Zang Xinxin, Dang Guanghui, Cai Zhuming, Shao Mingzhu, Tang Yangyang, Cao Jun, Cui Ziyin, Liu Siguo
State Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Street, Harbin 150069, China.
State Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Street, Harbin 150069, China; College of Animal Science and Technology, Jilin Agricultural University, Changchun, China.
Vet Microbiol. 2022 Oct;273:109529. doi: 10.1016/j.vetmic.2022.109529. Epub 2022 Aug 3.
Extracellular DNases/nucleases are important virulence factors in many bacteria. However, no DNase/nucleases have been reported in Mycobacterium avium subsp. paratuberculosis (MAP), which is a pathogen of paratuberculosis. Genome analyses of MAP K-10 revealed that the map3916c gene putatively encodes a nuclease. In this study, we show that MAP3916c is an extracellular nonspecific DNase requiring a divalent cation, especially Mg. The optimum DNase activity of MAP3916c was exhibited at 41 °C and pH 9.0. Site-directed mutagenesis studies indicated that 125-Histidine is necessary for MAP3916c DNase activity. In addition, MAP3916c DNase could destroy the neutrophil extracellular traps (NETs) induced by Phorbol 12-myristate 13-acetate in vitro and degrade the NETs induced by MAP K-10 upon infection. Furthermore, MAP3916c DNase promoted the colonization of MAP K-10, induced the formation of granulomas in the liver and small intestine and promoted the release of IL-1β, IL-6 and TNF-α inflammatory cytokines during the infection of mice. These results indicated that MAP3916c is relevant to NETs escape and the pathogenicity of MAP. It also provides a basis for further study of the function of nuclease activity on the MAP immune evasion.
细胞外脱氧核糖核酸酶/核酸酶是许多细菌中重要的毒力因子。然而，在副结核分枝杆菌（MAP）中尚未报道有脱氧核糖核酸酶/核酸酶，MAP是副结核病的病原体。对MAP K-10的基因组分析表明，map3916c基因可能编码一种核酸酶。在本研究中，我们表明MAP3916c是一种需要二价阳离子（尤其是镁离子）的细胞外非特异性脱氧核糖核酸酶。MAP3916c的最佳脱氧核糖核酸酶活性在41°C和pH 9.0时表现出来。定点诱变研究表明，125位组氨酸对于MAP3916c的脱氧核糖核酸酶活性是必需的。此外，MAP3916c脱氧核糖核酸酶可以在体外破坏佛波酯12-肉豆蔻酸酯13-乙酸酯诱导的中性粒细胞胞外陷阱（NETs），并在感染时降解MAP K-10诱导的NETs。此外，MAP3916c脱氧核糖核酸酶促进了MAP K-10的定植，在小鼠感染期间诱导肝脏和小肠中肉芽肿的形成，并促进白细胞介素-1β、白细胞介素-6和肿瘤坏死因子-α炎症细胞因子的释放。这些结果表明MAP3916c与NETs逃逸和MAP的致病性有关。它也为进一步研究核酸酶活性在MAP免疫逃避中的作用提供了基础。
