Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, 25240 Erzurum, Turkey; Erzurum Technical University, High Technology Research and Application Centre, Erzurum, Turkey.
Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, 25240 Erzurum, Turkey; Erzurum Technical University, High Technology Research and Application Centre, Erzurum, Turkey.
J Proteomics. 2021 Feb 20;233:104075. doi: 10.1016/j.jprot.2020.104075. Epub 2020 Dec 9.
In the present study, 120 fungal isolates were locally isolated from soil and selected according to their ability to antimicrobial activity. Then, selected isolates were tested for their ability to prevent biofilm formation and only one isolate (A01) showed an antibiofilm effect. The isolate A01 identified as Aspergillus tubingensis by sequencing of the 18S ITS region and a segment of β-tubulin gene. Then, 5 fractions were prepared from the culture filtrate of A. tubingensis A01 using the ultrafiltration technique to find active polypeptide fraction. The experiments revealed that one of them had an antibiofilm activity. The MALDI-TOF/MS analyses demonstrated that this polypeptide composed of 92 amino acids and had a molecular mass of 10,087 Da. The sequence alignment showed homology with hypothetical protein (OJI81679.1). The gene coding for this polypeptide consisting of 279 nucleotides, herein we called astucin, was cloned and sequenced from A. tubingensis A01 to confirm results. The MIC of the purified polypeptide was 32 m/L and 128 μg/mL and the MBIC was 2 and 8 μg/mL against Staphylococcus aureus and MRSA, respectively. The results demonstrated that the antimicrobial and antibiofilm activity of astucin, together with its lack of cytotoxicity, makes it an alternative for application in medicine. SIGNIFICANCE: Antibiotic resistance is a global problem and the emergence of antibiotic resistant bacteria reduce the effect the current treatment approaches. In this context, antimicrobial peptides stand out as potentional agents to combat bacterial infection especially, biofilm related infections. Importantly, this study have greatly considered our understanding for fungal derived antibiofilm polypeptides. In this study, traditional selection method combined with crystal violet assay is used to investigate antibiofilm polypeptides. We identified antibiofilm polypeptides purified from A. tubingensis A01. This protein shows antimicrobial and antibiofilm activity against S. aureus.
在本研究中，从土壤中分离出 120 株真菌，根据其抗菌活性选择了这些真菌。然后，对选定的分离物进行了防止生物膜形成的能力测试，只有一个分离物（A01）表现出抗生物膜的作用。通过对 18S ITS 区和一段β-微管蛋白基因的测序，鉴定该分离物为构巢曲霉。然后，通过超滤技术从构巢曲霉 A01 的培养滤液中制备了 5 个馏分，以寻找活性多肽馏分。实验表明，其中一个具有抗生物膜活性。MALDI-TOF/MS 分析表明，该多肽由 92 个氨基酸组成，分子量为 10087 Da。序列比对显示与假想蛋白（OJI81679.1）具有同源性。该多肽的基因编码由 279 个核苷酸组成，我们称之为 astucin，从构巢曲霉 A01 中克隆并测序以确认结果。纯化多肽的 MIC 为 32 m/L 和 128 μg/mL，对金黄色葡萄球菌和 MRSA 的 MBIC 分别为 2 和 8 μg/mL。结果表明，astucin 的抗菌和抗生物膜活性及其缺乏细胞毒性使其成为在医学中应用的替代物。意义：抗生素耐药性是一个全球性问题，抗生素耐药菌的出现降低了当前治疗方法的效果。在这种情况下，抗菌肽作为对抗细菌感染的潜在药物，特别是生物膜相关感染，显得尤为重要。重要的是，这项研究极大地加深了我们对真菌来源的抗生物膜多肽的理解。在这项研究中，传统的选择方法结合结晶紫法用于研究抗生物膜多肽。我们从构巢曲霉 A01 中鉴定出具有抗生物膜活性的多肽。该蛋白对金黄色葡萄球菌具有抗菌和抗生物膜活性。
