Deng Simin, Li Haojie, Zhou Chang, Fan Jingyan, Zhu Fuxin, Jin Gexuan, Xu Jiali, Xia Jing, Wang Jing, Nie Zheng, Zhou Rui, Song Houhui, Cheng Changyong
Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection & Internet Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Veterinary Medicine of Zhejiang A&F University, Hangzhou, Zhejiang, P.R. China.
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, P.R. China.
Virulence. 2024 Dec;15(1):2401963. doi: 10.1080/21505594.2024.2401963. Epub 2024 Sep 16.
() is an important swine bacterial pathogen and causes human infections, leading to a wide range of diseases. However, the role of 5'-nucleotidases in its virulence remains to be fully elucidated. Herein, we identified four cell wall-anchored 5'-nucleotidases (Snts) within , named SntA, SntB, SntC, and SntD, each displaying similar domains yet exhibiting low sequence homology. The malachite green reagent and HPLC assays demonstrated that these recombinant enzymes are capable of hydrolysing ATP, ADP, and AMP into adenosine (Ado), with the hierarchy of catalytic efficiency being SntC>SntB>SntA>SntD. Moreover, comprehensive enzymatic activity assays illustrated slight variances in substrate specificity, pH tolerance, and metal ion requirements, yet highlighted a conserved substrate-binding pocket, His-Asp catalytic dyad, metal, and phosphate-binding sites across Snts, with the exception of SntA. Through bactericidal assays and murine infection assays involving in site-mutagenesis strains, it was demonstrated that SntB and SntC collaboratively enhance bacterial survivability within whole blood and polymorphonuclear leukocytes (PMNs) the Ado-A2aR pathway , and within murine blood and organs . This suggests a direct correlation between enzymatic activity and enhancement of bacterial survival and virulence. Collectively, 5'-nucleotidases additively contribute to the generation of adenosine, influencing susceptibility within blood and PMNs, and enhancing survival within blood and organs . This elucidation of their integral functions in the pathogenic process of not only enhances our comprehension of bacterial virulence mechanisms, but also illuminates new avenues for therapeutic intervention aimed at curbing infections.
（）是一种重要的猪源细菌病原体，可导致人类感染，引发多种疾病。然而，5'-核苷酸酶在其毒力中的作用仍有待充分阐明。在此，我们在（）中鉴定出四种细胞壁锚定的5'-核苷酸酶（Snts），分别命名为SntA、SntB、SntC和SntD，它们各自显示出相似的结构域，但序列同源性较低。孔雀石绿试剂和高效液相色谱分析表明，这些重组酶能够将ATP、ADP和AMP水解为腺苷（Ado），催化效率的顺序为SntC>SntB>SntA>SntD。此外，综合酶活性分析表明，底物特异性、pH耐受性和金属离子需求存在轻微差异，但突出了Snts（除SntA外）中保守的底物结合口袋、His-Asp催化二元组、金属和磷酸盐结合位点。通过涉及位点诱变菌株的杀菌试验和小鼠感染试验，证明SntB和SntC通过Ado-A2aR途径协同增强细菌在全血和多形核白细胞（PMNs）以及小鼠血液和器官中的生存能力。这表明酶活性与细菌生存和毒力增强之间存在直接关联。总体而言，（）5'-核苷酸酶对腺苷的产生具有累加作用，影响血液和PMNs中的易感性，并增强在血液和器官中的生存能力。对它们在（）致病过程中整体功能的阐明不仅增强了我们对细菌毒力机制的理解，还为旨在控制（）感染的治疗干预开辟了新途径。
