Neto João X S, Pereira Mirella L, Oliveira Jose T A, Rocha-Bezerra Lady C B, Lopes Tiago D P, Costa Helen P S, Sousa Daniele O B, Rocha Bruno A M, Grangeiro Thalles B, Freire José E C, Monteiro-Moreira Ana Cristina O, Lobo Marina D P, Brilhante Raimunda S N, Vasconcelos Ilka M
Department of Biochemistry and Molecular Biology, Federal University of CearaFortaleza, Brazil.
Department of Biology, Federal University of CearaFortaleza, Brazil.
Front Microbiol. 2017 Jun 6;8:980. doi: 10.3389/fmicb.2017.00980. eCollection 2017.
species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from seeds and to evaluate its antifungal properties against species. The purified protein, named -CBP, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, -CBP presented 13,309 Da. However, by SDS-PAGE, -CBP migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of -CBP under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that -CBP exists in different oligomeric forms. Moreover, -CBP is a basic glycoprotein (pI 10.9) with 4.1% (m/m) sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from -CBP in solution, after LC-ESI-MS/MS, revealed similarity with other proteins, as the 2S albumin -CBP and flocculating proteins, and 2S albumins from different species. -CBP possesses antifungal activity against , , , and , with MIC and MIC values ranging between 9.45-37.90 and 155.84-260.29 μM, respectively. In addition, -CBP (18.90 μM) increased the cell membrane permeabilization and reactive oxygen species production in and promoted degradation of circular plasmid DNA (pUC18) from . The data presented in this study highlight the potential use of -CBP as an anticandidal agent, based on its ability to inhibit spp. growth with apparently low toxicity on mammalian cells.
某些物种是机会性致病菌，可感染免疫功能低下和/或免疫抑制的患者，尤其是在医院环境中，这不仅对健康构成重大威胁，还会增加死亡风险。除了耐受性良好的棘白菌素和三唑类药物外，大多数用于治疗念珠菌病的抗真菌药物都会引起副作用并导致耐药菌株的产生。传统治疗方法的一种有前景的替代方案是使用植物蛋白。Lam.是一种具有宝贵药用特性的植物，包括抗菌活性。这项工作旨在从种子中纯化一种几丁质结合蛋白，并评估其对某些物种的抗真菌特性。纯化后的蛋白名为-CBP，约占种子总蛋白的0.2%，在非变性聚丙烯酰胺凝胶电泳（native PAGE）上呈现为单一条带。通过质谱分析，-CBP的分子量为13,309 Da。然而，在十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）中，-CBP迁移为一条表观分子量为23,400 Da的单一带。在还原条件下对-CBP进行三羟甲基氨基甲烷-十二烷基硫酸钠聚丙烯酰胺凝胶电泳（Tricine-SDS-PAGE）显示出两条表观分子量分别为7,900和4,600 Da的蛋白带。总之，这些结果表明-CBP以不同的寡聚形式存在。此外，-CBP是一种碱性糖蛋白（pI 10.9），含糖量为4.1%（m/m），对兔和人红细胞不显示血凝和溶血活性。在液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）后，对溶液中-CBP的胰蛋白酶肽段序列进行的比较分析显示，它与其他某些蛋白相似，如2S白蛋白-CBP和絮凝蛋白，以及来自不同物种的2S白蛋白。-CBP对某些物种具有抗真菌活性，对[具体物种1]、[具体物种2]、[具体物种3]和[具体物种4]的最低抑菌浓度（MIC）和最低杀菌浓度（MIC）值分别在9.45 - 37.90和155.84 - 260.29 μM之间。此外，-CBP（18.90 μM）增加了[具体物种5]和[具体物种6]的细胞膜通透性和活性氧的产生，并促进了来自[具体物种7]的环状质粒DNA（pUC18）的降解。本研究中呈现的数据突出了-CBP作为抗念珠菌剂的潜在用途，基于其抑制某些念珠菌属物种生长的能力以及对哺乳动物细胞明显较低的毒性。
