A streptococcal Fic domain-containing protein disrupts blood-brain barrier integrity by activating moesin in endothelial cells

Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic pathogen capable of causing meningitis in humans. The mechanisms that enable pathogens to traverse the blood-brain barrier (BBB) are incompletely understood. Here, we investigated the role of a newly identified Fic domain-containing protein, BifA, in SEZ virulence. BifA was required for SEZ to cross the BBB and to cause meningitis in mice. BifA also enhanced SEZ translocation across human Brain Microvascular Endothelial Cell (hBMEC) monolayers. Purified BifA or its Fic domain-containing C-terminus alone were able to enter into hBMECs, leading to disruption of monolayer barrier integrity. A SILAC-based proteomic screen revealed that BifA binds moesin. BifA’s Fic domain was required for its binding to this regulator of host cell cytoskeletal processes. BifA treatment of hBMECs led to moesin phosphorylation and downstream RhoA activation. Inhibition of moesin activation or moesin depletion in hBMEC monolayers abrogated BifA-mediated increases in barrier permeability and SEZ’s capacity to translocate across monolayers. Thus, BifA activation of moesin appears to constitute a key mechanism by which SEZ disrupts endothelial monolayer integrity to penetrate the BBB.

马链球菌兽疫亚种（Streptococcus equi subsp. zooepidemicus，SEZ）是一种可引发人类脑膜炎的人畜共患病原菌。目前，病原体穿越血脑屏障（blood-brain barrier，BBB）的分子机制尚未完全明晰。本研究聚焦于新鉴定的含Fic结构域蛋白BifA在SEZ致病过程中的作用展开探究。实验结果显示，BifA是SEZ穿越血脑屏障并诱发小鼠脑膜炎所必需的关键因子。此外，BifA可增强SEZ对人脑微血管内皮细胞（human Brain Microvascular Endothelial Cell，hBMEC）单层的跨膜转运能力。纯化的BifA蛋白或其仅携带Fic结构域的C端片段即可直接进入hBMEC，进而破坏细胞单层的屏障完整性。基于SILAC的蛋白质组学筛选结果表明，BifA可结合膜突蛋白（moesin）。BifA的Fic结构域是其与该宿主细胞骨架调控蛋白相结合的必需结构。BifA处理hBMEC可诱导膜突蛋白发生磷酸化，并激活下游RhoA信号通路。抑制hBMEC单层中膜突蛋白的活化或敲低膜突蛋白的表达，均可阻断BifA介导的屏障通透性升高以及SEZ穿越细胞单层的能力。综上，BifA通过激活膜突蛋白，构成了SEZ破坏内皮细胞单层完整性以穿透血脑屏障的核心致病机制。