葡萄糖诱导前噬菌体Sa3XN活性溶原性增强金黄色葡萄球菌毒力的机制研究

本研究针对金黄色葡萄球菌前噬菌体Sa3int家族的生命周期调控机制这一科学问题，通过发现葡萄糖通过CcpA-cI通路调控ΦSa3XN活性溶原性转换的新机制，揭示了高血糖环境下细菌毒力增强的分子基础，为理解糖尿病易感金黄色葡萄球菌感染提供了新的理论依据。

在微生物与宿主的复杂博弈中，噬菌体与细菌的共生关系一直是科学家关注的焦点。金黄色葡萄球菌作为重要的机会致病菌，其基因组中普遍存在前噬菌体整合现象，其中Sa3int家族噬菌体尤为特殊——它们特异性整合于细菌的β-溶血素编码基因hlb位点，既破坏了细菌重要毒力因子表达，又携带着SCIN、SAK等免疫逃逸基因。这种看似矛盾的现象背后，隐藏着精妙的调控机制：前噬菌体可能作为"分子开关"，通过可控的整合-切出循环动态调节细菌基因表达，而不产生子代噬菌体颗粒，这一现象被称为"活性溶原性"。

尽管前期研究发现感染条件可促进Sa3int噬菌体丢失和Hlb阳性变异株出现，但调控这一特殊生命周期转换的环境信号和分子机制仍知之甚少。特别是在糖尿病等高血糖状态下，金黄色葡萄球菌感染往往更加严重，葡萄糖是否参与调控前噬菌体生命周期转换，进而影响细菌毒力，成为亟待解答的科学问题。

针对这一空白，陆军军医大学微生物学教研室李明教授、周仁杰教授和李刚教授团队在《Virology Journal》发表了最新研究成果。研究人员从耐甲氧西林金黄色葡萄球菌XN108株中鉴定出Sa3int家族噬菌体ΦSa3XN，系统阐明了葡萄糖通过碳代谢调控蛋白CcpA抑制cI阻遏蛋白表达，进而触发ΦSa3XN切出和Hlb表达增加的分子通路，并在高血糖小鼠模型中验证了该通路对皮肤感染严重程度的促进作用。

研究主要采用了以下关键技术：通过PHASTER生物信息学工具预测前噬菌体；利用组合PCR、qPCR和噬斑分析验证活性溶原性特征；采用电泳迁移率变动分析和GFP报告基因实验研究CcpA与cI启动子结合活性；通过链脲佐菌素诱导建立高血糖小鼠模型评估体内毒力。

Features of prophages in S. aureus

研究人员首先对耐甲氧西林金黄色葡萄球菌XN108菌株进行前噬菌体图谱分析，鉴定出6个推定前噬菌体(pp1-pp6)。其中pp5特异性整合于hlb基因内部，编码的整合酶与Sa3int代表株Φ13和ΦSa3mw高度同源，且携带scn、sak、sea等典型毒力基因，因此将其命名为ΦSa3XN。

SA3XN is capable of excision but unable to produce phage virions

通过特异性引物组合PCR证实ΦSa3XN可从染色体切出并环化，qPCR检测显示自发切出频率为4.18×10^-4，丝裂霉素C诱导后显著提高至4.62×10^-3。切出后hlb基因恢复表达，Western blot验证37.2 kDa的Hlb蛋白表达。然而，噬斑形成实验、透射电镜和DNase保护实验均表明，ΦSa3XN切出后不产生感染性噬菌体颗粒，符合活性溶原性特征。

P<0.001.(E) Hemolytic activity of XN108 and derivatives. Data are the mean±SD from three independent replicates and determined by one-way ANOVA with Tukey's multiple comparisons test,*P<0.0001.(F) Representative hemolysis of XN108 and derivatives cultured on Columbia blood agar.△Sa3XN forms a clear hemolytic ring.(G) SDS-PAGE analysis of whole exoproteins from overnight cultures of XN108 and△Sa3XN.The red arrow indicates the band between 35 and 40 kDa that was present in△Sa3XN but not in XN108.(H) Western blot analysis for Hlb expression in XN108 and△Sa3XN.Representative gel is shown from three inde-pendent experiments.LC:loading control.(I)TEM analysis for overnight culture supernatants of XN108 and DDSa3XN that treated with 1μg/mL MMC.No obvious difference for phage particles was detected between XN108 and△DSa3XN'>

Glucose promotes DSa3XN excision and enhances the hemolytic activity of S. aureus

2%葡萄糖处理显著提高ΦSa3XN切出频率约15.6倍，并增强细菌溶血活性，提示葡萄糖是调控前噬菌体生命周期的重要环境信号。

CcpA responds to glucose and directly modulates Sa3XN excision

机制研究表明，葡萄糖激活碳代谢调控蛋白CcpA，其直接结合于cI基因启动子区的cre位点(5'-ATGAAAATTTCGTATA-3')，抑制cI阻遏蛋白表达，从而解除对前噬菌体裂解周期的阻遏。电泳迁移率变动分析和GFP报告基因实验证实了CcpA对cI启动子的直接调控作用。CcpA基因敲除株中，葡萄糖诱导的ΦSa3XN切出和溶血活性增强效应被显著削弱。

P<0.01;P<0.001;ns,not sig-nificant.(B) Hemolytic activities of XN108 and DccpA as treated in(A).The hemolytic activity of untreated XN108 was set to 1.Data are the mean±SD from three independent replicates and determined by two-way ANOVA with Sidak's multiple comparisons test,P<0.001;P<0.0001;ns,not significant.(C)RT-qPCR analysis of ccpA,cl,and mor expression in XN108 after 2% glucose treatment. Data are the mean±SD from three independent replicates and analyzed by Student's t-test,P<0.05.(D) RT-qPCR analysis of cl/expression in XN108 and mutant DccpA.Data are the mean±SD from three independent replicates and determined by Student's t-test,P<0.01.(E) The frequency plot of CcpA-binding cre sites. Examples of known cre sites positioned in the promoter regions of corresponding genes are shown below.The predicted CcpA-binding site in cl promoter is highlighted in red characters.(F) EMSA analysis of specific binding of CcpA to cl promoter. A 200-fold excess of unlabeled probe(Pcl) was included as a competitor. Probes Biotin-ProcD and Biotin-PproC served as positive and negative controls, respectively. Representative gel from three independent experiments is shown.(G) Assessment of c/ promoter activity. S. aureus XN108 containing the GFP-based reporter vector was cultivated overnight that untreated or supplemented with 2%glucose, and fluorescence was measured and normalized to bacterial growth. Data are the mean±SD from three independent replicates and analyzed by Student's t-test,***P<0.0001'>

Sa3XN excision-restored Hlb augments S. aureus virulence in hyperglycemic mice

通过构建保留hlb天然终止密码子的△hlb^C突变株，研究人员在高血糖小鼠皮肤感染模型中证实，ΦSa3XN切出恢复的Hlb表达显著加重皮肤炎症反应和皮下脓肿形成。组织病理学分析显示XN108感染组炎症细胞浸润更明显，结缔组织排列更松散，血管充血更严重。

P<0.01;ns,not significant.(B) Hemolytic activities of XN108 and△h/bC as treated in(A).The hemolytic activity of untreated XN108 was set to 1.Data are the mean±SD from three independent replicates and determined by two-way ANOVA with Sidak's multiple comparisons test,P<0.0001;ns,not significant.(C) Schematic illustrating the animal experimental procedure.(D) The 6-h fasting blood glucose levels of mice before and after streptozotocin injection.Five mice successfully developed hyperglycemia with FBG more than 11.1 mmol/L(the dotted line).(E) Monitoring for abscess formation. Data are the mean±SD(n=5 mice) and analyzed by two-way ANOVA with Sidak's multiple comparisons test,P<0.05.(F) Representative images of mouse skin damage.(G) Representative HE staining of mouse skin tissues collected on day 6 post-infection.The red triangles indicate the infiltration of inflammatory cells, black triangles show the vascular congestion, and blue triangles point to the loosed arrangement of connective tissues.(H) The severity of the skin lesions in histological sections of(G) was numerically classified with ImageJ software, and the relative inflammatory cell infiltration area(%) was calculated and indicated.Three sections with the bars of 200 um were used.Statistical significance was determined by Student's t-test,P<0.05'>

本研究首次系统阐明了葡萄糖-CcpA-cI信号通路调控Sa3int家族噬菌体活性溶原性的分子机制，提出了前噬菌体作为环境响应型分子开关的新范式。研究发现不仅深化了对金黄色葡萄球菌与噬菌体共生关系的理解，更重要的是为解释糖尿病状态下细菌感染加重的临床现象提供了分子层面的机制证据。

该研究建立的"环境信号-细菌调控蛋白-前噬菌体生命周期-毒力表达"四层调控网络，为开发针对糖尿病合并金黄色葡萄球菌感染的新型治疗策略提供了潜在靶点。未来研究可进一步探索临床糖尿病标本中Sa3int噬菌体携带、Hlb表达与疾病严重程度的因果关系，推动基础研究发现向临床应用的转化。