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.
Highlights
The human-specific pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) is responsible for common localized infections such as pharyngitis and impetigo as well as less common, but potentially life threatening, conditions including necrotizing fasciitis and streptococcal toxic shock [1]
We find that production of the secreted pore-forming toxin streptolysin O (SLO) triggered targeted autophagy of GAS in human oropharyngeal keratinocytes, and enhanced GAS intracellular survival
Prolonged GAS intracellular survival may contribute to pharyngitis treatment failure, relapse, and chronic carriage
Summary
The human-specific pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) is responsible for common localized infections such as pharyngitis and impetigo as well as less common, but potentially life threatening, conditions including necrotizing fasciitis and streptococcal toxic shock [1]. It has been suggested that survival of the bacteria within pharyngeal or tonsillar epithelial cells in vivo may contribute to persistence, as intracellular organisms are protected from many antimicrobial agents and host immune effectors such as complement and antibodies. In support of this view, intracellular GAS have been identified by microscopy and isolated in cultures of surgical specimens of human tonsils, even after treatment of the excised tissue with antibiotics to kill extracellular bacteria [10]
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