Matin Abdul, Stins Monique, Kim Kwang Sik, Khan Naveed Ahmed
School of Biological and Chemical Sciences, Birkbeck College, University of London, London, UK.
FEMS Immunol Med Microbiol. 2006 Jun;47(1):83-91. doi: 10.1111/j.1574-695X.2006.00065.x.
Balamuthia mandrillaris is a recently identified protozoan pathogen that can cause fatal granulomatous encephalitis. However, the pathogenesis and pathophysiology of B. mandrillaris encephalitis remain unclear. Because proteases may play a role in the central nervous system (CNS) pathology, we used spectrophotometric, cytopathic and zymographic assays to assess protease activities of B. mandrillaris. Using two clinical isolates of B. mandrillaris (from human and baboon), we observed that B. mandrillaris exhibits protease activities. Zymographic assays revealed major protease bands of approximate molecular weights in the region of 40-50 kDa on sodium dodecyl sulfate-polyacrylamide gels using gelatin as substrate. The protease bands were inhibited with 1,10-phenanthroline, suggesting metallo-type proteases. The proteolytic activities were observed over a pH range of 5-11 with maximum activity at neutral pH and at 42 degrees C. Balamuthia mandrillaris proteases exhibit properties to degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue. This is shown by degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as other substrates such as casein and gelatin but not haemoglobin. However, these proteases exhibited a minimal role in B. mandrillaris-mediated host cell death in vitro using human brain microvascular endothelial cells (HBMECs). This was shown using broad-spectrum matrix metalloprotease inhibitors, GM 6001 and GM 1489, which had no effect on B. mandrillaris-mediated HBMEC cytotoxicity. This is the first demonstration that B. mandrillaris exhibits metalloproteases, which may play important role(s) in the ECM degradation and thus in CNS pathology.
曼氏巴贝斯虫是一种最近才被确认的原生动物病原体，可导致致命的肉芽肿性脑炎。然而，曼氏巴贝斯虫脑炎的发病机制和病理生理学仍不清楚。由于蛋白酶可能在中枢神经系统（CNS）病理过程中发挥作用，我们使用分光光度法、细胞病变法和酶谱分析法来评估曼氏巴贝斯虫的蛋白酶活性。使用曼氏巴贝斯虫的两个临床分离株（来自人类和狒狒），我们观察到曼氏巴贝斯虫表现出蛋白酶活性。酶谱分析显示，在以明胶为底物的十二烷基硫酸钠-聚丙烯酰胺凝胶上，主要蛋白酶条带的近似分子量在40-50 kDa范围内。这些蛋白酶条带被1,10-菲啰啉抑制，表明是金属蛋白酶类型。在pH值为5-11的范围内观察到蛋白水解活性，在中性pH值和42摄氏度时活性最高。曼氏巴贝斯虫蛋白酶具有降解细胞外基质（ECM）的特性，而细胞外基质为脑组织提供结构和功能支持。这通过I型和III型胶原蛋白（胶原性ECM的主要成分）、弹性蛋白（ECM的弹性纤维）、纤溶酶原（参与ECM的蛋白水解降解）以及其他底物如酪蛋白和明胶但不包括血红蛋白的降解得以证明。然而，在使用人脑微血管内皮细胞（HBMECs）进行的体外实验中，这些蛋白酶在曼氏巴贝斯虫介导的宿主细胞死亡中发挥的作用极小。使用广谱基质金属蛋白酶抑制剂GM 6001和GM 1489可证明这一点，它们对曼氏巴贝斯虫介导的HBMEC细胞毒性没有影响。这是首次证明曼氏巴贝斯虫具有金属蛋白酶，其可能在ECM降解中发挥重要作用，进而在CNS病理过程中发挥重要作用。
