Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage.
Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage.
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DOI：
10.1371/journal.pone.0075708
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Pidard D
中科院分区：
文献类型：
--
作者：
Beaufort N;Corvazier E;Mlanaoindrou S;de Bentzmann S;Pidard D
在脉管系统内，不受控制的细胞周蛋白水解可导致细胞与细胞和细胞与基质相互作用的破坏以及随后的凋亡诱导的细胞凋亡或失巢凋亡，从而导致炎性血管疾病，其中内皮是主要靶点。迄今为止，大多数研究都集中在内源性蛋白酶。然而，在血流感染期间，细菌蛋白酶也可触发内皮失巢凋亡。因此，我们研究了潜在的细胞凋亡活性的嗜血性机会致病菌，铜绿假单胞菌分泌的蛋白酶，特别是其主要的金属蛋白酶，LasB。为此，我们使用了分泌组的LasB表达假单胞菌株，PAO 1，并比较它与同基因，LasB缺陷株（PAO 1 paslasB），以及与纯化的LasB。对分泌器在来自脐静脉或脑微血管系统的培养的人内皮细胞上的凋亡活性进行测试。我们发现PAO 1分泌组容易诱导内皮细胞失巢凋亡，LasB阳性临床假单胞菌分离株的分泌组也是如此，而PAO 1 β lasB分泌组对内皮细胞粘附和活力的影响有限。值得注意的是，纯化的LasB再现了含有LasB的分泌体的大部分作用，并且在LasB选择性抑制剂phosphoramidon的存在下，这些作用急剧减少。通过细胞培养物的免疫荧光和/或免疫印迹分析，观察到与内皮细胞外基质、纤连蛋白和血管性血友病因子相关的几种蛋白质的早熟和广泛的LasB依赖性降解。此外，PAO 1分泌蛋白，但不是从PAO 1的分泌蛋白B，专门诱导快速的两个主要的内皮间连接组件，VE-钙粘蛋白和occludin，以及抗失巢凋亡，整合素相关的尿激酶受体，uPAR的内蛋白水解。作为外源性出血性蛋白酶的原型，假单胞菌LasB因此似乎不仅通过基质溶解诱导内皮失巢凋亡，如在许多促凋亡蛋白酶中观察到的，而且通过切割一些必需的细胞-细胞和细胞-基质粘附受体诱导内皮失巢凋亡，这些粘附受体与内皮屏障的维持有关。
Within the vasculature, uncontrolled pericellular proteolysis can lead to disruption of cell-to-cell and cell-to-matrix interactions and subsequent detachment-induced cell apoptosis, or anoikis, contributing to inflammatory vascular diseases, with the endothelium as the major target. Most studies so far have focused on endogenous proteinases. However, during bloodstream infections, bacterial proteinases may also trigger endothelial anoikis. We thus investigated the potential apoptotic activity of the proteinases secreted by the haematotropic opportunistic pathogen, Pseudomonas aeruginosa, and particularly its predominant metalloproteinase, LasB. For this, we used the secretome of the LasB-expressing pseudomonal strain, PAO1, and compared it with that from the isogenic, LasB-deficient strain (PAO1∆lasB), as well as with purified LasB. Secretomes were tested for apoptotic activity on cultured human endothelial cells derived from the umbilical vein or from the cerebral microvasculature. We found that the PAO1 secretome readily induced endothelial cell anoikis, as did secretomes of LasB-positive clinical pseudomonal isolates, while the PAO1∆lasB secretome had only a limited impact on endothelial adherence and viability. Notably, purified LasB reproduced most of the effects of the LasB-containing secretomes, and these were drastically reduced in the presence of the LasB-selective inhibitor, phosphoramidon. A precocious and extensive LasB-dependent degradation of several proteins associated with the endothelial extracellular matrix, fibronectin and von Willebrand factor, was observed by immunofluorescence and/or immunoblotting analysis of cell cultures. Moreover, the PAO1 secretome, but not that from PAO1∆lasB, specifically induced rapid endoproteolysis of two major interendothelial junction components, VE-cadherin and occludin, as well as of the anti-anoikis, integrin-associated urokinase receptor, uPAR. Taken as a prototype for exogenous haemorrhagic proteinases, pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier.
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