Role of group A streptococcus NADase in intracellular multiplication in human endothelial cells

• Main Authors: Shu-YingHsieh, 謝淑英
• Other Authors: Jiunn-Jong Wu
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/04077480483062125201

碩士 === 國立成功大學 === 分子醫學研究所 === 104 === Streptococcus pyogenes (Group A streptococcus, GAS) is an important human pathogen that causes various severe diseases, such as necrotizing fasciitis and streptococcal toxic shock syndrome. Although GAS is considered as an extracellular pathogen, several studies have shown that GAS can survive within intracellular niches as well. In our study group, an invasive strain NZ131 can multiply inside human microvascular endothelial cells (HMEC-1), but noninvasive strain SF370 cannot, suggesting that unknown molecules in the invasive strain may be involved. Previous studies demonstrated that streptolysin O (SLO) and its co-toxin NAD-glycohydrolase (NADase) could protect GAS from xenophagic killing in keratinocytes. Our laboratory showed that nga encodes NADase, prevents the clearance of acidified autophagolysosome. However, how NADase associates with the cellular clearance mechanism still unknown. In this thesis, I aimed to figure out the role of enzymatic activity and the metabolic effect of NADase in intracellular multiplication. First, polar effects on the downstream genes of nga were ruled out by real-time RT-PCR. The results confirmed that nga play a key role. As known endogenous IFS inhibites NADase activity and further influences intracellular survival. I investigated the enzymatic activity of NADase by constructing IFS overexpression strain. The results showed that NZ131 engineered to secrete IFS impaired intracellular survival, suggesting that GAS multiplication depended on NADase activity. Meanwhile, the ratio of NAD＋/NADH was decreased when NZ131 infected with HMEC-1. Since NADase is able to cleave the intracellular β-NAD+ to produce nicotinamide and adenosine diphosphoribose, these substrates were added to HMEC-1 infected with NZ131 and nga mutant to investigate how the metabolic effects of NADase influence the interaction between GAS and HMEC-1. The results showed the multiplication ability of NZ131 and nga mutant in endothelial cells remained reduced when treated with nicotinamide. With bafilomycin A1 and immunofluorescence, I found that nicotinamide enhanced autophagolysosome clearance mechanism by increased the intracellular NAD+. Taken together, NADase promotes GAS multiplication within endothelial cells through interfered cellular NAD+ homeostasis, the phenomena can be improved by treating with nicotinamide at high concentration.