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Characterization of Streptococcus pyogenes beta-NAD+ glycohydrolase: re-evaluation of enzymatic properties associated with pathogenesis.

ABSTRACT: The gram-positive pathogen Streptococcus pyogenes injects a beta-NAD(+) glycohydrolase (SPN) into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. In this compartment, SPN accelerates the death of the host cell by an unknown mechanism that may involve its beta-NAD(+)-dependent enzyme activities. SPN has been reported to possess the unique characteristic of not only catalyzing hydrolysis of beta-NAD(+), but also carrying out ADP-ribosyl cyclase and ADP-ribosyltransferase activities, making SPN the only beta-NAD(+) glycohydrolase that can catalyze all of these reactions. With the long term goal of understanding how these activities may contribute to pathogenesis, we have further characterized the enzymatic activity of SPN using highly purified recombinant protein. Kinetic studies of the multiple activities of SPN revealed that SPN possessed only beta-NAD(+) hydrolytic activity and lacked detectable ADP-ribosyl cyclase and ADP-ribosyltransferase activities. Similarly, SPN was unable to catalyze cyclic ADPR hydrolysis, and could not catalyze methanolysis or transglycosidation. Kinetic analysis of product inhibition by recombinant SPN demonstrated an ordered uni-bi mechanism, with ADP-ribose being released as a second product. SPN was unaffected by product inhibition using nicotinamide, suggesting that this moiety contributes little to the binding energy of the substrate. Upon transformation, SPN was toxic to Saccharomyces cerevisiae, whereas a glycohydrolase-inactive SPN allowed for viability. Taken together, these data suggest that SPN functions exclusively as a strict beta-NAD(+) glycohydrolase during pathogenesis.

SUBMITTER: Ghosh J

PROVIDER: S-EPMC2820796 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Characterization of Streptococcus pyogenes beta-NAD+ glycohydrolase: re-evaluation of enzymatic properties associated with pathogenesis.

Ghosh Joydeep J Anderson Patricia J PJ Chandrasekaran Sukantha S Caparon Michael G MG

The Journal of biological chemistry 20091215 8

The gram-positive pathogen Streptococcus pyogenes injects a beta-NAD(+) glycohydrolase (SPN) into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. In this compartment, SPN accelerates the death of the host cell by an unknown mechanism that may involve its beta-NAD(+)-dependent enzyme activities. SPN has been reported to possess the unique characteristic of not only catalyzing hydrolysis of beta-NAD(+), but also carrying out ADP-ribosyl cyclase and ADP-ribo ...[more]

PMID: 20018886

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Project description:UnlabelledVirulence 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-?B, 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.ImportanceMany 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.

Project description:The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus, GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of cotoxins streptolysin O (SLO) and NAD+-glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a β-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro, suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. IMPORTANCE NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus, the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.

Dataset Information

Characterization of Streptococcus pyogenes beta-NAD+ glycohydrolase: re-evaluation of enzymatic properties associated with pathogenesis.

Publications

Characterization of Streptococcus pyogenes beta-NAD+ glycohydrolase: re-evaluation of enzymatic properties associated with pathogenesis.

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