S100A10蛋白调节隐球菌穿越血脑屏障的分子机制研究

Cryptococcal invasion and traversal of the Blood-Brain Barrier (BBB) is a prerequisite for CNS infections. Our previous study revealed that the S100A10 in Murine Brain Microvascular Endothial Cell (MBMEC) was an important regulator in the neutropism of Cryptococcus neoformans. Thicker capsule and decreased budding-rate were observed in Cryptococcus neoformans engulfed by MBMEC with S100A10 downregulation, which might be associated with Ca2+ concentration change inside MBMEC due to S100A10 interference (iS100A10). However, the mechanisms by which calcium ion affects cryptococcal survival and propagation inside MBMEC, remain to be further understood. Our project aims to study the changes of morphology, intracellular Ca2+ concentration and transcriptome in Cryptococcus when coincubated with normal versus iS100A10 MBMEC, and identify the essential regulators of Cryptococcus in response to reduced calcium ion through the utilization of gene knock-out combined with phenotypic screening in vivo and ex vivo. Our study will provide new insights for understanding the pathogenic mechanisms involved in cryptococcal translocation of the BBB, and for exploring novel anti-cryptococcal strategies.

病原体穿越血脑屏障是隐球菌感染中枢神经系统的关键，研究分析一些与隐球菌侵入血脑屏障相关的致病因子及其调控机制是阐明隐球菌嗜中枢性的核心。由本课题组前期筛选并部分鉴定的S100A10蛋白就是这样一个重要的候选致病因子。研究发现新生隐球菌被S100A10沉默表达的脑血管内皮细胞吞噬后，荚膜明显增厚，出芽受到抑制，同时伴细胞内的钙离子浓度变化。但钙离子浓度如何影响新生隐球菌在血管内皮细胞内生存和繁殖的确切机制还不清楚。本项目拟通过新生隐球菌与小鼠脑微血管内皮细胞共培养，检测两组（iS100A10组与正常组）细胞内寄生隐球菌的菌体形态、菌内Ca2+浓度及转录组学变化，筛选出隐球菌应答S100A10沉默表达所致血管内皮细胞内钙离子浓度下降的关键调控因子，结合基因敲除与活体内外表型分析，明确S100A10蛋白调节隐球菌穿越血脑屏障的分子机制，初步探索S100A10功能通路作为抗隐球菌药物靶点的可行性。

新生隐球菌是临床最重要的中枢神经系统病原真菌之一，其穿越脑血管内皮细胞(BMECs)的分子机制尚有待于进一步明确。膜联蛋白Annexin A2 (AnxA2)是重要的宿主胞内信号转导蛋白，参与胞吞、膜泡转运等多种生物学过程。本研究首次论证了AnxA2在隐球菌入侵宿主血脑屏障过程中的双重调控作用。隐球菌感染启动了AnxA2在小鼠BMECs中的上调表达；AnxA2缺失或钙离子拮抗剂存在可导致真菌对BMECs的粘附效率增加，而最终穿越效率不受影响。AnxA2表达抑制可导致其配体蛋白S100A10显著表达下调，而S100A10缺失可显著降低隐球菌的穿越效率。此外，AnxA2在Tyr23的去磷酸化以及其下游Cofilin蛋白的去磷酸化均参与隐球菌穿越BMECs过程，主要调控隐球菌对宿主内皮细胞的粘附过程。因此，AnxA2在隐球菌入侵BMECs过程中发挥双重调控作用，主要通过Tyr23磷酸化调控隐球菌的粘附过程，而质膜外表面AnxA2蛋白主要通过调控其伴侣蛋白S100A10表达影响隐球菌穿越血脑屏障。我们的研究系统论证了AnxA2通路在隐球菌粘附和穿越血脑屏障过程中的重要作用，为后续探索阻断隐球菌CNS感染的临床干预靶点奠定了基础。