Kaneko Tomoyoshi, Osaka Toshifumi, Inagaki Minoru, Habe Kento, Okabe Takuma, Tsuneda Satoshi
Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
Phage Therapy Institute, Comprehensive Research Organization, Waseda University, Tokyo, Japan.
J Virol. 2025 Sep 11:e0106125. doi: 10.1128/jvi.01061-25.
Understanding the relationship between bacteriophage (phage) classification and target receptors is crucial for phage ecology and applied research. In this study, we compared 13 previously isolated phages based on physiological characteristics, whole-genome sequences, and tail fiber protein phylogenetics. We improved our previously proposed physiological clustering method by optimizing the bacterial panel for host range assessment, implementing appropriate distance metrics for mixed data types, and applying silhouette coefficient analysis for objective determination of optimal cluster numbers. We combined genomic analysis and lipopolysaccharide (LPS) structural analysis of phage-resistant strains to identify target receptors of the phages. Complementation experiments further confirmed the direct involvement of identified genes in phage reception. The results revealed that phylogenetically distinct phages target different sites in the LPS R-core region (modified by WaaV, WaaW, WaaT, and WaaY), membrane proteins (NfrB, TolA, YhaH), or flagella. Our analysis revealed that subtle chemical modifications of LPS (such as heptose phosphorylation) were shown to be important for phage recognition. Furthermore, physiological characteristics, tail fiber phylogenetics, and whole genome analysis independently classified the phages with high correlation to target receptor specificity. The addition of three phages with known receptors further validated our approach. Our results suggest that grouping based on physiological characteristics (such as lysis dynamics and host range) and genotypes (tail fiber phylogenetics or whole genome analysis) independently classified phages with high correlation to target receptor specificity. Here, we elucidated the diversity and specificity of phage target receptors, providing new insights into the classification of phages and phage-host interactions.
Phage therapy is gaining attention as an alternative treatment for antibiotic-resistant bacteria. Developing effective phage cocktails requires combining phages with different target receptors, but traditional methods for identifying target receptors are labor-intensive. This study demonstrates that E. coli phages targeting the TK001 strain with different target receptors can be grouped based on their physiological characteristics, tail fiber sequences, or whole genomes. Our approach was enhanced through systematic bacterial panel selection for host range assessment, optimized distance metrics for physiological characteristics, and objective cluster determination using silhouette coefficient analysis for all three classifications. This insight can be used to more efficiently create diverse phage cocktails. Additionally, we identified phages targeting diverse sites, including different regions of LPS, membrane proteins, and flagella. These findings deepen our understanding of phage host recognition mechanisms, enabling the rapid preparation of effective phage cocktails and contributing to new advancements in bacterial infection treatment.
了解噬菌体分类与靶标受体之间的关系对于噬菌体生态学和应用研究至关重要。在本研究中，我们基于生理特征、全基因组序列和尾丝蛋白系统发育学对13种先前分离的噬菌体进行了比较。我们通过优化用于宿主范围评估的细菌面板、对混合数据类型实施适当的距离度量以及应用轮廓系数分析来客观确定最佳聚类数，改进了我们先前提出的生理聚类方法。我们结合了噬菌体抗性菌株的基因组分析和脂多糖（LPS）结构分析来鉴定噬菌体的靶标受体。互补实验进一步证实了已鉴定基因直接参与噬菌体识别。结果表明，系统发育上不同的噬菌体靶向LPS R核心区域（由WaaV、WaaW、WaaT和WaaY修饰）、膜蛋白（NfrB、TolA、YhaH）或鞭毛中的不同位点。我们的分析表明，LPS的细微化学修饰（如庚糖磷酸化）对噬菌体识别很重要。此外，生理特征、尾丝系统发育学和全基因组分析独立地对噬菌体进行分类，与靶标受体特异性高度相关。添加三种具有已知受体的噬菌体进一步验证了我们的方法。我们的结果表明，基于生理特征（如裂解动力学和宿主范围）和基因型（尾丝系统发育学或全基因组分析）进行分组，独立地对与靶标受体特异性高度相关的噬菌体进行了分类。在这里，我们阐明了噬菌体靶标受体的多样性和特异性，为噬菌体分类和噬菌体-宿主相互作用提供了新的见解。
噬菌体疗法作为抗生素耐药菌的替代治疗方法正受到关注。开发有效的噬菌体鸡尾酒需要将具有不同靶标受体的噬菌体组合起来，但传统的鉴定靶标受体的方法劳动强度大。本研究表明，针对TK001菌株且具有不同靶标受体的大肠杆菌噬菌体可以根据其生理特征、尾丝序列或全基因组进行分组。我们的方法通过系统选择用于宿主范围评估的细菌面板、优化生理特征的距离度量以及对所有三种分类使用轮廓系数分析进行客观聚类确定而得到增强。这一见解可用于更有效地创建多样化的噬菌体鸡尾酒。此外，我们鉴定了靶向不同位点的噬菌体，包括LPS的不同区域、膜蛋白和鞭毛。这些发现加深了我们对噬菌体宿主识别机制的理解，能够快速制备有效的噬菌体鸡尾酒，并为细菌感染治疗的新进展做出贡献。
