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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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Xenophagy in oropharyngeal keratinocytes can be stimulated by SLO or SLS and is not associated with killing of intracellular GAS.A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain. B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material. C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05. D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.
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ppat-1003394-g002: Xenophagy in oropharyngeal keratinocytes can be stimulated by SLO or SLS and is not associated with killing of intracellular GAS.A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain. B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material. C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05. D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.

Mentions: In HeLa cells, secretion of SLO stimulates the formation of autophagosome-like vacuoles that surround intracellular GAS and appear to mediate bacterial killing, whereas GAS-associated autophagosomes were not observed in cells infected with an SLO-deficient strain [16], [17]. In light of our apparently contradictory finding that SLO production is associated with GAS resistance to intracellular killing in oropharyngeal keratinocytes, we investigated the induction of autophagy/xenophagy by GAS in keratinocytes expressing EGFP-LC3, a marker of the autophagosomal membrane. Unexpectedly, we observed LC3-positive vacuoles surrounding intracellular GAS not only in cells infected with parent strain 188 but also in cells harboring the SLO-deficient strain 188SLO- (Figure 2A). Electron microscopy confirmed these observations: strains 188 and 188SLO- were localized to double or multiple membrane-bound structures that also contained cytosolic material (Figure 2B). That both GAS strains were associated with autophagosome-like compartments indicated that SLO is not required for GAS to induce xenophagy in oropharyngeal keratinocytes. A similar pattern of co-localization was observed for cells infected with GAS strain JRS4 or JRS4SLO-: both strains were found in LC3-positive compartments (Figure S2A). By contrast, in HeLa cells, association of intracellular GAS with EGFP-LC3 was dependent on SLO, as 188SLO- did not associate with autophagosomes, a result in agreement with previous studies (Figure S2B) [16], [17]. Thus, in oropharyngeal keratinocytes, internalization of GAS is associated with induction of xenophagy, whether or not the internalized strain produces SLO.


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

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

Xenophagy in oropharyngeal keratinocytes can be stimulated by SLO or SLS and is not associated with killing of intracellular GAS.A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain. B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material. C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05. D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675196&req=5

ppat-1003394-g002: Xenophagy in oropharyngeal keratinocytes can be stimulated by SLO or SLS and is not associated with killing of intracellular GAS.A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain. B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material. C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05. D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.
Mentions: In HeLa cells, secretion of SLO stimulates the formation of autophagosome-like vacuoles that surround intracellular GAS and appear to mediate bacterial killing, whereas GAS-associated autophagosomes were not observed in cells infected with an SLO-deficient strain [16], [17]. In light of our apparently contradictory finding that SLO production is associated with GAS resistance to intracellular killing in oropharyngeal keratinocytes, we investigated the induction of autophagy/xenophagy by GAS in keratinocytes expressing EGFP-LC3, a marker of the autophagosomal membrane. Unexpectedly, we observed LC3-positive vacuoles surrounding intracellular GAS not only in cells infected with parent strain 188 but also in cells harboring the SLO-deficient strain 188SLO- (Figure 2A). Electron microscopy confirmed these observations: strains 188 and 188SLO- were localized to double or multiple membrane-bound structures that also contained cytosolic material (Figure 2B). That both GAS strains were associated with autophagosome-like compartments indicated that SLO is not required for GAS to induce xenophagy in oropharyngeal keratinocytes. A similar pattern of co-localization was observed for cells infected with GAS strain JRS4 or JRS4SLO-: both strains were found in LC3-positive compartments (Figure S2A). By contrast, in HeLa cells, association of intracellular GAS with EGFP-LC3 was dependent on SLO, as 188SLO- did not associate with autophagosomes, a result in agreement with previous studies (Figure S2B) [16], [17]. Thus, in oropharyngeal keratinocytes, internalization of GAS is associated with induction of xenophagy, whether or not the internalized strain produces SLO.

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