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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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SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.
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ppat-1003394-g005: SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.

Mentions: The data presented above suggest that optimal GAS survival within human oropharyngeal cells depends on production of both SLO and its co-toxin, NADase. However, these results did not address the mechanism by which toxin production inhibits intracellular killing. A previous study from our group showed that SLO-deficient GAS rapidly co-localized with lysosomal markers after internalization by keratinocytes, whereas SLO-producing GAS did not [14]. Since results of the current investigation showed that both parent strain 188 and SLO- and NADase-deficient GAS are contained within autophagosome-like vacuoles, we reasoned that the differential survival of these strains might reflect the effects of SLO and NADase on the functional maturation of GAS-containing autophagosomes rather than simply induction of xenophagy per se. Maturation of autophagosomes into degradative autolysosomes occurs by fusion with lysosomes, which can be detected in vitro by co-localization of LC3 with the lysosomal marker LAMP-1. We therefore investigated the temporal association of LAMP-1 with LC3-positive vacuoles containing GAS strains 188, 188SLO-, or 188NADase-. All three GAS strains associated with LC3 shortly after internalization by the host cell (Figure 5A). However, there was a marked difference in the rate of lysosomal fusion: in the case of parent strain 188, 21% of LC3-associated GAS were co-localized with LAMP-1 after 1 h. At 3 h, 28% were co-localized, and by 6 h post-infection, 44% of autophagosomes containing 188 had fused with lysosomes (Figure 5A,B). In contrast, 81% of LC3-associated 188SLO- GAS co-localized with LAMP-1 at 1 h post infection, 72% at 3 h post-infection, and 75% at 6 h (Figure 5A,B). LAMP-1 was also more rapidly associated with autophagosomes harboring 188NADase- compared to parent strain 188, although less so than the SLO-mutant: 27% of autophagosomes containing 188NADase- were positive for LAMP-1 at 1 h post-infection, 64% at 3 h, and 82% at 6 h (Figure 5A,B). We observed a similar pattern for the intracellular trafficking of GAS strains JRS4, JRS4SLO-, and JRS4NADase-. That is, JRS4SLO- and JRS4NADase- rapidly associated with both LC3 and LAMP-1, whereas the co-localization of LC3-positive compartments containing parent strain JRS4 was delayed (not shown). Taken together, these data indicate that the co-expression of SLO and NADase by GAS inhibits maturation of GAS-containing autophagosomes into degradative autolysosomes, and that this delay is associated with prolonged bacterial survival in infected keratinocytes.


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

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

SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675196&req=5

ppat-1003394-g005: SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.
Mentions: The data presented above suggest that optimal GAS survival within human oropharyngeal cells depends on production of both SLO and its co-toxin, NADase. However, these results did not address the mechanism by which toxin production inhibits intracellular killing. A previous study from our group showed that SLO-deficient GAS rapidly co-localized with lysosomal markers after internalization by keratinocytes, whereas SLO-producing GAS did not [14]. Since results of the current investigation showed that both parent strain 188 and SLO- and NADase-deficient GAS are contained within autophagosome-like vacuoles, we reasoned that the differential survival of these strains might reflect the effects of SLO and NADase on the functional maturation of GAS-containing autophagosomes rather than simply induction of xenophagy per se. Maturation of autophagosomes into degradative autolysosomes occurs by fusion with lysosomes, which can be detected in vitro by co-localization of LC3 with the lysosomal marker LAMP-1. We therefore investigated the temporal association of LAMP-1 with LC3-positive vacuoles containing GAS strains 188, 188SLO-, or 188NADase-. All three GAS strains associated with LC3 shortly after internalization by the host cell (Figure 5A). However, there was a marked difference in the rate of lysosomal fusion: in the case of parent strain 188, 21% of LC3-associated GAS were co-localized with LAMP-1 after 1 h. At 3 h, 28% were co-localized, and by 6 h post-infection, 44% of autophagosomes containing 188 had fused with lysosomes (Figure 5A,B). In contrast, 81% of LC3-associated 188SLO- GAS co-localized with LAMP-1 at 1 h post infection, 72% at 3 h post-infection, and 75% at 6 h (Figure 5A,B). LAMP-1 was also more rapidly associated with autophagosomes harboring 188NADase- compared to parent strain 188, although less so than the SLO-mutant: 27% of autophagosomes containing 188NADase- were positive for LAMP-1 at 1 h post-infection, 64% at 3 h, and 82% at 6 h (Figure 5A,B). We observed a similar pattern for the intracellular trafficking of GAS strains JRS4, JRS4SLO-, and JRS4NADase-. That is, JRS4SLO- and JRS4NADase- rapidly associated with both LC3 and LAMP-1, whereas the co-localization of LC3-positive compartments containing parent strain JRS4 was delayed (not shown). Taken together, these data indicate that the co-expression of SLO and NADase by GAS inhibits maturation of GAS-containing autophagosomes into degradative autolysosomes, and that this delay is associated with prolonged bacterial survival in infected keratinocytes.

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