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      The NADase-Negative Variant of the Streptococcus pyogenes Toxin NAD + Glycohydrolase Induces JNK1-Mediated Programmed Cellular Necrosis

      American Society of Microbiology

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          Virulence factors are often multifunctional and contribute to pathogenesis through synergistic mechanisms. For the human pathogen Streptococcus pyogenes, two factors that act synergistically are the S. pyogenes NAD + glycohydrolase (SPN) and streptolysin O (SLO). Through distinct mechanisms, SLO forms pores in host cell membranes and translocates SPN into the host cell cytosol. Two natural variants of SPN exist, one that exhibits NADase activity and one that lacks this function, and both versions are translocated and act in concert with SLO to cause an accelerated death response in epithelial cells. While NADase + SPN is known to trigger a metabolic form of necrosis through the depletion of NAD +, the mechanism by which NADase SPN induces cell death was unknown. In the studies described here, we examined the pathway of NADase cell death through analysis of activation patterns of mitogen-activated protein kinases (MAPKs). S. pyogenes infection resulted in activation of members of three MAPK subfamilies (p38, ERK, and JNK). However, only JNK was activated in an SLO-specific manner. NADase SPN induced necrosis in HeLa epithelial cells associated with depolarization of mitochondrial membranes, activation of NF-κB, and the generation of reactive oxygen species. Remarkably, RNA interference (RNAi) silencing of JNK protected cells from NADase -SPN-mediated necrosis, suggesting that NADase SPN triggers a form of programmed necrosis dependent on JNK signaling. Taken together, these data demonstrate that SPN acts with SLO to elicit necrosis through two different mechanisms depending on its NADase activity, i.e., metabolic (NADase +) or programmed (NADase ), leading to distinct inflammatory profiles.


          Many bacterial pathogens produce toxins that alter how infected host cells interact with the immune system. For Streptococcus pyogenes, two toxins, a NAD + glycohydrolase (SPN) and streptolysin O (SLO), act in combination to cause infected cells to die. However, there are two natural forms of SPN, and these variants cause dying cells to produce different types of signaling molecules. The NADase + form of SPN kills cells by depleting reserves of NAD + and cellular energy. The other form of SPN lacks this activity (NADase ); thus, the mechanism by which this variant induces toxicity was unknown. Here, we show that infected cells recognize NADase SPN through a specific signaling molecule called JNK, which causes these cells to undergo a form of cellular suicide known as programmed necrosis. This helps us to understand how different forms of toxins alter host cell signaling to help S. pyogenes cause different types of diseases.

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          Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases.

          TNFalpha is a pleiotropic cytokine that induces either cell proliferation or cell death. Inhibition of NF-kappaB activation increases susceptibility to TNFalpha-induced death, concurrent with sustained JNK activation, an important contributor to the death response. Sustained JNK activation in NF-kappaB-deficient cells was suggested to depend on reactive oxygen species (ROS), but how ROS affect JNK activation was unclear. We now show that TNFalpha-induced ROS, whose accumulation is suppressed by mitochondrial superoxide dismutase, cause oxidation and inhibition of JNK-inactivating phosphatases by converting their catalytic cysteine to sulfenic acid. This results in sustained JNK activation, which is required for cytochrome c release and caspase 3 cleavage, as well as necrotic cell death. Treatment of cells or experimental animals with an antioxidant prevents H(2)O(2) accumulation, JNK phosphatase oxidation, sustained JNK activity, and both forms of cell death. Antioxidant treatment also prevents TNFalpha-mediated fulminant liver failure without affecting liver regeneration.
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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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              ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.

              Apoptosis signal-regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H(2)O(2), and activates c-Jun NH(2)-terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF- and H(2)O(2)-induced sustained activations of JNK and p38 are lost in ASK1(-/-) embryonic fibroblasts, and that ASK1(-/-) cells are resistant to TNF- and H(2)O(2)-induced apoptosis. TNF- but not Fas-induced apoptosis requires ROS-dependent activation of ASK1-JNK/p38 pathways. Thus, ASK1 is selectively required for TNF- and oxidative stress-induced sustained activations of JNK/p38 and apoptosis.

                Author and article information

                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2 February 2016
                Jan-Feb 2016
                : 7
                : 1
                : e02215-15
                [1]Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA
                Author notes
                Address correspondence to Michael G. Caparon, caparon@ 123456wusm.wustl.edu .

                Present address: Sukantha Chandrasekaran, Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA.

                Editor Larry S. McDaniel, University of Mississippi Medical Center

                This article is a direct contribution from a Fellow of the American Academy of Microbiology.

                Copyright © 2016 Chandrasekaran and Caparon.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                : 28 December 2015
                : 5 January 2016
                Page count
                supplementary-material: 1, Figures: 6, Tables: 0, Equations: 0, References: 44, Pages: 9, Words: 8247
                Funded by: National Institutes of Health (NIH) http://dx.doi.org/10.13039/501100003653
                Award ID: AI064721
                Award Recipient : Michael G. Caparon
                Research Article
                Custom metadata
                January/February 2016

                Life sciences
                Life sciences


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