Gandra Rafael M, Giovanini Lucas, Branquinha Marta H, Santos André L S
Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes (LEAMER), Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
Programa de Pós-Graduação em Bioquímica (PPGBq), Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
Braz J Microbiol. 2025 Aug 18. doi: 10.1007/s42770-025-01765-z.
Candida parapsilosis is a common cause of candidiasis worldwide, with biofilm formation and secretion of aspartic proteases (Saps) as key virulence factors. Conversely, serine protease secretion by this fungus is poorly understood. In this study, we investigated the secretion of serine-type proteases by planktonic- and biofilm-forming cells of C. parapsilosis cultured in brain heart infusion (BHI) medium. Cell-free supernatant from the reference strain (ATCC 22019) was screened against various serine protease substrates, revealing pronounced activity toward N-benzoyl-Phe-Val-Arg-pNa (0.74 nmol pNA.mg.min), with optimal activity at pH 9.0 and temperatures between 32 °C and 40 °C. Proteolytic activity was significantly reduced by serine protease inhibitors PMSF (32.8%), TLCK (40.2%) and benzamidine (50.7%), while inhibitors of other protease classes had no effect, confirming its serine-type specificity. Notably, serine protease activity was detected in supernatants from cells grown in BHI but absent in those cultured in albumin-supplemented yeast carbon base medium, a known inducer of Saps, suggesting culture-dependent regulation of protease expression. Serine protease activity also increased over time, rising from 0.36 pNA.mg.min at 24-hour to 1.14 pNA.mg.min at 72-hour. Clinical isolates of C. parapsilosis exhibited significantly higher serine protease activity than the reference strain under optimal conditions. Serine-type protease activity was also detected in the supernatant of mature biofilms, showing a correlation with metabolic activity and biomass. Infection of Galleria mellonella larvae with C. parapsilosis isolates revealed no correlation between larval mortality and serine protease production. These findings suggest that C. parapsilosis serine proteases contribute to fungal growth and biofilm development, representing potential targets for antifungal intervention.
近平滑念珠菌是全球念珠菌病的常见病因，生物膜形成和天冬氨酸蛋白酶（Saps）分泌是其关键毒力因子。相反，人们对这种真菌分泌丝氨酸蛋白酶的情况了解甚少。在本研究中，我们调查了在脑心浸液（BHI）培养基中培养的浮游型和平板生物膜型近平滑念珠菌细胞分泌丝氨酸型蛋白酶的情况。针对参考菌株（ATCC 22019）的无细胞上清液，对各种丝氨酸蛋白酶底物进行筛选，结果显示其对N-苯甲酰-Phe-Val-Arg-pNa具有显著活性（0.74 nmol pNA·mg·min），在pH 9.0以及32℃至40℃之间的温度下活性最佳。丝氨酸蛋白酶抑制剂PMSF（32.8%）、TLCK（40.2%）和苯甲脒（50.7%）可显著降低蛋白水解活性，而其他蛋白酶类别的抑制剂则无此作用，证实了其丝氨酸型特异性。值得注意的是，在BHI中生长的细胞上清液中检测到丝氨酸蛋白酶活性，但在添加白蛋白的酵母碳源培养基（一种已知的Saps诱导剂）中培养的细胞上清液中未检测到，这表明蛋白酶表达存在培养依赖性调节。丝氨酸蛋白酶活性也随时间增加，从24小时时的0.36 pNA·mg·min升至72小时时的1.14 pNA·mg·min。在最佳条件下，近平滑念珠菌的临床分离株显示出比参考菌株显著更高的丝氨酸蛋白酶活性。在成熟生物膜的上清液中也检测到丝氨酸型蛋白酶活性，显示出与代谢活性和生物量的相关性。用近平滑念珠菌分离株感染大蜡螟幼虫，结果显示幼虫死亡率与丝氨酸蛋白酶产生之间无相关性。这些发现表明，近平滑念珠菌丝氨酸蛋白酶有助于真菌生长和生物膜形成，是抗真菌干预的潜在靶点。
