Cient strain from human dendritic cells as compared to the wild type strain seems to be advantageous because the hemolytic activity of pneumolysin was not found to be essential for invasive disease in their experimental setup. Using a murine model of pneumococcal bacteremia, Harvey et al. found an enhanced in vivo survival of the pneumolysin deficient strain [27]. Independent observations from both groups about the pneumolysin expression dependent survival of S. pneumoniae support our findings suggesting that a low or absent hemolysin activity may, for some 
streptococcal strains, be critical for its survival within professional phagocytes. In our experiments, we could demonstrate that the survival of S. 842-07-9 agalactiae is dependent on the uptake into THP-1 macrophages. We were able to show that entry of S. agalactiae into THP-1 Arg8-vasopressin supplier macrophages is inhibited by Cytochalasin D in a dose dependent manner, indicating that the uptake of S. agalactiae occurs through phagocytosis, a mechanism that actively involves actin polymerization (Fig. 3). A similar finding was made by Valentin-Weigand et al. for serotype III Group B streptococci using the murineThe GBS ?Hemolysin and Intracellular SurvivalFigure 5. S. agalactiae does not multiply within macrophages. THP-1 macrophages were infected with hemolytic (BSU 98) and nonhemolytic (BSU 453) bacteria for 90 min. After this, antibiotics were added into the medium for the rest of the assay. Macrophages were lysed at indicated time points and lysate were plated. Data shown are the values from five independent experiments with median. Data is considered extremely significant for p values ,0.001 (***). doi:10.1371/journal.pone.0060160.gmacrophage-like cell line J774 [28]. Our results also support the findings of Fettuciari et al. who showed in a recent publication that the S. agalactiae b-hemolysin activates calpain leading to a severe disturbance of the cytoskeleton and may thus prevent the uptake of S. agalactiae into eukaryotic cells [29]. Our fluorescence microscopy data clearly distinguishes between intracellular and extracellular bacteria. However, the current study does not address the location of bacteria within the macrophages. Previous electron micrograph and fluorescence microscopic studies on various GBS serotypes have reported thelocation of GBS within the membrane-bound vacuole in a variety of host-cell types, including epithelial cells [17], endothelial cells [30] [31], dendritic cells [32] and macrophages [33] [29] [34]. In Listeria monocytogenes, the hemolysin is important in phagosomal escape and hemolysin negative mutants are thus impaired in intracellular survival [35]. However, in a recent investigation of the subcellular localization of S. agalactiae within J774 macrophages, no indication for the escape of S. agalactiae from the phagosome was observed [33]. The vast majority of S. agalactiae could be observed within phagosomes and survival of S. agalactiaeFigure 6. Intracellular S. agalactiae induced cytokine expression in THP-1 macrophages. THP-1 macrophages were infected with hemolytic (BSU 98) and nonhemolytic (BSU 453) GBS at a MOI of 1:1 at indicated time points. Extracellular bacteria were killed by antibiotics for an additional 1 h. A) IL-8 and B) TNF-a levels were measured in the supernatant by ELISA. Uninfected cells served as control. Data shown are the mean 6 SD of three independent experiments. doi:10.1371/journal.pone.0060160.gThe GBS ?Hemolysin and Intracellular SurvivalFigure.Cient strain from human dendritic cells as compared to the wild type strain seems to be advantageous because the hemolytic activity of pneumolysin was not found to be essential for invasive disease in their experimental setup. Using a murine model of pneumococcal bacteremia, Harvey et al. found an enhanced in vivo survival of the pneumolysin deficient strain [27]. Independent observations from both groups about the pneumolysin expression dependent survival of S. pneumoniae support our findings suggesting that a low or absent hemolysin activity may, for some streptococcal strains, be critical for its survival within professional phagocytes. In our experiments, we could demonstrate that the survival of S. agalactiae is dependent on the uptake into THP-1 macrophages. We were able to show that entry of S. agalactiae into THP-1 macrophages is inhibited by Cytochalasin D in a dose dependent manner, indicating that the uptake of S. agalactiae occurs through phagocytosis, a mechanism that actively involves actin polymerization (Fig. 3). A similar finding was made by Valentin-Weigand et al. for serotype III Group B streptococci using the murineThe GBS ?Hemolysin and Intracellular SurvivalFigure 5. S. agalactiae does not multiply within macrophages. THP-1 macrophages 
were infected with hemolytic (BSU 98) and nonhemolytic (BSU 453) bacteria for 90 min. After this, antibiotics were added into the medium for the rest of the assay. Macrophages were lysed at indicated time points and lysate were plated. Data shown are the values from five independent experiments with median. Data is considered extremely significant for p values ,0.001 (***). doi:10.1371/journal.pone.0060160.gmacrophage-like cell line J774 [28]. Our results also support the findings of Fettuciari et al. who showed in a recent publication that the S. agalactiae b-hemolysin activates calpain leading to a severe disturbance of the cytoskeleton and may thus prevent the uptake of S. agalactiae into eukaryotic cells [29]. Our fluorescence microscopy data clearly distinguishes between intracellular and extracellular bacteria. However, the current study does not address the location of bacteria within the macrophages. Previous electron micrograph and fluorescence microscopic studies on various GBS serotypes have reported thelocation of GBS within the membrane-bound vacuole in a variety of host-cell types, including epithelial cells [17], endothelial cells [30] [31], dendritic cells [32] and macrophages [33] [29] [34]. In Listeria monocytogenes, the hemolysin is important in phagosomal escape and hemolysin negative mutants are thus impaired in intracellular survival [35]. However, in a recent investigation of the subcellular localization of S. agalactiae within J774 macrophages, no indication for the escape of S. agalactiae from the phagosome was observed [33]. The vast majority of S. agalactiae could be observed within phagosomes and survival of S. agalactiaeFigure 6. Intracellular S. agalactiae induced cytokine expression in THP-1 macrophages. THP-1 macrophages were infected with hemolytic (BSU 98) and nonhemolytic (BSU 453) GBS at a MOI of 1:1 at indicated time points. Extracellular bacteria were killed by antibiotics for an additional 1 h. A) IL-8 and B) TNF-a levels were measured in the supernatant by ELISA. Uninfected cells served as control. Data shown are the mean 6 SD of three independent experiments. doi:10.1371/journal.pone.0060160.gThe GBS ?Hemolysin and Intracellular SurvivalFigure.
