In murine models. The identification of these bacterial gene products, and their specific interactions with the host immune system, will allow greater understanding of the pathogenesis of invasive pneumococcal infections and identify potential points at which intervention may be possible to reduce morbidity and mortality.Materials and Methods Bacterial strains and mediaS. pneumoniae TIGR4, a Title Loaded From File capsular type 4 strain and an isogenic mutant deleted at idtr (described below) were used in all experiments. Bacteria from stocks stored at 280uC were used to inoculate chemically-defined medium (CDM) (JRH Bioscience, 1531364 Lenexa, KS) [39] supplemented with 0.1 choline, 0.25 sodium bicarbonate and 0.073 cysteine. Iron-depleted CDM was prepared by treatment with 3 w/v Chelex-100H (Bio-Rad, Hercules, CA) for 20 h. Chelex-treated CDM was supplemented with MnSO4, MgSO4, and CaCl2 to a final concentration as that in CDM. All media were sterilized by filtration and stored at 4uC. Todd Hewitt yeast extract medium (THY) used in transformation of TIGR4 was made by adding 0.2 glucose, 0.2 CaCl2, and 0.02 bovine serum albumin (BSA) to THY medium and adjusted to pH 7.2?.4 [40]. The trimethoprim resistance gene tmp was isolated from E. coli cells containing the pkoT plasmid [41]. Trimethoprim (Tmp) was used at 50 mg/ml to select for transformants.Standard error of the mean. doi:10.1371/journal.pone.0055157.tcytokines tested are associated with a poor prognosis in sepsis patients or animal models of sepsis [30?5]. Combined high levels of IL-10 and IL-6 are associated with a very high risk of death in sepsis patients [36]. This difference was not related to a faster growth rate and higher bacterial burden with TIGR4, as both wild-type and mutant were at the same approximate density in the blood at the time of cytokine sampling. These results imply that idtr not only modulates the bacterial virulence but also modulates the host response to pneumococcal infection. The mechanisms by which this modulation occurs remain to be determined. It is likely that idtr controls genes which encode pneumococcal surface-exposed components or other factors which interact with the host immune system. To our knowledge, this is the first report indicating a role of iron dependent transcription regulator in host immune response to pneumococcal infections. The role of iron-regulated bacterial genes in modulation of host responses has been reported for other Gram-positive pathogens. In Staphylococcus aureus the inactivation of fur is reported to be associated with increased nitric oxide sensitivity [37]. In Mycobacterium smegmatis, insertional inactivation of ideR (a homolog of dtxR and idtr) was shown to decrease production of manganese superoxide dismutase and catalase/ peroxidase 24786787 (katG), and increase susceptibility to killing by H2O2 [38]. IDTR has an important role in virulence and gene expression and its function is likely related to the form and quantity of available iron at different anatomic sites of the host. Invasive disease in humans follows Title Loaded From File translocation of pneumococci from mucosal surfaces of the nasopharynx to the lower respiratory tract and, in some cases, dissemination via blood. Environmental conditions are markedly different at each location and the concentrations of certain nutrients necessary for pneumococcal growth almost certainly function, by various pathways, to regulate bacterial gene expression. Future work will define the role of IDTR on global protein expr.In murine models. The identification of these bacterial gene products, and their specific interactions with the host immune system, will allow greater understanding of the pathogenesis of invasive pneumococcal infections and identify potential points at which intervention may be possible to reduce morbidity and mortality.Materials and Methods Bacterial strains and mediaS. pneumoniae TIGR4, a capsular type 4 strain and an isogenic mutant deleted at idtr (described below) were used in all experiments. Bacteria from stocks stored at 280uC were used to inoculate chemically-defined medium (CDM) (JRH Bioscience, 1531364 Lenexa, KS) [39] supplemented with 0.1 choline, 0.25 sodium bicarbonate and 0.073 cysteine. Iron-depleted CDM was prepared by treatment with 3 w/v Chelex-100H (Bio-Rad, Hercules, CA) for 20 h. Chelex-treated CDM was supplemented with MnSO4, MgSO4, and CaCl2 to a final concentration as that in CDM. All media were sterilized by filtration and stored at 4uC. Todd Hewitt yeast extract medium (THY) used in transformation of TIGR4 was made by adding 0.2 glucose, 0.2 CaCl2, and 0.02 bovine serum albumin (BSA) to THY medium and adjusted to pH 7.2?.4 [40]. The trimethoprim resistance gene tmp was isolated from E. coli cells containing the pkoT plasmid [41]. Trimethoprim (Tmp) was used at 50 mg/ml to select for transformants.Standard error of the mean. doi:10.1371/journal.pone.0055157.tcytokines tested are associated with a poor prognosis in sepsis patients or animal models of sepsis [30?5]. Combined high levels of IL-10 and IL-6 are associated with a very high risk of death in sepsis patients [36]. This difference was not related to a faster growth rate and higher bacterial burden with TIGR4, as both wild-type and mutant were at the same approximate density in the blood at the time of cytokine sampling. These results imply that idtr not only modulates the bacterial virulence but also modulates the host response to pneumococcal infection. The mechanisms by which this modulation occurs remain to be determined. It is likely that idtr controls genes which encode pneumococcal surface-exposed components or other factors which interact with the host immune system. To our knowledge, this is the first report indicating a role of iron dependent transcription regulator in host immune response to pneumococcal infections. The role of iron-regulated bacterial genes in modulation of host responses has been reported for other Gram-positive pathogens. In Staphylococcus aureus the inactivation of fur is reported to be associated with increased nitric oxide sensitivity [37]. In Mycobacterium smegmatis, insertional inactivation of ideR (a homolog of dtxR and idtr) was shown to decrease production of manganese superoxide dismutase and catalase/ peroxidase 24786787 (katG), and increase susceptibility to killing by H2O2 [38]. IDTR has an important role in virulence and gene expression and its function is likely related to the form and quantity of available iron at different anatomic sites of the host. Invasive disease in humans follows translocation of pneumococci from mucosal surfaces of the nasopharynx to the lower respiratory tract and, in some cases, dissemination via blood. Environmental conditions are markedly different at each location and the concentrations of certain nutrients necessary for pneumococcal growth almost certainly function, by various pathways, to regulate bacterial gene expression. Future work will define the role of IDTR on global protein expr.
