Intranasal or intravenous infection (Figure 2A and B). Although there was a significant survival 23388095 advantage in mice infected i.v. with Didtr all mice eventually succumbed to infection. The kinetics of bacterial cell growth did not 114311-32-9 chemical information appear to vary greatly between TIGR4 and Didtr following i.v. infection (Figure 3). However, loss of idtr markedly attenuates the ability of pneumococcus to invade and cause fatal bacteremia from the nasopharyngeal epithelial surface.Results Role of idtr in pneumococcal growth in vitroThe role of idtr in vitro in the presence or absence of free iron was examined. TIGR4 and Didtr exhibited similar growth kinetics in chemically-defined medium (CDM) and iron-depleted CDM. The deletion mutant had a shorter lag phase than TIGR4 but both attained similar cell density at stationary phase. Also, Didtr entered the exponential phase of growth slightly faster than TIGR4 in both CDM and iron-depleted CDM (Figure 1A). The microscopic appearance of Didtr cells was strikingly different from TIGR4. The mutant formed aggregates and clusters as contrasted with short chains and diplococci of the parent wild-type TIGR4 (Figure 1B).Expression of selected virulence genes in vitro and in vivoExpression of several well characterized and putative virulence genes in Didtr and TIGR4 in vitro and in vivo was evaluated. Transcripts of cps4A, pspA, ply, hemolysin, and non-heme ferritin were up-regulated in Didtr cells, while those of exfoliative toxin, iron ABC transporter and pavA remained essentially unchanged in vitro. Transcription of nanB was markedly MedChemExpress Mirin repressed in the deletion mutant (Figure 4A). The expression of these genes in vivo varied significantly at the three anatomical sites examined. During nasopharyngeal coloni-Figure 1. Growth of TIGR4 and Didtr and Gram stain morphology of Didtr in vitro. The growth of TIGR4 and Didtr in CDM and iron depleted CDM was monitored by measuring absorbance at 600 nm. B) The morphology of Didtr was observed in (I) Iron depleted CDM (II) CDM by Gram staining. The results shown are average of three independent experiments cells grown in iron. doi:10.1371/journal.pone.0055157.gRole of idtr in Pneumococcal InfectionsFigure 3. Average bacterial counts from mouse blood TIGR4 and Didtr. A group of 5 mice each were infected intravenously with 105 CFU of TIGR4 or Didtr. Blood samples at different time points were plated to determine bacterial counts. The error bars represent standard error of mean. **Significantly decreased as compared to TIGR4 infected blood counts (P,0.01). doi:10.1371/journal.pone.0055157.gFigure 2. Survival of mice infected with TIGR4 and Didtr. CBA/ CaHN-Btkxid/J mice were inoculated (A) intranasally with 106 CFU and (B) intravenously with 105 CFU of TIGR4 and Didtr. Kaplan Meier curves shown are a representative of triplicate experiments (n = 5 in each experiment). doi:10.1371/journal.pone.0055157.gzation, the transcription of all ten genes was up-regulated in the mutant. During pneumonia transcription of the genes in the mutant was less than that during colonization but still higher when compared to that in TIGR4. During bacteremia transcription was unchanged or slightly repressed in the mutant as compared to TIGR4 cells (Figure 4B).Affect of idtr on host cytokine response in intravenous sepsis modelBased on studies in humans and animal models, a panel of 14 cytokines (Table 1) were chosen [18?4] to evaluate the effect of idtr on the host innate immune response. At 48 hours after.Intranasal or intravenous infection (Figure 2A and B). Although there was a significant survival 23388095 advantage in mice infected i.v. with Didtr all mice eventually succumbed to infection. The kinetics of bacterial cell growth did not appear to vary greatly between TIGR4 and Didtr following i.v. infection (Figure 3). However, loss of idtr markedly attenuates the ability of pneumococcus to invade and cause fatal bacteremia from the nasopharyngeal epithelial surface.Results Role of idtr in pneumococcal growth in vitroThe role of idtr in vitro in the presence or absence of free iron was examined. TIGR4 and Didtr exhibited similar growth kinetics in chemically-defined medium (CDM) and iron-depleted CDM. The deletion mutant had a shorter lag phase than TIGR4 but both attained similar cell density at stationary phase. Also, Didtr entered the exponential phase of growth slightly faster than TIGR4 in both CDM and iron-depleted CDM (Figure 1A). The microscopic appearance of Didtr cells was strikingly different from TIGR4. The mutant formed aggregates and clusters as contrasted with short chains and diplococci of the parent wild-type TIGR4 (Figure 1B).Expression of selected virulence genes in vitro and in vivoExpression of several well characterized and putative virulence genes in Didtr and TIGR4 in vitro and in vivo was evaluated. Transcripts of cps4A, pspA, ply, hemolysin, and non-heme ferritin were up-regulated in Didtr cells, while those of exfoliative toxin, iron ABC transporter and pavA remained essentially unchanged in vitro. Transcription of nanB was markedly repressed in the deletion mutant (Figure 4A). The expression of these genes in vivo varied significantly at the three anatomical sites examined. During nasopharyngeal coloni-Figure 1. Growth of TIGR4 and Didtr and Gram stain morphology of Didtr in vitro. The growth of TIGR4 and Didtr in CDM and iron depleted CDM was monitored by measuring absorbance at 600 nm. B) The morphology of Didtr was observed in (I) Iron depleted CDM (II) CDM by Gram staining. The results shown are average of three independent experiments cells grown in iron. doi:10.1371/journal.pone.0055157.gRole of idtr in Pneumococcal InfectionsFigure 3. Average bacterial counts from mouse blood TIGR4 and Didtr. A group of 5 mice each were infected intravenously with 105 CFU of TIGR4 or Didtr. Blood samples at different time points were plated to determine bacterial counts. The error bars represent standard error of mean. **Significantly decreased as compared to TIGR4 infected blood counts (P,0.01). doi:10.1371/journal.pone.0055157.gFigure 2. Survival of mice infected with TIGR4 and Didtr. CBA/ CaHN-Btkxid/J mice were inoculated (A) intranasally with 106 CFU and (B) intravenously with 105 CFU of TIGR4 and Didtr. Kaplan Meier curves shown are a representative of triplicate experiments (n = 5 in each experiment). doi:10.1371/journal.pone.0055157.gzation, the transcription of all ten genes was up-regulated in the mutant. During pneumonia transcription of the genes in the mutant was less than that during colonization but still higher when compared to that in TIGR4. During bacteremia transcription was unchanged or slightly repressed in the mutant as compared to TIGR4 cells (Figure 4B).Affect of idtr on host cytokine response in intravenous sepsis modelBased on studies in humans and animal models, a panel of 14 cytokines (Table 1) were chosen [18?4] to evaluate the effect of idtr on the host innate immune response. At 48 hours after.
