Secott T E, Lin T L, Wu C C
Department of Veterinary Pathobiology, Purdue University, 406 S. University St., West Lafayette, IN 47907, USA.
Infect Immun. 2004 Jul;72(7):3724-32. doi: 10.1128/IAI.72.7.3724-3732.2004.
Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the alpha5, alphaV, beta1, and beta3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells.
培养的上皮细胞对副结核分枝杆菌的有效附着和摄取需要一种纤连蛋白（FN）附着蛋白同源物（FAP-P）的表达，该同源物介导副结核分枝杆菌与FN的结合。副结核分枝杆菌对派伊尔结的侵袭是通过M细胞发生的，与其他肠道上皮细胞不同，M细胞在其腔面表达整合素。我们试图确定副结核分枝杆菌的FAP-P与可溶性FN之间的相互作用是否能使副结核分枝杆菌在体内通过这些表面整合素靶向并侵袭M细胞。将野生型和反义FAP-P突变型副结核分枝杆菌菌株单独注射或与阻断肽或抗体共同注射到小鼠肠袢中，然后进行免疫荧光显微镜检查以评估副结核分枝杆菌对M细胞的靶向和侵袭情况。未调理的副结核分枝杆菌优先侵袭小鼠肠袢中的M细胞。用FN预处理副结核分枝杆菌后，M细胞侵袭增强了2.6倍。与对照菌株相比，副结核分枝杆菌反义FAP-P突变体对M细胞的侵袭减少了77%至90%。对应于FN的RGD和协同位点整合素识别区域的肽分别阻断了副结核分枝杆菌对M细胞侵袭的75%和45%，而连接段1肽无抑制作用。针对α5、αV、β1和β3整合素亚基的抗体抑制M细胞侵袭的程度为52%至73%。结果表明，副结核分枝杆菌在体内对M细胞的靶向和侵袭主要是由副结核分枝杆菌的FAP-P与M细胞上的整合素之间形成的FN桥介导的。
