School of Medicine, Southern University of Science and Technology / Shenzhen Key Laboratory for Gene Regulation and Systems Biology, Southern University of Science and Technology, Shenzhen 518055, Guangdong, PR China; School of Biomedical Sciences, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong; Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, PR China.
School of Medicine, Southern University of Science and Technology / Shenzhen Key Laboratory for Gene Regulation and Systems Biology, Southern University of Science and Technology, Shenzhen 518055, Guangdong, PR China; Shenzhen Third People's Hospital, National Clinical Research Center for Infectious Disease, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518112, PR China.
Microbiol Res. 2024 Jan;278:127535. doi: 10.1016/j.micres.2023.127535. Epub 2023 Oct 22.
C-di-GMP is a bacterial second messenger implicated in the regulation of many key functions including antibiotic tolerance and biofilm formation. Our understanding of how c-di-GMP exerts its action via receptors to modulate different biological functions is still limited. Here we used a c-di-GMP affinity pull-down assay coupled to LC-MS/MS to identify c-di-GMP-binding proteins in the opportunistic pathogen Stenotrophomonas maltophilia. This analysis identified Smlt3238 (SodA), a protein of the superoxide dismutase family, as a c-di-GMP-binding protein. Microscale thermophoresis showed that purified SodA protein bound c-di-GMP with an estimated dissociation constant (Kd) value of 141.5 μM. Using various in vivo and in vitro experiments, we demonstrated that c-di-GMP modulates the enzyme activity of SodA directly. Circular dichroism experiments revealed that SodA protein gradually altered its basic structure with increasing levels of c-di-GMP. Phenotypic experiments conducted in the presence of a range of intracellular c-di-GMP levels showed that SodA function is modulated by c-di-GMP. The findings thus identify a novel c-di-GMP binding protein that governs oxidative stress tolerance in S. maltophilia.
c-di-GMP 是一种细菌第二信使，参与调节许多关键功能，包括抗生素耐药性和生物膜形成。我们对 c-di-GMP 通过受体发挥作用以调节不同生物学功能的方式的理解仍然有限。在这里，我们使用 c-di-GMP 亲和下拉测定法结合 LC-MS/MS 来鉴定机会性病原体嗜麦芽寡养单胞菌中的 c-di-GMP 结合蛋白。该分析鉴定出 Smlt3238（SodA），一种超氧化物歧化酶家族的蛋白质，为 c-di-GMP 结合蛋白。微量热泳动实验表明，纯化的 SodA 蛋白与 c-di-GMP 的结合解离常数（Kd）值约为 141.5 μM。通过各种体内和体外实验，我们证明 c-di-GMP 直接调节 SodA 的酶活性。圆二色性实验表明，随着 c-di-GMP 水平的升高，SodA 蛋白逐渐改变其基本结构。在存在一系列细胞内 c-di-GMP 水平的表型实验中，表明 SodA 功能受 c-di-GMP 调节。因此，该研究确定了一种新型的 c-di-GMP 结合蛋白，该蛋白控制嗜麦芽寡养单胞菌的氧化应激耐受性。
