E-043. Streptolysin O-Mediated Inhibition of Phagosomal Acidification Reduces Processing of Group A Streptococcus Antigens by Human Dendritic Cells
Background: The cholesterol-binding cytolysin streptolysin O (SLO) prevents internalization of group A Streptococcus (GAS) and delivery to lysosomes in epithelial cells, resulting in enhanced intracellular bacterial survival. In antigen-presenting cells (APC), lysosomal degradation is critical for the generation of immunogenic peptides recognized by T cells. Therefore, bacterial manipulation of lysosomal function in APC not only affects direct bacterial killing, but also influences the development of the adaptive immune response. We hypothesized that SLO-mediated inhibition of lysosomal function prevents processing of streptococcal antigens by human dendritic cells (DC). Methods: Human peripheral blood monocyte-derived DC were exposed to a virulent GAS strain or an isogenic SLO-deficient mutant strain in the presence or absence of the vacuolar proton pump inhibitor bafilomycin. At different time points, samples were collected, lysed, and intracellular degradation of the streptococcal surface-associated M protein was analyzed by immunoblot. Lysosomal fusion following ingestion of GAS was determined by immunostaining of fixed and permeabilized DC. Phagosomal pH was measured by flow cytometric analysis of live DC following exposure to FITC-labeled GAS. Results: Internalization of the SLO-deficient GAS strain resulted in rapid M-protein degradation (<1 h) by acidification-dependent DC proteases. In striking contrast, intact M-protein was still detectable 7 h post-internalization of the SLO+ GAS strain. While both intracellular wild-type SLO+ and mutant SLO-deficient GAS strains co-localized with the lysosomal markers LAMP-1 and CatS, SLO+ GAS was exposed to a significantly less acidic pH (6.2 ± 0.2) than SLO- GAS (5.0 ± 0.1). A cell-free assay in which purified M-protein was digested with DC lysates at different pH values revealed that a pH ≤5 is required for optimal M-protein degradation, and showed no effect of SLO in the absence of cell compartmentalization. Conclusion: SLO-mediated inhibition of phagosomal acidification, rather than lysosomal evasion, prevents M-protein degradation by DC exposed to GAS.