Kansau Imad, Berger Cédric, Hospital Maxime, Amsellem Raymonde, Nicolas Valérie, Servin Alain L, Bernet-Camard Marie-Françoise
Unité 510 INSERM, Faculté de Pharmacie Paris XI, F-92296 Châtenay-Malabry, France.
Infect Immun. 2004 Jul;72(7):3733-42. doi: 10.1128/IAI.72.7.3733-3742.2004.
We undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements. We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells). Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the alpha5beta1 integrin, which plays a pivotal role in Afa/Dr diffusely adhering Escherichia coli bacterial entry, is mobilized around adhering Dr-positive bacteria. We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-positive bacteria. We also observed that extracting membrane cholesterol with methyl-beta-cyclodextrin (MBCD) did not affect the recruitment of CD55, GM1, or beta1 integrin to adhering Dr-positive bacteria. In contrast, extracting or changing membrane-bound cholesterol by means of drugs that modify lipid rafts (MBCD, filipin III, or mevalonate plus lovastatin plus MBCD) inhibited the entry of Dr-positive IH11128 both into cells that expressed VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) and into those that did not (parental Caco-2 cells). Finally, restoring cholesterol within the cell membrane of MBCD-treated cells restored Dr-positive IH11128 internalization.
我们对Dr阳性细菌侵入上皮细胞的机制进行了研究。我们的研究结果表明，Dr阳性细菌通过一种拉链样机制进入细胞，该机制独立于Dr诱导的F-肌动蛋白动员以及控制Dr诱导的F-肌动蛋白重排的信号分子。我们还观察到，Dr阳性的IH11128细菌进入小窝标志物VIP21/小窝蛋白呈阳性的细胞（HeLa和Caco-2/Cav-1细胞）的程度与进入不表达该标志物的细胞（亲代Caco-2细胞）相同。通过荧光标记和共聚焦激光扫描显微镜，我们提供了证据表明，在黏附步骤中，在Afa/Dr弥漫性黏附大肠杆菌细菌进入过程中起关键作用的α5β1整合素会在黏附的Dr阳性细菌周围发生动员。我们发现，Afa/Dr黏附素的受体、糖基磷脂酰肌醇锚定的CD55；脂筏标志物神经节苷脂GM1；以及VIP21/小窝蛋白都会在黏附的Dr阳性细菌周围聚集。我们还观察到，用甲基-β-环糊精（MBCD）提取膜胆固醇并不影响CD55、GM1或β1整合素向黏附的Dr阳性细菌的聚集。相反，通过改变脂筏的药物（MBCD、制霉菌素III或甲羟戊酸加洛伐他汀加MBCD）提取或改变膜结合胆固醇会抑制Dr阳性的IH11128进入表达VIP21/小窝蛋白的细胞（HeLa和Caco-2/Cav-1细胞）以及不表达该蛋白的细胞（亲代Caco-2细胞）。最后，在MBCD处理的细胞的细胞膜内恢复胆固醇可恢复Dr阳性的IH11128的内化。
