Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells.
Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells.
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DOI：
10.1111/omi.12136
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发表时间：
2016-02
影响因子：
3.7
通讯作者：
Lally ET
中科院分区：
文献类型：
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作者：
Balashova N;Dhingra A;Boesze-Battaglia K;Lally ET
研究表明，伴放线聚集杆菌白细胞毒素（LtxA）通过溶酶体介导的机制杀死携带LFA-1（CD 11 a/CD 18）的人免疫细胞。溶酶体是膜结合的细胞器，其含有能够分解生物分子的酸性水解酶阵列。溶酶体膜双层将pH敏感性酶限制在最佳酸性（pH 4.8）环境内，从而保护微碱性细胞溶质（pH 6.8-7.5）。在目前的研究中，我们探讨了LTxA诱导的细胞溶解对两种不同的K562红白血病细胞系中溶酶体完整性的影响。用CD 11 a和CD 18 cDNA稳定转染K562-puro/LFA-1细胞以在细胞表面上表达LFA-1，而不表达LFA-1的K562-puro用作对照。用100 ng/ml LtxA处理后，通过活细胞成像结合延时共聚焦显微镜和流式细胞术分析细胞。使用pH敏感指示剂（pHrodo®，Life Technologies），我们证明了毒素导致K562-puro/LFA-1细胞中细胞内pH的降低，在处理的前15分钟内是明显的。该过程与胞质溶胶中溶酶体的消失相关，如通过吖啶橙子和LysoTracker® Red DND-99（Life Technologies）染色所确定的。在K562-puro细胞中或当热灭活毒素加入K562-puro/LFA-1中时未观察到这些变化。我们的研究结果表明，LtxA诱导溶酶体损伤，胞浆酸化，这是随后的细胞死亡K562-puro/LFA-1细胞。
Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human LFA-1(CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8–7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng/ml LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH sensitive indicator (pHrodo®, Life Technologies) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker® Red DND-99 (Life Technologies) staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells.