The Efficiency of Bacteriophage Lytic Enzymes in the Course of Bacterial Ghost Generation

Pub Date : 2022-12-25 DOI:10.3103/s0891416822030077

M. E. Platonov, A. S. Vagaiskaya, A. S. Trunyakova, D. V. Grinenko, V. N. Gerasimov, S. V. Dentovskaya, A. P. Anisimov

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Abstract

Bacterial ghosts (BGs) are gram-negative bacteria cell membranes without cytoplasmic content obtained by lysis as a result of cell-wall perforation mediated by the φX174 bacteriophage E protein. Introducing into a lytic plasmid lysis gene from other bacteriophages, which target alternative molecular targets, in addition to the E protein gene of bacteriophage φX174, can lead to an increase in the efficiency of lysis and, in some cases, to an increase in the immunogenicity of preparations. Escherichia coli strains carrying plasmids with various combinations of genes encoding protein E of phage φ174 with cassettes of lytic genes of the “choline–endolysin” systems of phages λ or L-413C, providing different degrees of destruction of the cell wall, were obtained for the subsequent selection of the most promising lytic structures. The formation of E. coli ghosts and the release of cell contents was confirmed by transmission electron microscopy. When the cultivation temperature was increased from 28 to 42°C, the lysis of E. coli cultures DH5α/pEYR'-E, DH5α/pEYR'-Y-K, DH5α/pEYR'-E-Y-K, and DH5α/pEYR'-E-Sam7-R-Rz was observed. Lysis was practically not detected during the growth of the DH5α/pEYR'-S-R-Rz culture, as in the control strain DH5α/pEYR'. The results of the analysis of ultrathin sections of BGs preparations by transmission electron microscopy (TEM) were comparable with the data on optical density and the results of inoculation of induced cultures of engineered strains, and also made it possible to assess the fine structure of bacterial cells carrying various combinations of lytic phage genes. BGs of gram-negative bacteria are promising for the creation of highly effective inactivated candidate vaccines that could replace currently available bacterial heat-inactivated and formol vaccines.

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噬菌体裂解酶在细菌幽灵生成过程中的效率

摘要细菌幽灵(bacterial ghosts, BGs)是由φX174噬菌体E蛋白介导的细胞壁穿孔，通过裂解获得的无细胞质含量的革兰氏阴性细菌细胞膜。除了噬菌体φX174的E蛋白基因外，从其他靶向其他分子靶点的噬菌体中引入裂解质粒裂解基因，可以提高裂解效率，在某些情况下，还可以提高制剂的免疫原性。大肠杆菌菌株携带噬菌体φ174蛋白E编码基因与噬菌体λ或L-413C“胆碱-内溶素”系统裂解基因的不同组合质粒，提供不同程度的细胞壁破坏，为后续选择最有希望的裂解结构提供了基础。透射电镜证实了大肠杆菌幽灵的形成和细胞内容物的释放。当培养温度从28℃升高到42℃时，观察到大肠杆菌培养物DH5α/pEYR'-E、DH5α/pEYR'-Y-K、DH5α/pEYR'-E-Y-K和DH5α/pEYR'-E- sam7 - r - rz的裂解情况。与对照菌株DH5α/pEYR’一样，在DH5α/pEYR’-S-R-Rz培养过程中几乎没有检测到裂解。透射电镜(TEM)超薄切片分析结果与光密度数据和工程菌株诱导培养的接种结果相当，也可以评估携带不同裂解噬菌体基因组合的细菌细胞的精细结构。革兰氏阴性细菌的BGs有望创造出高效的灭活候选疫苗，可以取代目前可用的细菌热灭活疫苗和福尔摩疫苗。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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