dos Santos André Luis Souza, de Carvalho Isabela Miller, da Silva Bianca Alcântara, Portela Maristela Barbosa, Alviano Celuta Sales, de Araújo Soares Rosangela Maria
Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
FEMS Immunol Med Microbiol. 2006 Mar;46(2):209-20. doi: 10.1111/j.1574-695X.2005.00023.x.
Candida albicans expresses a vast number of hydrolytic enzymes, playing roles in several phases of yeast-host interactions. Here, we identified two novel extracellular peptidase classes in C. albicans. Using gelatin-sodium dodecyl sulfate polyacrylamide gel electrophoresis two gelatinolytic activities were detected at physiological pH: a 60-kDa metallopeptidase, completely blocked by 1,10-phenanthroline, and a 50-kDa serine peptidase inhibited by phenylmethylsulfonyl fluoride. In an effort to establish a probable functional implication for these novel peptidase classes, we demonstrated that the 50-kDa secretory serine peptidase was active over a broad pH range (5.0-7.2) and was capable to hydrolyze some soluble human serum proteins and extracellular matrix components. Conversely, when this isolate was grown in yeast carbon base supplemented with bovine serum albumin, a secretory aspartyl peptidase activity was measured, instead of metallo- and serine peptidases, suggesting that distinct medium composition induces different expression of released peptidases in C. albicans. Additionally, we showed by quantitative proteolytic measurement, flow cytometry and immunoblotting assays that the brain heart infusion medium might repress the Sap1-3 production. Collectively, our results showed for the first time the capability of an extracellular proteolytic enzyme other than aspartic-type peptidases to cleave a broad spectrum of relevant host proteinaceous substrates by the human pathogen C. albicans.
白色念珠菌表达大量水解酶，在酵母与宿主相互作用的几个阶段发挥作用。在此，我们在白色念珠菌中鉴定出两类新的细胞外肽酶。使用明胶 - 十二烷基硫酸钠聚丙烯酰胺凝胶电泳在生理pH值下检测到两种明胶酶活性：一种60 kDa的金属肽酶，被1,10 - 菲咯啉完全阻断，以及一种50 kDa的丝氨酸肽酶，被苯甲基磺酰氟抑制。为了确定这些新肽酶类可能的功能意义，我们证明50 kDa的分泌型丝氨酸肽酶在较宽的pH范围（5.0 - 7.2）内具有活性，并且能够水解一些可溶性人血清蛋白和细胞外基质成分。相反，当该分离株在补充有牛血清白蛋白的酵母碳基中生长时，检测到的是分泌型天冬氨酸肽酶活性，而不是金属肽酶和丝氨酸肽酶，这表明不同的培养基组成会诱导白色念珠菌中释放的肽酶产生不同的表达。此外，我们通过定量蛋白水解测量、流式细胞术和免疫印迹分析表明，脑心浸液培养基可能会抑制Sap1 - 3的产生。总体而言，我们的结果首次表明，除了天冬氨酸型肽酶外，人类病原体白色念珠菌的一种细胞外蛋白水解酶能够切割广泛的相关宿主蛋白质底物。
