. The exposure to raw milk has a dramatic impact on MAP, resulting in a more invasive and virulent phenotype. The MAP1203 gene is upregulated over 28-fold after exposure of the bacterium to milk. In this study, the role of MAP1203 in binding and invasion of the bovine epithelial cells was investigated. By over-expressing the native MAP1203 gene and two clones of deletion mutant in the signal sequence and of missense mutations changing the integrin domain from RGD into RDE, we demonstrate that MAP1203 plays a role in increasing binding in more than 50% and invasion in 35% of bovine MDBK epithelial cells during early phase of infection. Furthermore, results obtained suggest that MAP1203 is a surface-exposed protein in MAP and the signal sequence is required for processing and expression of functional protein on the surface of the bacterium. Using the protein pull-down assay and far-Western blot, we also demonstrate that MAP1203 interacts with the host dihydropyrimidinase-related protein 2 and glyceraldehyde 3-phosphate dehydrogenase proteins, located on the membrane of epithelial cell and involved in the remodeling of the cytoskeleton. Our data suggests that MAP1203 plays a significant role in the initiation of MAP infection of the bovine epithelium.
亚种（MAP）是约翰氏病的病原体，是一种慢性的、最终致命的肠炎，影响着全球反刍动物群体。MAP 的一种传播方式是经口传播，即幼畜从受感染母畜的初乳和乳汁中摄取细菌。接触生奶对 MAP 有显著影响，导致更具侵袭性和毒力的表型。暴露于生奶后，细菌中的 MAP1203 基因的表达上调了 28 倍以上。在这项研究中，研究了 MAP1203 在结合和侵袭牛上皮细胞中的作用。通过过表达天然 MAP1203 基因和信号序列缺失突变的两个克隆以及将整合素结构域从 RGD 突变为 RDE 的错义突变，我们证明 MAP1203 在感染早期增加了超过 50%的结合和 35%的牛 MDBK 上皮细胞的侵袭。此外，结果表明 MAP1203 是 MAP 表面暴露的蛋白，信号序列是细菌表面加工和表达功能蛋白所必需的。通过使用蛋白下拉测定和远 Western blot，我们还证明了 MAP1203 与宿主二氢嘧啶酶相关蛋白 2 和甘油醛 3-磷酸脱氢酶蛋白相互作用，这些蛋白位于上皮细胞膜上，参与细胞骨架的重塑。我们的数据表明，MAP1203 在 MAP 感染牛上皮细胞的初始阶段发挥重要作用。
