Shadrin V S, Machulin A V, Dorofeeva L V, Chernyshov S V, Mikoulinskaia G V
Branch of Shemyakin & Ovchinnikov's Institute of Bioorganic Chemistry RAS, Pushchino, Russia.
Skryabin's Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center 'Pushchino Scientific Center for Biological Research of the, Russian Academy of Sciences', Pushchino, Russia.
J Appl Microbiol. 2021 Jun;130(6):1902-1912. doi: 10.1111/jam.14910. Epub 2020 Nov 10.
The objective of this work was to study the antibacterial specificity and antibacterial effect of endolysins isolated from colibacteriophages RB43, RB49 and T5-as manifested on the exponential and stationary cell cultures of diverse bacteria depending on the growth stage, structure of peptidoglycan (PG) and antibiotic resistance.
Enzyme activity was assayed by the spectrophotometric method. Antimicrobial activity was estimated by the number of colony forming units (CFUs), with the results represented as logarithmic units. Morphological examination of bacterial cells was conducted using phase-contrast and scanning electron microscopy. The enzymes EndoT5, endolysin of bacteriophage T5, EndoRB43, endolysin of bacteriophage RB43 and EndoRB49, endolysin of bacteriophage RB49 turned out to be much less bacteriospecific than the corresponding Escherichia coli phages; they lysed bacteria of the genera Bacillus, Cellulomonas and Sporosarcina, whose PGs had different structures (A1γ, A4α and A4β) and chemical modifications (amidation). The specific lytic activity of phage enzymes was independent of the antibiotic resistance of bacterial cells and was higher when the cells were in the exponential, rather than stationary, growth phase. The analysis of morphological changes showed that the intermediate stage of the endolysin-induced lysis of bacterial cells was the formation of spheroplasts and protoplasts.
Endolysins of colibacteriophages RB49, RB43 and T5 have a wide spectrum of antibacterial action, which includes a number of diverse micro-organisms with different PG structures.
This is a study of the bacterial selectivity of enzymes degrading bacterial cell wall in relation to the chemical structure of PG. It is shown that endolysins of bacteriophages RB49 and RB43 efficiently lyse cell wall of Gram-positive bacteria of the genus Bacillus and Gram-negative bacteria of the genus Pseudomonas (including an antibiotic-resistant strain). The number of bacterial cells is reduced by 3-6 orders of magnitude, which indicates good prospects for using these enzymes in biotechnology.
本研究旨在探讨从大肠杆菌噬菌体RB43、RB49和T5中分离出的内溶素的抗菌特异性和抗菌效果，具体表现为其对不同细菌的指数生长期和稳定期细胞培养物的作用，这取决于细菌的生长阶段、肽聚糖（PG）结构和抗生素抗性。
采用分光光度法测定酶活性。通过菌落形成单位（CFU）数量评估抗菌活性，结果以对数单位表示。使用相差显微镜和扫描电子显微镜对细菌细胞进行形态学检查。结果表明，噬菌体T5的内溶素EndoT5、噬菌体RB43的内溶素EndoRB43和噬菌体RB49的内溶素EndoRB49的细菌特异性远低于相应的大肠杆菌噬菌体；它们能裂解芽孢杆菌属、纤维单胞菌属和嗜盐芽孢杆菌属的细菌，这些细菌的PG具有不同结构（A1γ、A4α和A4β）和化学修饰（酰胺化）。噬菌体酶的特异性裂解活性与细菌细胞的抗生素抗性无关，且在细胞处于指数生长期而非稳定期时更高。形态学变化分析表明，内溶素诱导细菌细胞裂解的中间阶段是原生质球和原生质体的形成。
大肠杆菌噬菌体RB49、RB43和T5的内溶素具有广泛的抗菌作用，包括许多具有不同PG结构的微生物。
本研究是关于降解细菌细胞壁的酶对PG化学结构的细菌选择性研究。结果表明，噬菌体RB49和RB43的内溶素能有效裂解芽孢杆菌属革兰氏阳性菌和假单胞菌属革兰氏阴性菌（包括一株抗生素抗性菌株）的细胞壁。细菌细胞数量减少3至6个数量级，这表明这些酶在生物技术领域具有良好的应用前景。
