Department of Chemical and Biological Engineering, and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, United States.
Department of Chemical and Biological Engineering, and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, United States.
J Biotechnol. 2021 Mar 20;330:27-34. doi: 10.1016/j.jbiotec.2021.02.010. Epub 2021 Feb 27.
The bacteriolysin lysostaphin (Lst) and endolysin PlyPH are potent modular lytic enzymes with activity against clinically-relevant Gram-positive Staphylococcus aureus and Bacillus cereus, respectively. Both enzymes possess an N-terminal catalytic domain and C-terminal binding domain, with the latter conferring significant enzyme specificity. Lst and PlyPH show reduced activity in the presence of bacterial growth-supporting conditions, such as complex media. Here, we hypothesize that Lst and PlyPH bind poorly to their targets in growth media, which may influence their use in antimicrobial applications in the food industry, as therapeutics, and for control of microbial communities. To this end, binding of isolated Lst and PlyPH binding domains to target bacteria was quantified in the presence of three increasingly complex media - phosphate buffered saline (PBS), defined growth medium (AAM) and undefined complex medium (TSB) by surface plasmon resonance (SPR) and flow cytometry. Evaluation of binding kinetics by SPR demonstrated that PlyPH binding was particularly sensitive to medium composition, with 8-fold lower association and 3.4-fold lower dissociation rate constants to B. cereus in TSB compared to PBS. Flow cytometry studies indicated a decrease in the binding-dependent fluorescent populations of S. aureus and B. cereus, for lysostaphin binding domain and PlyPH binding domain, respectively, in TSB compared to PBS. Enzyme binding behavior was consistent with the enzymes' catalytic activity in the three media, thereby suggesting that compromised enzyme binding could be responsible for poor activity in more complex growth media.
溶菌酶 lysostaphin（Lst）和内溶素 PlyPH 是两种强效的模块化溶酶，分别对临床相关的革兰氏阳性葡萄球菌金黄色葡萄球菌和蜡状芽孢杆菌具有活性。这两种酶都具有一个 N 端催化结构域和 C 端结合结构域，后者赋予了酶显著的特异性。Lst 和 PlyPH 在含有支持细菌生长的条件（如复杂培养基）下活性降低。在这里，我们假设 Lst 和 PlyPH 在生长培养基中的靶标结合不良，这可能会影响它们在食品工业中的抗菌应用、作为治疗剂以及控制微生物群落中的应用。为此，我们通过表面等离子体共振（SPR）和流式细胞术，在三种越来越复杂的培养基 - 磷酸盐缓冲盐水（PBS）、定义生长培养基（AAM）和未定义复杂培养基（TSB）中，定量了分离的 Lst 和 PlyPH 结合结构域与靶细菌的结合。SPR 评估的结合动力学表明，PlyPH 的结合对培养基组成特别敏感，与 PBS 相比，TSB 中 PlyPH 与 B. cereus 的结合常数降低了 8 倍，解离常数降低了 3.4 倍。流式细胞术研究表明，与 PBS 相比，Lst 结合结构域和 PlyPH 结合结构域与金黄色葡萄球菌和蜡状芽孢杆菌的结合依赖性荧光群体分别减少了 TSB。酶结合行为与三种培养基中酶的催化活性一致，从而表明在更复杂的生长培养基中，酶结合不良可能是活性降低的原因。
