Dataset Information

The tuberculosis necrotizing toxin is an NAD+ and NADP+ glycohydrolase with distinct enzymatic properties.

ABSTRACT: Upon host infection, Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) into the cytosol of infected macrophages, leading to host cell death by necroptosis. TNT hydrolyzes NAD+ in the absence of any exogenous cofactor, thus classifying it as a ?-NAD+ glycohydrolase. However, TNT lacks sequence similarity with other NAD+ hydrolyzing enzymes and lacks the essential motifs involved in NAD+ binding and hydrolysis by these enzymes. In this study, we used NMR to examine the enzymatic activity of TNT and found that TNT hydrolyzes NADP+ as fast as NAD+ but does not cleave the corresponding reduced dinucleotides. This activity of TNT was not inhibited by ADP-ribose or nicotinamide, indicating low affinity of TNT for these reaction products. A selection assay for nontoxic TNT variants in Escherichia coli identified four of six residues in the predicted NAD+-binding pocket and four glycine residues that form a cradle directly below the NAD+-binding site, a conserved feature in the TNT protein family. Site-directed mutagenesis of residues near the predicted NAD+-binding site revealed that Phe727, Arg757, and Arg780 are essential for NAD+ hydrolysis by TNT. These results identify the NAD+-binding site of TNT. Our findings also show that TNT is an NAD+ glycohydrolase with properties distinct from those of other bacterial glycohydrolases. Because many of these residues are conserved within the TNT family, our findings provide insights into understanding the function of the >300 TNT homologs.

SUBMITTER: Tak U

PROVIDER: S-EPMC6398120 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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The tuberculosis necrotizing toxin is an NAD+ and NADP+ glycohydrolase with distinct enzymatic properties.

Tak Uday U Vlach Jiri J Garza-Garcia Acely A William Doreen D Danilchanka Olga O de Carvalho Luiz Pedro Sório LPS Saad Jamil S JS Niederweis Michael M

The Journal of biological chemistry 20181228 9

Upon host infection, Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) into the cytosol of infected macrophages, leading to host cell death by necroptosis. TNT hydrolyzes NAD+ in the absence of any exogenous cofactor, thus classifying it as a β-NAD+ glycohydrolase. However, TNT lacks sequence similarity with other NAD+ hydrolyzing enzymes and lacks the essential motifs involved in NAD+ binding and hydrolysis by these e ...[more]

PMID: 30593509

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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:Group A Streptococcus (Streptococcus pyogenes or GAS) causes pharyngitis, severe invasive infections, and the post-infectious syndromes of glomerulonephritis and rheumatic fever. GAS can be internalized and killed by epithelial cells in vitro, a process that may contribute to local innate defense against pharyngeal infection. Secretion of the pore-forming toxin streptolysin O (SLO) by GAS has been reported to stimulate targeted autophagy (xenophagy) upon internalization of the bacteria by epithelial cells. Whereas this process was associated with killing of GAS in HeLa cells, studies in human keratinocytes found SLO production enhanced intracellular survival. To reconcile these conflicting observations, we now report in-depth investigation of xenophagy in response to GAS infection of human oropharyngeal keratinocytes, the predominant cell type of the pharyngeal epithelium. We found that SLO expression was associated with prolonged intracellular survival; unexpectedly, expression of the co-toxin NADase was required for this effect. Enhanced intracellular survival was lost upon deletion of NADase or inactivation of its enzymatic activity. Shortly after internalization of GAS by keratinocytes, SLO-mediated damage to the bacteria-containing vacuole resulted in exposure to the cytosol, ubiquitination of GAS and/or associated vacuolar membrane remnants, and engulfment of GAS in LC3-positive vacuoles. We also found that production of streptolysin S could mediate targeting of GAS to autophagosomes in the absence of SLO, a process accompanied by galectin 8 binding to damaged GAS-containing endosomes. Maturation of GAS-containing autophagosome-like vacuoles to degradative autolysosomes was prevented by SLO pore-formation and by SLO-mediated translocation of enzymatically active NADase into the keratinocyte cytosol. We conclude that SLO stimulates xenophagy in pharyngeal keratinocytes, but the coordinated action of SLO and NADase prevent maturation of GAS-containing autophagosomes, thereby prolonging GAS intracellular survival. This novel activity of NADase to block autophagic killing of GAS in pharyngeal cells may contribute to pharyngitis treatment failure, relapse, and chronic carriage.

Dataset Information

The tuberculosis necrotizing toxin is an NAD+ and NADP+ glycohydrolase with distinct enzymatic properties.

Publications

The tuberculosis necrotizing toxin is an NAD<sup>+</sup> and NADP<sup>+</sup> glycohydrolase with distinct enzymatic properties.

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