Amino acid sequences of SLCTs used in this study.

Phage therapy is a promising alternative to antibiotics for treating Clostridioides difficile infections. Despite its fundamental importance, the initial and most critical step of C. difficile phage infection cycle - the precise recognition of a host receptor - remains poorly understood. The surface-layer protein SlpA was previously identified as a general phage receptor in C. difficile, but the molecular determinants of phage-S-layer interactions remain unclear. We investigated the structural features of SlpA required for phage recognition by engineering and expressing modified isoforms in FM2.5 and R20291 ΔslpA strains. By deleting specific SlpA domains and creating chimeric constructs from different isoforms, we assessed the capacity of seven phages to adsorb and infect the complemented strains. Our results demonstrate that both the LMW and HMW fragments of the S-layer protein contribute to phage specificity in an isoform-dependent manner. In addition, the LMW D2 domain is frequently required, but not always essential for productive infection. Several phages were also able to infect some of the complemented strains despite poor or delayed adsorption, highlighting differences in receptor binding. Interestingly, some phages adsorbed efficiently but failed to infect, showing that the capacity to bind to a host is not always predictive of a successful infection. Our findings reveal the complexity of phage-host interactions in C. difficile and provide new insights into the structural features of the S-layer protein that govern phage binding. These findings will be instrumental in guiding the future design of phage cocktails to target a broad spectrum of C. difficile clinical isolates.

噬菌体疗法（phage therapy）是治疗艰难梭菌（Clostridioides difficile）感染的极具前景的抗生素替代方案。尽管其研究价值至关重要，但艰难梭菌噬菌体感染周期中最基础且最关键的步骤——宿主受体的精准识别——仍知之甚少。此前研究已将S层蛋白SlpA（surface-layer protein SlpA）鉴定为艰难梭菌的通用噬菌体受体，但噬菌体与S层相互作用的分子决定簇仍不明确。本研究通过在FM2.5与R20291 ΔslpA菌株中工程化改造并表达修饰后的蛋白异构体，探究了噬菌体识别所需的SlpA结构特征。通过删除SlpA的特定结构域并构建来自不同异构体的嵌合构建体，我们评估了7株噬菌体吸附并感染互补菌株的能力。研究结果表明，S层蛋白的低分子量（low molecular weight, LMW）与高分子量（high molecular weight, HMW）片段均以异构体依赖的方式参与噬菌体特异性的调控。此外，LMW D2结构域通常是实现有效感染所必需的，但并非绝对不可或缺。部分噬菌体即便吸附能力较弱或吸附延迟，仍可感染部分互补菌株，这凸显了受体结合过程中的差异。有趣的是，部分噬菌体虽可高效吸附宿主却无法完成感染，这表明结合宿主的能力并非总能预测感染是否成功。本研究揭示了艰难梭菌中噬菌体-宿主相互作用的复杂性，并为调控噬菌体结合的S层蛋白结构特征提供了新的见解。上述研究结果将为未来设计可靶向广谱艰难梭菌临床分离株的噬菌体鸡尾酒（phage cocktails）提供重要指导。