Local host defenses limit proliferation and systemic spread of pathogenic bacteria from sites of mucosal colonization. For pathogens such as streptococci that fail to grow intracellularly, internalization and killing by epithelial cells contribute to the control of bacterial growth and dissemination. Here, we show that group A Streptococcus (GAS), the agent of streptococcal sore throat and invasive soft tissue infections, evades internalization and intracellular killing by pharyngeal epithelial cells. Production of the cholesterol-binding cytotoxin streptolysin O (SLO) prevented internalization of GAS into lysosomes. In striking contrast, GAS rendered defective in production of SLO were internalized directly or rapidly transported into lysosomes, where they were killed by a pH-dependent mechanism. Because SLO is the prototype of cholesterol-dependent cytolysins produced by many Grampositive bacteria, cytolysin-mediated evasion of lysosomal killing may be a general mechanism to protect such pathogens from clearance by host epithelial cells.Streptococcus pyogenes ͉ streptolysin O ͉ virulence E pithelial cells of mucosal surfaces constitute an early line of defense against infecting microorganisms. These cells protect the host not only by acting as a physical barrier but also by secreting antimicrobial molecules and by recruiting professional immune cells to the site of infection (1-5). Also, epithelial cells have the capacity to internalize and kill bacteria. Extracellular pathogens such as streptococci, Staphylococcus aureus, and Pseudomonas aeruginosa can be internalized by epithelial cells but do not appear to multiply in the intracellular environment (6-11). Rather, internalized bacteria are killed over time. Therefore, to persist at the mucosal surface, extracellular bacterial pathogens must evade internalization and killing by epithelial cells.In this study, we investigated the effects of streptolysin O (SLO) on the interactions of group A Streptococcus (GAS), the agent of streptococcal sore throat and severe, invasive, or ''flesh-eating'' infections, with human oropharyngeal epithelial cells. SLO is the prototype of a family of cholesterol-binding cytotoxins produced by many pathogenic Gram-positive bacteria including Streptococcus pneumoniae (pneumolysin), Listeria monocytogenes (listeriolysin O), Clostridium perfringens (perfringolysin O), and Bacillus anthracis (anthrolysin) (12-14). The cholesterol-binding cytotoxins share the property of poreforming activity for a broad range of cholesterol-containing cell membranes (12, 15). However, it remains unknown whether they serve a common functional role in pathogen-host biology for the diverse species that produce them. Here, we report that SLO is a critical modulator of GAS internalization, intracellular trafficking, and bacterial killing by human oropharyngeal keratinocytes. We found that SLO prevented direct uptake of GAS into lysosomes and efficient pH-dependent intracellular killing. On the basis of these findings, we propose that SLO enhances GAS surviv...

SummaryGroup A Streptococcus (GAS) colonizes the oropharynx and damaged skin. To cause local infection or severe invasive syndromes the bacteria must gain access into deeper tissues. Host cell death may facilitate this process. GAS internalization has been identified to induce apoptosis. We now report an alternate mechanism of GAS-mediated apoptosis of primary human keratinocytes, initiated by extracellular GAS and involving dysregulation of intracellular calcium to produce endoplasmic reticulum stress. Two bacterial virulence factors are required for effective induction of apoptosis by extracellular GAS: (i) hyaluronic acid capsule that inhibits bacterial internalization and (ii) secreted cytolysin, streptolysin O (SLO), that forms transmembrane pores that permit extracellular calcium influx into the cytosol. Induction of keratinocyte apoptosis by wild-type GAS was accompanied by cell detachment and loss of epithelial integrity, a phenomenon not observed with GAS deficient in capsule or SLO. We propose that cell signalling initiated by extracellular GAS compromises the epithelial barrier by inducing premature keratinocyte differentiation and apoptosis, thereby facilitating GAS invasion of deeper tissues.