Jacob Tobias, Lee Rebecca J, Engel Joanne N, Machen Terry E
Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, California 94720-3200, USA.
Infect Immun. 2002 Nov;70(11):6399-408. doi: 10.1128/IAI.70.11.6399-6408.2002.
Modulation of cytosolic (intracellular) Ca(2+) concentration (Ca(i)) may be an important host response when airway epithelial cells are exposed to Pseudomonas aeruginosa. We measured Ca(i) in Calu-3 cells exposed from the apical or basolateral surface to cytotoxic and noncytotoxic strains of P. aeruginosa. Apical addition of either noncytotoxic strains or cytotoxic strains failed to affect Ca(i) over a 3-h time period, nor were changes observed after basolateral addition of noncytotoxic strains. In contrast, basolateral addition of cytotoxic strains caused a slow increase in Ca(i) from 100 nM to 200 to 400 nM. This increase began after 20 to 50 min and persisted for an additional 30 to 75 min, at which time the cells became nonviable. P. aeruginosa-induced increases in Ca(i) were blocked by the addition of the Ca channel blocker La(3+) to the basolateral but not to the apical chamber. Likewise, replacing the basolateral but not the apical medium with Ca-free solution prevented P. aeruginosa-mediated changes in Ca(i). With isogenic mutants of PA103, we demonstrated that the type III secretion apparatus, the type III-secreted effector ExoU, and type IV pili were necessary for increased Ca(i). We propose that translocation of ExoU through the basolateral surface of polarized airway epithelial cells via the type III secretion apparatus leads to release of Ca stored in the endoplasmic reticulum and activation of Ca channels in the basolateral membranes of epithelial cells.
当气道上皮细胞暴露于铜绿假单胞菌时，调节胞质（细胞内）钙离子浓度（Ca(i)）可能是一种重要的宿主反应。我们测量了从顶端或基底外侧表面暴露于铜绿假单胞菌细胞毒素和非细胞毒素菌株的Calu-3细胞中的Ca(i)。顶端添加非细胞毒素菌株或细胞毒素菌株在3小时内均未影响Ca(i)，基底外侧添加非细胞毒素菌株后也未观察到变化。相比之下，基底外侧添加细胞毒素菌株导致Ca(i)从100 nM缓慢增加到200至400 nM。这种增加在20至50分钟后开始，并持续另外30至75分钟，此时细胞变得无法存活。在基底外侧而非顶端腔室中添加钙通道阻滞剂La(3+)可阻断铜绿假单胞菌诱导的Ca(i)增加。同样，用无钙溶液替代基底外侧而非顶端培养基可防止铜绿假单胞菌介导的Ca(i)变化。对于PA103的同基因突变体，我们证明III型分泌装置、III型分泌效应子ExoU和IV型菌毛对于Ca(i)增加是必需的。我们提出，ExoU通过III型分泌装置穿过极化气道上皮细胞的基底外侧表面，导致内质网中储存的钙释放，并激活上皮细胞基底外侧膜中的钙通道。
