摘要

肠毒素K1（EntK1），肠毒素EJ97（EntEJ97）和LsbB是三种序列相关的无前导细菌素。然而，LsbB仅杀死乳酸球菌，而EntK1和EntEJ97靶向更宽的谱，EntK1对屎肠球菌尤其活跃，包括医院内多药耐药性菌株。 EntK1的NMR研究表明它具有与LsbB非常相似的结构 - 两者都具有两亲性N-末端α-螺旋和非结构化C-末端。然而，EntK1中的α-螺旋比LsbB长约3-4个残基。发现对EntEJ97和EntK1高度耐药的肠球菌突变体在rseP内具有突变，该基因编码应激反应膜结合Zn依赖性蛋白酶。肠球菌rseP的异源表达使得对肺炎链球菌耐药的细胞对EntK1和EntEJ97敏感，表明RseP可能作为EntK1和EntEJ97的受体。它还表明，粪肠球菌RseP中保守的蛋白水解活性位点对于EntK1和EntEJ97活性部分需要，因为其保守残基（HExxH）的丙氨酸取代降低了克隆对细菌素的敏感性。已知RseP参与细菌应激反应。正如预期的那样，当暴露于较高（胁迫）的生长温度时，例如在45°C时，在rseP内具有突变的抗性突变体的生长受到严重影响，其中野生型细胞仍然生长良好。这些发现使我们能够设计出一种结合细菌素和更高温度的有效杀死细菌素敏感细菌并阻止抗性细胞发育的跨栏策略。

Enterocin K1 (EntK1), enterocin EJ97 (EntEJ97), and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium, including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB – both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3–4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP, a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45∘C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells.

https://www.frontiersin.org/articles/10.3389/fmicb.2017.00774/full