Arked i) are intracellular, whereas purchase LED-209 others (marked e) are extracellular. OnFigure 1. Survival of S. agalactiae and b-hemolysin expression in professional phagocytes. The monocyte-derived macrophage cell line THP-1 or freshly isolated granulocytes were infected with hemolytic (BSU 98) and nonhemolytic (BSU 453) bacteria at a MOI of 1:1 for indicated time points. A) Intracellular bacteria were quantified after killing the extracellular bacteria using Penicillin (1 mg/ml) and Gentamicin (100 mg/ml) for additional 1 h. B) Total viable bacteria after incubation with granulocytes without killing of extracellular bacteria. Data shown are the mean 6 SD of six independent experiments. Data is considered extremely significant for p values ,0.001 (***). doi:10.1371/journal.pone.0060160.gThe GBS ?Hemolysin and Intracellular SurvivalFigure 2. Effect of bacterial cell mediated cytotoxicity as measured by LDH Cytotoxicity Assay. A ) THP-1 macrophages were infected at indicated multiplicity of infections and time points to measure the LDH release into the supernatant. The amount of LDH released is proportional to the percentage of lysed eukaryotic cells. D) Human granulocytes were infected at indicated multiplicity of infections for 2 h to measure the LDH release into the supernatant. High control corresponds to maximum lysis achieved using 2 of Triton X-100. Uninfected cells served as control. Data shown are the mean 6 SD of three independent experiments. Data is considered significant for p values ,0.05 (*). doi:10.1371/journal.pone.0060160.gthe basis of qualitative comparison, analysis of the infected cells illustrate that within THP-1 macrophages multiple chains of the nonhemolytic bacteria were found more often than in macrophages infected with the hemolytic strain.Cytokine Induction by Type Ia Group B StreptococciThe strength and efficiency of the immune response of the host is dependent on the release of proinflammatory cytokines. We investigated the induction of TNF-a and IL-8 from S. agalactiae infected THP-1 macrophages. Both BSU 98 and BSU 453 induce marked production of IL-8; however there was no overall difference in the release by macrophages (Fig. 6A). Nevertheless, the production of TNF-a from the infected macrophages in response to S. agalactiae is delayed. However a significant difference in the 23727046 ability of the two S. agalactiae JSI124 chemical information strains to produce TNF-a was observed after 3 hours of incubation, suggesting a functional role of TNF-a in S. agalactiae pathogenesis (Fig. 6B).Intracellular S. agalactiae MultiplicationPrevious literature showed that hemolytic S. agalactiae strains do not multiply within the eukaryotic host cell [17]. To analyze if the higher colony counts of S. agalactiae strain BSU 453 in our assays were caused by intracellular multiplication of the nonhemolytic mutant, we 
tested the ability of both strains to multiply within the THP-1 macrophages. As shown in Fig. 5, no significant increase in intracellular CFU was observed between 1 and 5 h of infection. At 24 h, no viable bacteria were recovered, indicating that both S. agalactiae strains did not multiply and are eventually killed by the macrophages. These data confirm the enhanced intracellular bacterial counts of BSU 453 in human macrophages, without evidence of a significant intracellular multiplication within phagocytes.Induction of Proinflammatory Cytokines by S. agalactiae Cell Wall PreparationsS. agalactiae molecules located on the cell surface or secreted into t.Arked i) are intracellular, whereas others (marked e) are extracellular. OnFigure 1. Survival of S. agalactiae and b-hemolysin expression in professional phagocytes. The monocyte-derived macrophage cell line THP-1 or freshly isolated granulocytes were infected with hemolytic (BSU 98) and nonhemolytic (BSU 453) bacteria at a MOI of 1:1 for indicated time points. A) Intracellular bacteria were quantified after killing the extracellular bacteria using Penicillin (1 mg/ml) and Gentamicin (100 mg/ml) for additional 1 h. B) Total viable bacteria after incubation with granulocytes without killing of extracellular bacteria. Data shown are the mean 6 SD of six independent experiments. Data is considered extremely significant for p values ,0.001 (***). doi:10.1371/journal.pone.0060160.gThe GBS ?Hemolysin and Intracellular SurvivalFigure 2. Effect of bacterial cell mediated cytotoxicity as measured by LDH Cytotoxicity Assay. A ) THP-1 macrophages were infected at indicated multiplicity of infections and time points to measure the LDH release into the supernatant. The amount of LDH released is proportional to the percentage of lysed eukaryotic cells. D) Human granulocytes were infected at indicated multiplicity of infections for 2 h to measure the LDH release into the supernatant. High control corresponds to maximum lysis achieved using 2 of Triton X-100. Uninfected cells served as control. Data shown are the mean 6 SD of three independent experiments. Data is considered significant for p values ,0.05 (*). doi:10.1371/journal.pone.0060160.gthe basis of qualitative comparison, analysis of the infected cells illustrate that within THP-1 macrophages multiple chains of the nonhemolytic bacteria were found more often than in macrophages infected with the hemolytic strain.Cytokine Induction by Type Ia Group B StreptococciThe strength and efficiency of the immune response of the host is dependent on the release of proinflammatory cytokines. We investigated the induction of TNF-a and IL-8 from S. agalactiae infected THP-1 macrophages. Both BSU 98 and BSU 453 induce marked production of IL-8; however there was no overall difference in the release by macrophages (Fig. 6A). Nevertheless, 
the production of TNF-a from the infected macrophages in response to S. agalactiae is delayed. However a significant difference in the 23727046 ability of the two S. agalactiae strains to produce TNF-a was observed after 3 hours of incubation, suggesting a functional role of TNF-a in S. agalactiae pathogenesis (Fig. 6B).Intracellular S. agalactiae MultiplicationPrevious literature showed that hemolytic S. agalactiae strains do not multiply within the eukaryotic host cell [17]. To analyze if the higher colony counts of S. agalactiae strain BSU 453 in our assays were caused by intracellular multiplication of the nonhemolytic mutant, we tested the ability of both strains to multiply within the THP-1 macrophages. As shown in Fig. 5, no significant increase in intracellular CFU was observed between 1 and 5 h of infection. At 24 h, no viable bacteria were recovered, indicating that both S. agalactiae strains did not multiply and are eventually killed by the macrophages. These data confirm the enhanced intracellular bacterial counts of BSU 453 in human macrophages, without evidence of a significant intracellular multiplication within phagocytes.Induction of Proinflammatory Cytokines by S. agalactiae Cell Wall PreparationsS. agalactiae molecules located on the cell surface or secreted into t.