MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China.
PLoS Pathog. 2023 Mar 13;19(3):e1011227. doi: 10.1371/journal.ppat.1011227. eCollection 2023 Mar.
GntR transcription factor of Streptococcus suis serotype 2 (SS2) is a potential substrate protein of STK, but the regulation mechanisms of GntR phosphorylation are still unclear. This study confirmed that STK phosphorylated GntR in vivo, and in vitro phosphorylation experiments showed that STK phosphorylated GntR at Ser-41. The phosphomimetic strain (GntR-S41E) had significantly reduced lethality in mice and reduced bacterial load in the blood, lung, liver, spleen, and brain of infected mice compared to wild-type (WT) SS2. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that the promoter of nox was bound by GntR. The phosphomimetic protein GntR-S41E cannot bind to the promoter of nox, and the nox transcription levels were significantly reduced in the GntR-S41E mutant compared to WT SS2. The virulence in mice and the ability to resist oxidative stress of the GntR-S41E strain were restored by complementing transcript levels of nox. NOX is an NADH oxidase that catalyzes the oxidation of NADH to NAD+ with the reduction of oxygen to water. We found that NADH is likely accumulated under oxidative stress in the GntR-S41E strain, and higher NADH levels resulted in increased amplified ROS killing. In total, we report GntR phosphorylation could inhibit the transcription of nox, which impaired the ability of SS2 to resist oxidative stress and virulence.
猪链球菌 2 型（SS2）的 GntR 转录因子是 STK 的潜在底物蛋白，但 GntR 磷酸化的调控机制尚不清楚。本研究证实 STK 可在体内磷酸化 GntR，体外磷酸化实验表明 STK 可使 GntR 的丝氨酸 41 位磷酸化。与野生型（WT）SS2 相比，磷酸化模拟株（GntR-S41E）在小鼠中的致死率显著降低，感染小鼠血液、肺、肝、脾和脑中的细菌载量也降低。电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）实验表明，nox 的启动子与 GntR 结合。磷酸化模拟蛋白 GntR-S41E 不能与 nox 的启动子结合，与 WT SS2 相比，GntR-S41E 突变株中 nox 的转录水平显著降低。通过补充 nox 的转录水平，可恢复 GntR-S41E 菌株在小鼠中的毒力和抵抗氧化应激的能力。NOX 是一种 NADH 氧化酶，可催化 NADH 氧化为 NAD+，同时将氧还原为水。我们发现，在 GntR-S41E 菌株中，NADH 可能在氧化应激下积累，较高的 NADH 水平导致扩增的 ROS 杀伤增加。总之，我们报告 GntR 磷酸化可抑制 nox 的转录，从而削弱 SS2 抵抗氧化应激和毒力的能力。
