Oliveira Aline Ferreira, Fernandes Fabricio Freitas, Mariano Vânia Sammartino, Almeida Fausto, Ruas Luciana Pereira, Oliveira Leandro Licursi, Oliver Constance, Jamur Maria Celia, Roque-Barreira Maria Cristina
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil.
Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brasil.
PLoS One. 2017 Aug 28;12(8):e0184010. doi: 10.1371/journal.pone.0184010. eCollection 2017.
Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-β-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72-96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation.
据报道，巴西副球孢子菌酵母表达副球孢子蛋白，这是一种结合GlcNAc的蛋白，具有N-乙酰-β-D-葡萄糖胺酶（NAGase）活性。高度特异性的抗副球孢子蛋白抗体先前已被用于检测副球孢子蛋白在酵母中的定位，并在体外抑制其生长。在本研究中，抗副球孢子蛋白抗体被用于通过扫描共聚焦显微镜来表征副球孢子蛋白在巴西副球孢子菌菌丝、从菌丝到酵母的过渡形式以及成熟酵母中的分布。在菌丝阶段，副球孢子蛋白主要在菌丝尖端被检测到，在那里它呈现点状分布，并与细胞壁相关。在温度从26°C转变为37°C后的最初48小时内，分化菌丝中的副球孢子蛋白表达主要在出芽区域被检测到，即侧向突起处以及新的子细胞内部。在温度转变72 - 96小时后，有更多的厚垣孢子在其表面和/或内部表达高浓度的副球孢子蛋白。120小时后，酵母细胞成为主要形式，其细胞质广泛地被副球孢子蛋白染色，而小麦胚凝集素（WGA）染色在其外壁上占主导。在37°C下培养10天后，母酵母细胞和子酵母细胞的内部以及出芽区域都被副球孢子蛋白强烈染色。不同真菌形式中mRNA表达的比较表明，PCN转录本虽然在所有评估的形态形式中都能检测到，但在菌丝和酵母到菌丝的过渡形式中含量更高。总之，副球孢子蛋白在所有巴西副球孢子菌形态型中的分布模式支持了普遍的观点，即它在真菌生长和二态性转变中起重要作用。
