Laboratorio de Microbiología y Proteómica, Instituto de Oftalmología "Fundación de Asistencia Privada Conde de Valenciana", Mexico City, Mexico; Laboratorio de Estructura de Proteínas, Instituto Nacional de Medicina Genómica, Mexico City, Mexico; Laboratorio de Microbiología Molecular, Universidad Autónoma de Querétaro, Qro, Mexico.
Laboratorio de Estructura de Proteínas, Instituto Nacional de Medicina Genómica, Mexico City, Mexico.
Microb Pathog. 2019 May;130:232-241. doi: 10.1016/j.micpath.2019.03.001. Epub 2019 Mar 6.
Biofilms are structures that confer adaptive ability to and facilitate the virulence of fungal pathogens. Certain multi-functional proteins have been shown to be involved in fungal pathogenesis and these proteins may also be implicated in biofilm formation. The aim of this study was to identify a fungal agent isolated from the human cornea, to analyze the ability of this organism to form biofilms in vitro and to investigate protein expression in this condition. The fungus was identified by phylogenetic inference analysis. Biofilm formation and structure were evaluated by colorimetric methods and by optical and electron microscopy. We also resolved proteins obtained from biofilms and planktonic cultures by two-dimensional gel electrophoresis and identified those proteins by mass spectrometry. The fungus was identified as Fusarium falciforme. Colorimetric analysis and microscopy revealed that the highest level of biofilm formation was obtained at a concentration of 1 × 10 conidia/mL with 96 h of incubation at 28 °C. The biofilm architecture consisted of an extracellular matrix that embedded fungal filaments. We found nineteen proteins that were over-expressed in biofilms, as compared with planktonic cultures, and six proteins with unique expression in biofilms. Among the more abundant proteins identified were: transketolase, a putative antigen 1, enolase, phosphoglycerate kinase and ATP-citrate synthase. Some of these proteins are involved in basal metabolism, function as multi-functional proteins or have been described as potential virulence factors. We focused on the expression in biofilm of the enzyme, enolase, which was determined by real-time PCR. Our findings provide a perspective on the proteins associated with the formation of biofilms in vitro by an F. falciforme keratitis isolate.
生物膜赋予真菌病原体适应能力并促进其毒力。某些多功能蛋白已被证明参与真菌发病机制，这些蛋白也可能与生物膜形成有关。本研究旨在鉴定一种从人角膜分离的真菌剂，分析该生物体在体外形成生物膜的能力，并研究该条件下的蛋白表达。通过系统发育推断分析鉴定真菌。通过比色法和光学及电子显微镜评估生物膜形成和结构。我们还通过二维凝胶电泳解析来自生物膜和浮游培养物的蛋白质，并通过质谱鉴定这些蛋白质。该真菌被鉴定为镰孢弯孢菌。比色分析和显微镜观察显示，在 28°C 孵育 96 小时，浓度为 1×10 个分生孢子/mL 时，生物膜形成水平最高。生物膜结构由嵌入真菌丝的细胞外基质组成。与浮游培养物相比，我们发现生物膜中表达上调的蛋白有 19 种，而在生物膜中具有独特表达的蛋白有 6 种。鉴定出的较丰富蛋白包括：转酮醇酶、假定抗原 1、烯醇酶、磷酸甘油酸激酶和 ATP-柠檬酸合酶。其中一些蛋白参与基础代谢，作为多功能蛋白发挥作用，或被描述为潜在的毒力因子。我们关注的是在生物膜中表达的酶烯醇酶，通过实时 PCR 确定。我们的研究结果为体外形成生物膜的 F. falciforme 角膜炎分离株相关蛋白提供了一个视角。
