Hsieh Cheng-Lu, Hsieh Shu-Ying, Huang Hsuan-Min, Lu Shiou-Ling, Omori Hiroko, Zheng Po-Xing, Ho Yen-Ning, Cheng Yi-Lin, Lin Yee-Shin, Chiang-Ni Chuan, Tsai Pei-Jane, Wang Shu-Ying, Liu Ching-Chuan, Noda Takeshi, Wu Jiunn-Jong
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Front Microbiol. 2020 Feb 11;11:117. doi: 10.3389/fmicb.2020.00117. eCollection 2020.
Group A streptococcus (GAS) is a versatile pathogen that causes a wide spectrum of diseases in humans. Invading host cells is a known strategy for GAS to avoid antibiotic killing and immune recognition. However, the underlying mechanisms of GAS resistance to intracellular killing need to be explored. Endothelial HMEC-1 cells were infected with GAS, methicillin-resistant (MRSA) and Typhimurium under nicotinamide (NAM)-supplemented conditions. The intracellular NAD level and cell viability were respectively measured by NAD quantification kit and protease-based cytotoxicity assay. Moreover, the intracellular bacteria were analyzed by colony-forming assay, transmission electron microscopy, and confocal microscopy. We found that supplementation with exogenous nicotinamide during infection significantly inhibited the growth of intracellular GAS in endothelial cells. Moreover, the NAD content and NAD/NADH ratio of GAS-infected endothelial cells were dramatically increased, whereas the cell cytotoxicity was decreased by exogenous nicotinamide treatment. After knockdown of the autophagy-related ATG9A, the intracellular bacterial load was increased in nicotinamide-treated endothelial cells. The results of Western blot and transmission electron microscopy also revealed that cells treated with nicotinamide can increase autophagy-associated LC3 conversion and double-membrane formation during GAS infection. Confocal microscopy images further showed that more GAS-containing vacuoles were colocalized with lysosome under nicotinamide-supplemented conditions than without nicotinamide treatment. In contrast to GAS, supplementation with exogenous nicotinamide did not effectively inhibit the growth of MRSA or Typhimurium in endothelial cells. These results indicate that intracellular NAD homeostasis is crucial for controlling intracellular GAS infection in endothelial cells. In addition, nicotinamide may be a potential new therapeutic agent to overcome persistent infections of GAS.
A组链球菌（GAS）是一种多能病原体，可在人类中引发多种疾病。侵入宿主细胞是GAS避免被抗生素杀灭和免疫识别的一种已知策略。然而，GAS对细胞内杀伤产生抗性的潜在机制仍有待探索。在内皮HMEC-1细胞中，于添加烟酰胺（NAM）的条件下感染GAS、耐甲氧西林金黄色葡萄球菌（MRSA）和鼠伤寒沙门氏菌。分别通过NAD定量试剂盒和基于蛋白酶的细胞毒性测定法测量细胞内NAD水平和细胞活力。此外，通过集落形成测定、透射电子显微镜和共聚焦显微镜分析细胞内细菌。我们发现，感染期间补充外源性烟酰胺可显著抑制内皮细胞中细胞内GAS的生长。此外，经外源性烟酰胺处理后，感染GAS的内皮细胞的NAD含量和NAD/NADH比值显著增加，而细胞毒性降低。自噬相关蛋白ATG9A敲低后，烟酰胺处理的内皮细胞中细胞内细菌载量增加。蛋白质印迹和透射电子显微镜结果还显示，烟酰胺处理的细胞在GAS感染期间可增加自噬相关的LC3转化和双膜形成。共聚焦显微镜图像进一步显示，与未用烟酰胺处理的情况相比，在补充烟酰胺的条件下，更多含GAS的液泡与溶酶体共定位。与GAS不同，补充外源性烟酰胺并不能有效抑制内皮细胞中MRSA或鼠伤寒沙门氏菌的生长。这些结果表明，细胞内NAD稳态对于控制内皮细胞中的细胞内GAS感染至关重要。此外，烟酰胺可能是克服GAS持续性感染的一种潜在新型治疗剂。
