Taheri-Anganeh Mortaza, Khatami Seyyed Hossein, Jamali Zeinab, Movahedpour Ahmad, Ghasemi Younes, Savardashtaki Amir, Mostafavi-Pour Zohreh
Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
Recombinant Protein Laboratory, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Mol Biol Res Commun. 2019 Dec;8(4):151-158.
Methicillin-resistant (MRSA) is a challenging infectious agent worldwide. The ever growing antibiotic resistance has made the researchers to look for new anti-staphylococcal agents. Autolysins are staphylococcal enzymes that lyse bacterial cell wall for cell division. Autolysins can be used as novel enzybiotics (enzymes have antibiotic effects) for staphylococcal infections. LytU is a newly explored autolysin. SH3b is a potent cell wall binding domain that can be fused to lytic enzymes to increase their activity. The aim of this study was to design a novel and efficient fusion enzybiotic that could lyse staphylococcal cell wall peptidoglycan by disrupting the bacteria. LytU-SH3b fusion construct was synthesized and LytU was amplified through the construct, using overhang PCR. The fusion and native forms that had his-tag were synthesized by recombinant technology in BL21 (DE3) strain and purified utilizing Ni-NTA agarose beads. LytU and LytU-SH3b activity and potency were assessed using plate lysis assay, turbidity reduction assay and minimal inhibitory concentration (MIC) tests. All these tests showed that LytU-SH3b has more activity and potency than LytU. LytU-SH3b has MIC 421 fold lesser than LytU. Finally, LytU-SH3b is a novel and efficient recombinant enzybiotic that can lyse MRSA as an alternative to chemical small molecule antibiotics.
耐甲氧西林金黄色葡萄球菌（MRSA）是一种在全球范围内具有挑战性的感染源。不断增长的抗生素耐药性促使研究人员寻找新的抗葡萄球菌药物。自溶素是葡萄球菌的酶，可裂解细菌细胞壁以进行细胞分裂。自溶素可用作治疗葡萄球菌感染的新型酶抗生素（具有抗生素作用的酶）。LytU是一种新发现的自溶素。SH3b是一种有效的细胞壁结合结构域，可与裂解酶融合以提高其活性。本研究的目的是设计一种新型高效的融合酶抗生素，通过破坏细菌来裂解葡萄球菌细胞壁肽聚糖。合成了LytU-SH3b融合构建体，并通过该构建体使用重叠PCR扩增LytU。带有组氨酸标签的融合形式和天然形式通过重组技术在BL21（DE3）菌株中合成，并使用镍-亚氨基二乙酸琼脂糖珠进行纯化。使用平板裂解试验、浊度降低试验和最低抑菌浓度（MIC）测试评估LytU和LytU-SH3b的活性和效力。所有这些测试表明，LytU-SH3b比LytU具有更高的活性和效力。LytU-SH3b的MIC比LytU低421倍。最后，LytU-SH3b是一种新型高效的重组酶抗生素，可作为化学小分子抗生素的替代品裂解MRSA。
