Induction of Brain Microvascular Endothelial Cell Urokinase Expression by <em>Cryptococcus neoformans</em> Facilitates Blood-Brain Barrier Invasion

The invasive ability of the blood-borne fungal pathogen Cryptococcus neoformans can be enhanced through interactions with host plasma components, such as plasminogen. Previously we showed by in vitro studies that plasminogen coats the surface of C. neoformans and is converted to the active serine protease, plasmin, by host plasminogen activators. Viable, but not formaldehyde- or sodium azide-killed, cryptococcal strains undergo brain microvascular endothelial cell-dependent plasminogen-to-plasmin activation, which results in enhanced, plasmin-dependent cryptococcal invasion of primary bovine brain microvascular endothelial cells and fungal ability to degrade plasmin substrates. In the present work, brain microvascular endothelial cells cultured with viable, but not killed, cryptococcal strains led to significant increases in both urokinase mRNA transcription and cell-associated urokinase protein expression. Soluble urokinase was also detected in conditioned medium from brain microvascular endothelial cells cultured with viable, but not killed, C. neoformans. Exposure of plasminogen pre-coated viable C. neoformans to conditioned medium from strain-matched brain microvascular endothelial cell-fungal co-cultures resulted in plasminogen-to-plasmin activation and plasmin-dependent cryptococcal invasion. siRNA-mediated silencing of urokinase gene expression or the use of specific inhibitors of urokinase activity abrogated both plasminogen-to-plasmin activation on C. neoformans and cryptococcal-brain microvascular endothelial cell invasion. Our results suggest that pathogen exploitation of the host urokinase-plasmin(ogen) system may contribute to C. neoformans virulence during invasive cryptococcosis.血源性真菌病原体新生隐球菌（Cryptococcus neoformans）的侵袭能力，可通过与宿主血浆成分（如纤溶酶原（plasminogen））的相互作用得到增强。此前我们通过体外研究证实，纤溶酶原可包裹新生隐球菌的菌体表面，并在宿主纤溶酶原激活剂的催化下转化为活性丝氨酸蛋白酶——纤溶酶（plasmin）。仅活的隐球菌菌株，而非经甲醛或叠氮钠灭活的菌株，能够依赖脑微血管内皮细胞（brain microvascular endothelial cell）完成纤溶酶原向纤溶酶的活化过程；该过程可提升依赖纤溶酶的新生隐球菌对原代牛脑微血管内皮细胞的侵袭能力，同时增强真菌降解纤溶底物的能力。在本研究中，与活的隐球菌菌株共培养的脑微血管内皮细胞，其尿激酶（urokinase）的mRNA转录水平与细胞结合型尿激酶蛋白的表达量均显著升高；在该共培养体系的条件培养基中，同样可检测到可溶性尿激酶，而与灭活菌株共培养的内皮细胞则未检测到此物质。将预先包被纤溶酶原的活新生隐球菌，暴露于菌株匹配的脑微血管内皮细胞-真菌共培养体系的条件培养基中，可触发纤溶酶原向纤溶酶的活化，并介导依赖纤溶酶的隐球菌侵袭作用。通过小干扰RNA（siRNA）介导的尿激酶基因沉默，或使用尿激酶活性特异性抑制剂，均可阻断新生隐球菌表面的纤溶酶原活化过程，同时抑制隐球菌对脑微血管内皮细胞的侵袭能力。本研究结果表明，病原体对宿主尿激酶-纤溶酶（原）系统的利用，可能在侵袭性隐球菌病的病程中促进新生隐球菌的毒力表达。