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Streptolysin O and its co-toxin NAD-glycohydrolase protect group A Streptococcus from Xenophagic killing.

O'Seaghdha M, Wessels MR - PLoS Pathog. (2013)

Bottom Line: Whereas this process was associated with killing of GAS in HeLa cells, studies in human keratinocytes found SLO production enhanced intracellular survival.We found that SLO expression was associated with prolonged intracellular survival; unexpectedly, expression of the co-toxin NADase was required for this effect.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
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.

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Expression of active NADase plays a central role in the intracellular survival of GAS in oropharyngeal keratinocytes.A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001. NADase western blot (B) and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).
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ppat-1003394-g003: Expression of active NADase plays a central role in the intracellular survival of GAS in oropharyngeal keratinocytes.A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001. NADase western blot (B) and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).

Mentions: SLO is expressed from an operon that also encodes NADase and its intracellular inhibitor, Immunity Factor for Streptococcus pyogenes NADase (IFS) [25], [26]. SLO and NADase are functionally linked in that SLO is required for the translocation of NADase across cholesterol-containing membranes [27], [28]. Since SLO-negative strains of GAS are unable to translocate NADase, the reduced intracellular survival of such strains could be due to the absence of SLO or to failure to translocate NADase into keratinocytes, or both. To specifically address the potential role of NADase, we tested intracellular survival of strain 188NADase-, an isogenic mutant that produces SLO, but not NADase [27]. Deletion of NADase was associated with an increase in bacterial uptake: the mean number of intracellular GAS was 8.7×103 CFU/monolayer at 2 h for strain 188NADase- compared to 3.1×103 CFU/monolayer for strain 188, in agreement with previously published observations [14], [27]. Intracellular survival of the NADase-negative strain was reduced by approximately 20-fold compared to parent strain 188 (Figures 1A and 3A). NADase expression also enhanced intracellular survival of GAS strain JRS4 (Figure 1C).


Streptolysin O and its co-toxin NAD-glycohydrolase protect group A Streptococcus from Xenophagic killing.

O'Seaghdha M, Wessels MR - PLoS Pathog. (2013)

Expression of active NADase plays a central role in the intracellular survival of GAS in oropharyngeal keratinocytes.A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001. NADase western blot (B) and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675196&req=5

ppat-1003394-g003: Expression of active NADase plays a central role in the intracellular survival of GAS in oropharyngeal keratinocytes.A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001. NADase western blot (B) and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).
Mentions: SLO is expressed from an operon that also encodes NADase and its intracellular inhibitor, Immunity Factor for Streptococcus pyogenes NADase (IFS) [25], [26]. SLO and NADase are functionally linked in that SLO is required for the translocation of NADase across cholesterol-containing membranes [27], [28]. Since SLO-negative strains of GAS are unable to translocate NADase, the reduced intracellular survival of such strains could be due to the absence of SLO or to failure to translocate NADase into keratinocytes, or both. To specifically address the potential role of NADase, we tested intracellular survival of strain 188NADase-, an isogenic mutant that produces SLO, but not NADase [27]. Deletion of NADase was associated with an increase in bacterial uptake: the mean number of intracellular GAS was 8.7×103 CFU/monolayer at 2 h for strain 188NADase- compared to 3.1×103 CFU/monolayer for strain 188, in agreement with previously published observations [14], [27]. Intracellular survival of the NADase-negative strain was reduced by approximately 20-fold compared to parent strain 188 (Figures 1A and 3A). NADase expression also enhanced intracellular survival of GAS strain JRS4 (Figure 1C).

Bottom Line: Whereas this process was associated with killing of GAS in HeLa cells, studies in human keratinocytes found SLO production enhanced intracellular survival.We found that SLO expression was associated with prolonged intracellular survival; unexpectedly, expression of the co-toxin NADase was required for this effect.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Boston Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus