Table_1_NAD-Glycohydrolase Depletes Intracellular NAD+ and Inhibits Acidification of Autophagosomes to Enhance Multiplication of Group A Streptococcus in Endothelial Cells.docx

Group A Streptococcus (GAS) is a human pathogen causing a wide spectrum of diseases, from mild pharyngitis to life-threatening necrotizing fasciitis. GAS has been shown to evade host immune killing by invading host cells. However, how GAS resists intracellular killing by endothelial cells is still unclear. In this study, we found that strains NZ131 and A20 have higher activities of NADase and intracellular multiplication than strain SF370 in human endothelial cells (HMEC-1). Moreover, nga mutants of NZ131 (SW957 and SW976) were generated to demonstrate that NADase activity is required for the intracellular growth of GAS in endothelial cells. We also found that intracellular levels of NAD+ and the NAD+/NADH ratio of NZ131-infected HMEC-1 cells were both lower than in cells infected by the nga mutant. Although both NZ131 and its nga mutant were trapped by LC3-positive vacuoles, only nga mutant vacuoles were highly co-localized with acidified lysosomes. On the other hand, intracellular multiplication of the nga mutant was increased by bafilomycin A1 treatment. These results indicate that NADase causes intracellular NAD+ imbalance and impairs acidification of autophagosomes to escape autophagocytic killing and enhance multiplication of GAS in endothelial cells.

A群链球菌（Group A Streptococcus, GAS）是一类可引发多种疾病的人类致病菌，感染谱涵盖轻度咽炎至危及生命的坏死性筋膜炎。已有研究证实，A群链球菌可通过侵入宿主细胞逃避免疫杀伤，但目前其如何抵抗内皮细胞的胞内杀伤机制仍不明确。本研究发现，菌株NZ131与A20在人微血管内皮细胞系（HMEC-1）中的NAD酶（NADase）活性及胞内增殖能力均高于菌株SF370。此外，我们构建了NZ131的nga突变株（SW957与SW976），实验证明NAD酶活性是A群链球菌在内皮细胞中实现胞内增殖的必要条件。我们还观察到，感染NZ131的HMEC-1细胞内的烟酰胺腺嘌呤二核苷酸（NAD+）水平及NAD+/NADH比值均低于感染nga突变株的细胞。尽管NZ131及其nga突变株均被微管相关蛋白1轻链3（LC3）阳性囊泡包裹，但仅nga突变株的囊泡可与酸化溶酶体发生高度共定位。经巴佛洛霉素A1（bafilomycin A1）处理后，nga突变株的胞内增殖能力显著提升。综上，本研究结果表明，NAD酶可引发胞内NAD+失衡，损伤自噬体的酸化过程，帮助A群链球菌逃避自噬杀伤并提升其在内皮细胞中的增殖能力。