Escobedo Susana, Pérez de Pipaon Mikel, Rendueles Claudia, Rodríguez Ana, Martínez Beatriz
Instituto de Productos Lacteos de Asturias (IPLA), CSIC, Villaviciosa, Spain.
Front Microbiol. 2023 Jan 20;14:1106049. doi: 10.3389/fmicb.2023.1106049. eCollection 2023.
Bacteriophages are a nuisance in the production of fermented dairy products driven by starter bacteria and strategies to reduce the risk of phage infection are permanently sought. Bearing in mind that the bacterial cell wall plays a pivotal role in host recognition and lysis, our goal was to elucidate to which extent modifications in the cell wall may alter endolysin activity and influence the outcome of phage infection in . Three lactococcal endolysins with distinct catalytic domains (CHAP, amidase and lysozyme) from phages 1,358, p2 and c2 respectively, were purified and their exolytic activity was tested against lactococcal mutants either overexpressing or lacking genes involved in the cell envelope stress (CES) response or in modifying peptidoglycan (PG) composition. After recombinant production in , Lys1358 (CHAP) and LysC2 (muramidase) were able to lyse lactococcal cells in turbidity reduction assays, but no activity of LysP2 was detected. The degree of PG acetylation, namely C--acetylation and de--acetylation influenced the exolytic activity, being LysC2 more active against cells depleted of the PG deacetylase PgdA and the acetyl transferase OatA. On the contrary, both endolysins showed reduced activity on cells with an induced CES response. By measuring several growth parameters of phage c2 on these lactococcal mutants (lytic score, efficiency of plaquing, plaque size and one-step curves), a direct link between the exolytic activity of its endolysin and phage performance could not be stablished.
噬菌体在由发酵剂细菌驱动的发酵乳制品生产中是一个麻烦，人们一直在寻求降低噬菌体感染风险的策略。鉴于细菌细胞壁在宿主识别和裂解中起关键作用，我们的目标是阐明细胞壁的修饰在多大程度上可能改变内溶素活性并影响噬菌体感染的结果。分别从噬菌体1358、p2和c2中纯化了三种具有不同催化结构域（CHAP、酰胺酶和溶菌酶）的乳球菌内溶素，并针对过表达或缺乏参与细胞壁应激（CES）反应或修饰肽聚糖（PG）组成的基因的乳球菌突变体测试了它们的外溶活性。在大肠杆菌中重组生产后，Lys1358（CHAP）和LysC2（胞壁质酶）能够在浊度降低试验中裂解乳球菌细胞，但未检测到LysP2的活性。PG乙酰化程度，即C-乙酰化和去乙酰化，影响外溶活性，LysC2对缺乏PG脱乙酰酶PgdA和乙酰转移酶OatA的细胞更具活性。相反，两种内溶素在具有诱导的CES反应的细胞上均表现出降低的活性。通过测量噬菌体c2在这些乳球菌突变体上的几个生长参数（裂解评分、噬菌斑形成效率、噬菌斑大小和一步生长曲线），无法确定其内溶素的外溶活性与噬菌体性能之间的直接联系。
