The Metalloprotease, Mpr1, Engages AnnexinA2 to Promote the Transcytosis of Fungal Cells across the Blood-Brain Barrier.
The Metalloprotease, Mpr1, Engages AnnexinA2 to Promote the Transcytosis of Fungal Cells across the Blood-Brain Barrier.
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DOI：
10.3389/fcimb.2017.00296
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发表时间：
2017
影响因子：
5.7
通讯作者：
Gelli A
中科院分区：
文献类型：
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作者：
Na Pombejra S;Salemi M;Phinney BS;Gelli A
真核病原体在突破血脑屏障（BBB）和侵入中枢神经系统（CNS）方面表现出多种机制。在导致哺乳动物疾病的真菌类中，只有一些穿过构成血脑屏障的大脑微血管内皮细胞侵入大脑。新型隐球菌是真菌性脑膜脑炎的主要病因，它通过胞吞作用或吞噬单核细胞直接穿过血脑屏障。我们之前确定了Mpr1，一种分泌的真菌金属蛋白酶，促进真菌细胞与脑微血管内皮细胞的结合，我们证实了CnMPR1的单一表达赋予酿酒酵母穿越血脑屏障的能力。本研究利用CnMPR1赋予酿酒酵母的功能增益（即Sc<CnMPR1>菌株）来鉴定可能位于血脑屏障表面的Mpr1靶点。生物素标记BBB表面蛋白后，LC-MS/MS鉴定Sc<CnMPR1>-相关蛋白。在鉴定的62个蛋白中，有几个与细胞骨架内吞相关，包括AnnexinA2 （AnxA2）。利用AnxA2活性被阻断的人血脑屏障体外模型，我们发现缺乏AnxA2活性阻止了葡萄球菌在血脑屏障上的运动（即Sc<CnMPR1>菌株的胞活），但出乎意料的是，TEM分析显示，Sc<CnMPR1>的关联或内化并不需要AnxA2。此外，AnxA2和Sc<CnMPR1>的共定位表明，血脑屏障的成功穿越依赖于axna2 - mpr1介导的相互作用。总的来说，这些数据表明AnxA2在人脑微血管内皮细胞的真菌胞吞作用中起核心作用。Sc<CnMPR1>的运动和胞外分泌依赖于涉及AnxA2的膜运输事件，但这些事件似乎与宿主细胞表面的AnxA2的作用无关。我们提出Mpr1活性促进脑微血管内皮细胞的细胞骨架重塑，从而参与AnxA2以促进血脑屏障的真菌胞吞作用。
Eukaryotic pathogens display multiple mechanisms for breaching the blood-brain barrier (BBB) and invading the central nervous system (CNS). Of the fungal spp., that cause disease in mammals, only some cross brain microvascular endothelial cells which constitute the BBB, and invade the brain. Cryptococcus neoformans, the leading cause of fungal meningoencephalitis, crosses the BBB directly by transcytosis or by co-opting monocytes. We previously determined that Mpr1, a secreted fungal metalloprotease, facilitates association of fungal cells to brain microvascular endothelial cells and we confirmed that the sole expression of CnMPR1 endowed S. cerevisiae with an ability to cross the BBB. Here, the gain of function conferred onto S. cerevisiae by CnMPR1 (i.e., Sc<CnMPR1> strain) was used to identify targets of Mpr1 that might reside on the surface of the BBB. Following biotin-labeling of BBB surface proteins, Sc<CnMPR1>-associated proteins were identified by LC-MS/MS. Of the 62 proteins identified several were cytoskeleton-endocytosis-associated including AnnexinA2 (AnxA2). Using an in vitro model of the human BBB where AnxA2 activity was blocked, we found that the lack of AnxA2 activity prevented the movement of S. cerevisiae across the BBB (i.e., transcytosis of Sc<CnMPR1> strain) but unexpectedly, TEM analysis revealed that AnxA2 was not required for the association or the internalization of Sc<CnMPR1>. Additionally, the co-localization of AnxA2 and Sc<CnMPR1> suggest that successful crossing of the BBB is dependent on an AxnA2-Mpr1-mediated interaction. Collectively the data suggest that AnxA2 plays a central role in fungal transcytosis in human brain microvascular endothelial cells. The movement and exocytosis of Sc<CnMPR1> is dependent on membrane trafficking events that involve AnxA2 but these events appear to be independent from the actions of AnxA2 at the host cell surface. We propose that Mpr1 activity promotes cytoskeleton remodeling in brain microvascular endothelial cells and thereby engages AnxA2 in order to facilitate fungal transcytosis of the BBB.