Vázquez Roberto, Gutiérrez Diana, Grimon Dennis, Fernández Lucía, García Pilar, Rodríguez Ana, Briers Yves
Department of Biotechnology, Ghent University, Ghent, Belgium.
Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain.
Probiotics Antimicrob Proteins. 2024 Jul 30. doi: 10.1007/s12602-024-10309-0.
Endolysins, proteins encoded by phages to lyse their hosts and release their progeny, have evolved to adapt to the structural features of each host. The endolysins from Staphylococcus-infecting phages typically feature complex architectures with two enzymatically active domains (EADs) and one cell wall-binding domain (CBD) belonging to the bacterial SH3 (SH3b) superfamily. This study focuses on three SH3b-like CBDs from representative staphylococcal phage endolysins (LysRODI, LysC1C and LysIPLA5) that were structurally and functionally characterized. While RODI_CBD and C1C_CBD were assigned to the well-known SH3_5 family, a new family, SH3b_T (PF24246), was identified using the CBD from LysIPLA5 as a model. GFP-fused CBDs were created to assess their differential binding to a collection of staphylococcal strains. IPLA5_CBD showed enhanced binding to Staphylococcus epidermidis, while RODI_CBD and C1C_CBD exhibited distinct binding profiles, with RODI_CBD targeting Staphylococcus aureus specifically and C1C_CBD displaying broad binding. Sequence comparisons suggested that a few differences in key amino acids could be responsible for the latter binding difference. The CBDs modulated the activity spectrum of synthetic EAD-CBD combinations in accordance with the previous binding profiles, but in a manner that was also dependent on the EAD present in the fusion protein. These results serve as a context for the diversity and versatility of SH3b domains in staphylococcal endolysins, providing insights on how (i) the CBDs from this superfamily have diverged to adapt to diverse bacterial ligands in spite of sharing a common fold; and (ii) the evolution of specificity relies on the EAD-CBD combination rather than solely the CBD.
内溶素是噬菌体编码的用于裂解宿主并释放其后代的蛋白质，它们已经进化以适应每个宿主的结构特征。感染葡萄球菌的噬菌体产生的内溶素通常具有复杂的结构，包含两个酶活性结构域（EAD）和一个属于细菌SH3（SH3b）超家族的细胞壁结合结构域（CBD）。本研究聚焦于来自代表性葡萄球菌噬菌体内溶素（LysRODI、LysC1C和LysIPLA5）的三种SH3b样CBD，对其进行了结构和功能表征。虽然RODI_CBD和C1C_CBD被归类到著名的SH3_5家族，但以LysIPLA5的CBD为模型鉴定出了一个新家族SH3b_T（PF24246）。构建了绿色荧光蛋白融合的CBD，以评估它们与一系列葡萄球菌菌株的差异结合情况。IPLA5_CBD对表皮葡萄球菌的结合增强，而RODI_CBD和C1C_CBD表现出不同的结合谱，RODI_CBD特异性靶向金黄色葡萄球菌，C1C_CBD表现出广泛结合。序列比较表明，关键氨基酸的一些差异可能导致了后者的结合差异。CBD根据先前的结合谱调节合成EAD-CBD组合的活性谱，但方式也取决于融合蛋白中存在的EAD。这些结果为葡萄球菌内溶素中SH3b结构域的多样性和多功能性提供了背景，深入了解了（i）尽管该超家族的CBD具有共同的折叠，但它们如何分化以适应不同的细菌配体；以及（ii）特异性的进化如何依赖于EAD-CBD组合而不是仅依赖于CBD。
