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Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression.
BMC Biotechnology ( IF 3.5 ) Pub Date : 2019-12-21 , DOI: 10.1186/s12896-019-0594-7
Rosemarie W Hammond 1 , Steven M Swift 2 , Juli A Foster-Frey 2 , Natalia Y Kovalskaya 1, 3 , David M Donovan 2
Affiliation  

BACKGROUND Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and a cause of necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of drug-resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains. RESULTS An Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) containing a polyhistidine tag was expressed in E. coli. In addition, The E. coli optimized endolysin gene was engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X (PVX)-based transient expression vector. PlyCP41p accumulated to ~ 1% total soluble protein (100μg/gm f. wt. leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal systemic expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue. CONCLUSION We demonstrated that functionally active bacteriophage PlyCP41 endolysin can be produced in systemically infected plant tissue with potential for use of crude plant sap as an effective antimicrobial agent against C. perfringens.

中文翻译:

使用基于病毒的系统表达在植物中优化生产具有生物活性的产气荚膜梭菌糖基水解酶噬菌体内溶素 PlyCP41。

背景技术产气荚膜梭菌是一种革兰氏阳性、厌氧、杆状细菌,是人类食源性细菌疾病的第三大原因,也是家禽坏死性肠炎的原因。它可以通过抗生素来控制,广泛使用抗生素可能会导致耐药细菌的产生。噬菌体编码的内溶素可降解细菌细胞壁中的肽聚糖,是抗生素的潜在替代品。噬菌体内溶素已被鉴定对多种梭菌菌株表现出抗菌活性。结果编码含有多组氨酸标签的糖基水解酶内溶素(PlyCP41)的大肠杆菌密码子优化基因在大肠杆菌中表达。此外,大肠杆菌优化的内溶素基因被设计用于在植物中表达(PlyCP41p),并且植物密码子优化的基因(PlyCP41pc)均含有多组氨酸标签,使用马铃薯病毒X(PVX)在本塞姆塞姆氏烟草植物中表达基于瞬时表达载体。PlyCP41p 积累了约 1% 的总可溶性蛋白(100μg/gm f. wt. 叶组织),对植物细胞没有任何明显的毒性作用,并且纯化的蛋白和植物汁液均含有在平板裂解中裂解的产气荚膜梭菌菌株 Cp39化验。PlyCP41p 在感染后 2 周达到最佳全身表达。PlyCP41pc 在感染组织中的累积水平并不高于 PlyCP41p。结论 我们证明,具有功能活性的噬菌体 PlyCP41 内溶素可以在系统感染的植物组织中产生,具有利用粗植物汁液作为针对产气荚膜梭菌的有效抗菌剂的潜力。
更新日期:2020-04-22
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