Arroyo-Moreno Sara, Begley Máire, Dembicka Kornelia, Coffey Aidan
Department of Biological Sciences, Munster Technological University, Cork T12 P928, Ireland.
APC Microbiome Institute, University College, Cork T12 YT20, Ireland.
Antibiotics (Basel). 2021 Jun 16;10(6):722. doi: 10.3390/antibiotics10060722.
Bacteriophage endolysins and their derivatives have strong potential as antibacterial agents considering the increasing prevalence of antibiotic resistance in common bacterial pathogens. The peptidoglycan degrading peptidase CHAPk, a truncated derivate of staphylococcal phage K endolysin (LysK), has proven efficacy in preventing and disrupting staphylococcal biofilms. Nevertheless, the concentration of CHAPk required to eliminate populations of stationary-phase cells was previously found to be four-fold higher than that for log-phase cells. Moreover, CHAPk-mediated lysis of stationary-phase cells was observed to be slower than for log-phase cultures. In the present study, we report the fusion of a 165 amino acid fragment containing CHAPk with a 136 amino acid fragment containing the cell-binding domain of the bacteriocin lysostaphin to create a chimeric enzyme designated CHAPk-SH3blys in the vector pET28a. The chimeric protein was employed in concentrations as low as 5 μg/mL, producing a reduction in turbidity in 7-day-old cultures, whereas the original CHAPk required at least 20 μg/mL to achieve this. Where 7-day old liquid cultures were used, the chimeric enzyme exhibited a 16-fold lower MIC than CHAPk. In terms of biofilm prevention, a concentration of 1 μg/mL of the chimeric enzyme was sufficient, whereas for CHAPk, 125 μg/mL was needed. Moreover, the chimeric enzyme exhibited total biofilm disruption when 5 μg/mL was employed in 4-h assays, whereas CHAPk could only partially disrupt the biofilms at this concentration. This study demonstrates that the cell-binding domain from lysostaphin can make the phage endolysin CHAPk more effective against sessile staphylococcal cells.
鉴于常见细菌病原体中抗生素耐药性日益普遍，噬菌体溶菌酶及其衍生物作为抗菌剂具有巨大潜力。肽聚糖降解肽酶CHAPk是葡萄球菌噬菌体K溶菌酶（LysK）的截短衍生物，已被证明在预防和破坏葡萄球菌生物膜方面有效。然而，先前发现消除稳定期细胞群体所需的CHAPk浓度比对数期细胞高四倍。此外，观察到CHAPk介导的稳定期细胞裂解比对数期培养物慢。在本研究中，我们报告了将包含CHAPk的165个氨基酸片段与包含细菌素溶葡萄球菌素细胞结合域的136个氨基酸片段融合，在载体pET28a中创建了一种嵌合酶，命名为CHAPk-SH3blys。该嵌合蛋白的使用浓度低至5μg/mL，可使7日龄培养物的浊度降低，而原始的CHAPk至少需要20μg/mL才能达到此效果。在使用7日龄液体培养物时，嵌合酶的最低抑菌浓度（MIC）比CHAPk低16倍。在生物膜预防方面，1μg/mL的嵌合酶浓度就足够了，而CHAPk则需要125μg/mL。此外，在4小时试验中使用5μg/mL时，嵌合酶表现出完全的生物膜破坏，而CHAPk在此浓度下只能部分破坏生物膜。这项研究表明，溶葡萄球菌素的细胞结合域可以使噬菌体溶菌酶CHAPk对固着的葡萄球菌细胞更有效。
