Table 3_Mycobacteriophage Yasnaya_Polyana and its engineered lytic derivative: specificity of regulatory motifs and lytic potential.xlsx

IntroductionThe growing prevalence of multidrug-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) highlights the urgent need for alternative therapeutic approaches. Mycobacteriophages, viruses that selectively infect mycobacteria, have emerged as promising tools. Here, we report the isolation and characterization of a new subcluster K4 phage, Yasnaya_Polyana, with a focus on its regulatory motifs and engineered lytic variant.
MethodsThe phage was isolated by enrichment on Mycobacterium smegmatis mc(2)155, followed by genome sequencing and functional annotation. Start-Associated Sequences (SAS) and Extended SAS (ESAS) were analyzed in silico across 188 cluster K phages. A lytic derivative, YPΔ47, was engineered by deleting the repressor gene and characterized in terms of morphology, stability, infection dynamics, and host range.
ResultsYasnaya_Polyana exhibited siphovirus morphology and high genetic similarity with other subcluster K4 phages. Regulatory motif analysis revealed a reduced abundance of SAS and ESAS elements in subcluster K4 phages, including Yasnaya_Polyana, along with specific ESAS sequence deviations. The engineered YPΔ47 mutant retained morphology and infection parameters comparable to the wild-type phage but exhibited a decline in lysogeny frequency (from 18% to <0.01%), confirming a lytic phenotype. Host range analysis revealed limited activity of YPΔ47 against NTM, while the phage demonstrated a high efficiency of plating (EOP = 10−1) on M. tuberculosis H37Rv and effectively lysed clinical isolates.
DiscussionThese findings suggest that Yasnaya Polyana, and apparently other subcluster K4 phages, harbor distinct regulatory features that may reflect divergent transcriptional control strategies. Moreover, YPΔ47 shows potential as a candidate for phage therapy targeting mycobacterial infections.

引言：多重耐药结核分枝杆菌与非结核分枝杆菌（nontuberculous mycobacteria, NTM）的流行率日益攀升，凸显了开发新型治疗手段的迫切需求。分枝杆菌噬菌体（mycobacteriophages）——一类特异性侵染分枝杆菌的病毒——已成为颇具前景的研究工具。本研究报道了一株新型K4亚群噬菌体Yasnaya_Polyana的分离与鉴定，并重点聚焦其调控基序与工程化裂解突变株。

方法：本研究通过在耻垢分枝杆菌mc²155中富集分离得到该噬菌体，随后开展基因组测序与功能注释。通过生物信息学手段，对188株K群噬菌体的起始相关序列（Start-Associated Sequences, SAS）及延伸型SAS（Extended SAS, ESAS）进行分析。通过敲除阻遏基因构建裂解型衍生株YPΔ47，并对其形态学特征、稳定性、感染动力学及宿主范围进行表征。

结果：Yasnaya_Polyana属于长尾噬菌体科，与其他K4亚群噬菌体具有较高的遗传相似性。调控基序分析显示，包括Yasnaya_Polyana在内的K4亚群噬菌体中，SAS与ESAS元件的丰度较低，且存在特定的ESAS序列变异。工程化突变株YPΔ47的形态学特征与感染参数与野生型噬菌体基本一致，但溶原化频率从18%降至0.01%以下，证实其具备裂解表型。宿主范围分析表明，YPΔ47对非结核分枝杆菌的裂解活性有限；该噬菌体在结核分枝杆菌H37Rv上的铺板效率（efficiency of plating, EOP）可达10⁻¹，且可有效裂解临床分离菌株。

讨论：本研究结果提示，Yasnaya_Polyana及其他K4亚群噬菌体具有独特的调控特征，这可能反映了差异化的转录调控策略。此外，YPΔ47有望成为针对分枝杆菌感染的噬菌体治疗候选菌株。