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      Nicotinamide Increases Intracellular NAD + Content to Enhance Autophagy-Mediated Group A Streptococcal Clearance in Endothelial Cells

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          Abstract

          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 Staphylococcus aureus (MRSA) and Salmonella 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 S. 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.

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          Disease manifestations and pathogenic mechanisms of group a Streptococcus.

          Streptococcus pyogenes, also known as group A Streptococcus (GAS), causes mild human infections such as pharyngitis and impetigo and serious infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. Furthermore, repeated GAS infections may trigger autoimmune diseases, including acute poststreptococcal glomerulonephritis, acute rheumatic fever, and rheumatic heart disease. Combined, these diseases account for over half a million deaths per year globally. Genomic and molecular analyses have now characterized a large number of GAS virulence determinants, many of which exhibit overlap and redundancy in the processes of adhesion and colonization, innate immune resistance, and the capacity to facilitate tissue barrier degradation and spread within the human host. This improved understanding of the contribution of individual virulence determinants to the disease process has led to the formulation of models of GAS disease progression, which may lead to better treatment and intervention strategies. While GAS remains sensitive to all penicillins and cephalosporins, rising resistance to other antibiotics used in disease treatment is an increasing worldwide concern. Several GAS vaccine formulations that elicit protective immunity in animal models have shown promise in nonhuman primate and early-stage human trials. The development of a safe and efficacious commercial human vaccine for the prophylaxis of GAS disease remains a high priority.
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            Crosstalk Between Mammalian Autophagy and the Ubiquitin-Proteasome System

            Autophagy and the ubiquitin–proteasome system (UPS) are the two major intracellular quality control and recycling mechanisms that are responsible for cellular homeostasis in eukaryotes. Ubiquitylation is utilized as a degradation signal by both systems, yet, different mechanisms are in play. The UPS is responsible for the degradation of short-lived proteins and soluble misfolded proteins whereas autophagy eliminates long-lived proteins, insoluble protein aggregates and even whole organelles (e.g., mitochondria, peroxisomes) and intracellular parasites (e.g., bacteria). Both the UPS and selective autophagy recognize their targets through their ubiquitin tags. In addition to an indirect connection between the two systems through ubiquitylated proteins, recent data indicate the presence of connections and reciprocal regulation mechanisms between these degradation pathways. In this review, we summarize these direct and indirect interactions and crosstalks between autophagy and the UPS, and their implications for cellular stress responses and homeostasis.
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              NAD + Depletion Triggers Macrophage Necroptosis, a Cell Death Pathway Exploited by Mycobacterium tuberculosis

              Mycobacterium tuberculosis (Mtb) kills infected macrophages by inhibiting apoptosis and promoting necrosis. The tuberculosis necrotizing toxin (TNT) is a secreted nicotinamide adenine dinucleotide (NAD+) glycohydrolase that induces necrosis in infected macrophages. Here, we show that NAD+ depletion by TNT activates RIPK3 and MLKL, key mediators of necroptosis. Notably, Mtb bypasses the canonical necroptosis pathway since neither TNF-α nor RIPK1 are required for macrophage death. Macrophage necroptosis is associated with depolarized mitochondria and impaired ATP synthesis, known hallmarks of Mtb-induced cell death. These results identify TNT as the main trigger of necroptosis in Mtb-infected macrophages. Surprisingly, NAD+ depletion itself was sufficient to trigger necroptosis in a RIPK3- and MLKL-dependent manner by inhibiting the NAD+ salvage pathway in THP-1 cells or by TNT expression in Jurkat T cells. These findings suggest avenues for host-directed therapies to treat tuberculosis and other infectious and age-related diseases in which NAD+ deficiency is a pathological factor.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                11 February 2020
                2020
                : 11
                : 117
                Affiliations
                [1] 1Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University , Tainan, Taiwan
                [2] 2Institute of Molecular Medicine, College of Medicine, National Cheng Kung University , Tainan, Taiwan
                [3] 3Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University , Tainan, Taiwan
                [4] 4Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University , Osaka, Japan
                [5] 5Research Institute for Microbial Diseases, Osaka University , Osaka, Japan
                [6] 6Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University , Tainan, Taiwan
                [7] 7Department of Biotechnology and Laboratory Science in Medicine, School of Biomedical Science and Engineering, National Yang-Ming University , Taipei, Taiwan
                [8] 8Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University , Tainan, Taiwan
                [9] 9Department of Microbiology & Immunology, College of Medicine, Chang Gung University , Taoyuan, Taiwan
                [10] 10Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital , Taoyuan, Taiwan
                [11] 11Department of Pediatrics, College of Medicine, National Cheng Kung University and Hospital , Tainan, Taiwan
                Author notes

                Edited by: Diego Robledo, The University of Edinburgh, United Kingdom

                Reviewed by: Blanca Estela Garcia-Perez, National Polytechnic Institute (Mexico), Mexico; Maghnus N O’Seaghdha, Suffolk University, United States

                *Correspondence: Jiunn-Jong Wu, jjwu1019@ 123456ym.edu.tw

                This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2020.00117
                7026195
                32117141
                221991bd-f9c7-48b9-bd02-61c423737a33
                Copyright © 2020 Hsieh, Hsieh, Huang, Lu, Omori, Zheng, Ho, Cheng, Lin, Chiang-Ni, Tsai, Wang, Liu, Noda and Wu.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 10 October 2019
                : 20 January 2020
                Page count
                Figures: 7, Tables: 0, Equations: 0, References: 61, Pages: 13, Words: 0
                Funding
                Funded by: Ministry of Science and Technology, Taiwan 10.13039/501100004663
                Award ID: MOST105-2320-B006-009
                Award ID: MOST106-2320-B010-039
                Award ID: MOST 107-2320-B010-021
                Award ID: MOST 108-2320-B010-004
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                group a streptococcus (gas),nicotinamide (nam),nad+ homeostasis,intracellular survival,endothelial cells (ecs)

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