Ession both in vitro and within a host and undoubtedly expand our understanding the complete subset of genes which are controlled either directly or indirectly byidtr mutant constructionAn idtr mutant was constructed using PCR ligation mutagenesis as described by [42]. A schematic representation of the mutant construction is outlined in Figure 5. Briefly, tmp was amplified from pkoT plasmid DNA (primers T1 and T2) and the flanking regions of idtr were amplified from TIGR4 genomic DNA (primers I1 and I2, I3 and I4) described in table 2. The PCR amplified and purified I1-I2, I3-I4 and the tmpr cassette were subjected to single and double digestion by HindiIII and BamHI respectively according to the manufacturer’s protocol (Promega, Madison, WI), The digested PCR products were ligated using T4 DNA ligase (Promega, Madison, WI). The resulting construct (,2 kb) was amplified using primers I1-I4 and was used to transform TIGR4 as previously described [25]. The double recombination event was selected by plating on plates containing 50 mg/ml of Tmp. Identification of Tmp-resistant mutants was confirmed by both PCR analysis and DNA sequencing.Animal models of pneumococcal infectionAll animal studies were performed on either 10?2 wk old CBA/CaHN-Btkxid/J or C57BL/6 mice obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in the VA animal facility. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional 1527786 Animal Care and Use Committee at the VA Licochalcone-A Medical Center (assurance number: A31011). For all in vivo studies pneumococcal strains were administered either intranasally (i.n.) or intravenously (i.v.) as noted. Following infection animals were observed every twelve hours for signs of piloerection, inability to eat or drink, or failure to withdraw from threatening stimuli. Animals were weighed daily and any animal which exhibited any aforementioned behavior, or lost more than 15 of pre-infection body weight, was euthanized. For intranasalRole of idtr in Pneumococcal InfectionsFigure 5. Schematic representation of Didtr construction. H-HindIII, B-BamHI. T1, T2 amplify the tmp cassette (495 bp); T1 and T2 have H and B at 59 end. I1, I2 and I3, I4 amplify 59 and 39 end of idtr. I2 and I3 have H and B at 59 end. 15857111 I1, I2 amplify a 945 bp product and I3, I4 amplify a product of 489 bp. doi:10.1371/journal.pone.MedChemExpress JI-101 0055157.ginfection, a suspension of mid-exponential phase TIGR4 or Didtr (106 CFU) in PBS was delivered into the nares of anesthetized mice (20 ml per mouse) as previously described [43]. At this volume and cell number pneumococci remain localized to the nasopharynx. Intravenous inoculation was performed by injecting 105 CFU of TIGR4 or Didtr in 100 ml of PBS into the tail vein. Inocula for each experiment were confirmed by serial dilution and plate counting.were performed by plating on to blood agar plates (BAP) and testing for a-hemolysis and optochin sensitivity to check the purity and identity of the cultures. Cultures of Didtr were terminally subcultured on plates containing 50 mg/ml Tmp.In vivo growth of TIGR4 and DidtrBlood samples were collected by retro-orbital bleeding from mice inoculated intravenously with either TIGR4 or Didtr at 3, 6, 12, 24, 36 and 48 h after infection. Bacterial density was determined by plating serially diluted blood samples on BAP and incubating 18?4 hr.Ession both in vitro and within a host and undoubtedly expand our understanding the complete subset of genes which are controlled either directly or indirectly byidtr mutant constructionAn idtr mutant was constructed using PCR ligation mutagenesis as described by [42]. A schematic representation of the mutant construction is outlined in Figure 5. Briefly, tmp was amplified from pkoT plasmid DNA (primers T1 and T2) and the flanking regions of idtr were amplified from TIGR4 genomic DNA (primers I1 and I2, I3 and I4) described in table 2. The PCR amplified and purified I1-I2, I3-I4 and the tmpr cassette were subjected to single and double digestion by HindiIII and BamHI respectively according to the manufacturer’s protocol (Promega, Madison, WI), The digested PCR products were ligated using T4 DNA ligase (Promega, Madison, WI). The resulting construct (,2 kb) was amplified using primers I1-I4 and was used to transform TIGR4 as previously described [25]. The double recombination event was selected by plating on plates containing 50 mg/ml of Tmp. Identification of Tmp-resistant mutants was confirmed by both PCR analysis and DNA sequencing.Animal models of pneumococcal infectionAll animal studies were performed on either 10?2 wk old CBA/CaHN-Btkxid/J or C57BL/6 mice obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in the VA animal facility. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional 1527786 Animal Care and Use Committee at the VA Medical Center (assurance number: A31011). For all in vivo studies pneumococcal strains were administered either intranasally (i.n.) or intravenously (i.v.) as noted. Following infection animals were observed every twelve hours for signs of piloerection, inability to eat or drink, or failure to withdraw from threatening stimuli. Animals were weighed daily and any animal which exhibited any aforementioned behavior, or lost more than 15 of pre-infection body weight, was euthanized. For intranasalRole of idtr in Pneumococcal InfectionsFigure 5. Schematic representation of Didtr construction. H-HindIII, B-BamHI. T1, T2 amplify the tmp cassette (495 bp); T1 and T2 have H and B at 59 end. I1, I2 and I3, I4 amplify 59 and 39 end of idtr. I2 and I3 have H and B at 59 end. 15857111 I1, I2 amplify a 945 bp product and I3, I4 amplify a product of 489 bp. doi:10.1371/journal.pone.0055157.ginfection, a suspension of mid-exponential phase TIGR4 or Didtr (106 CFU) in PBS was delivered into the nares of anesthetized mice (20 ml per mouse) as previously described [43]. At this volume and cell number pneumococci remain localized to the nasopharynx. Intravenous inoculation was performed by injecting 105 CFU of TIGR4 or Didtr in 100 ml of PBS into the tail vein. Inocula for each experiment were confirmed by serial dilution and plate counting.were performed by plating on to blood agar plates (BAP) and testing for a-hemolysis and optochin sensitivity to check the purity and identity of the cultures. Cultures of Didtr were terminally subcultured on plates containing 50 mg/ml Tmp.In vivo growth of TIGR4 and DidtrBlood samples were collected by retro-orbital bleeding from mice inoculated intravenously with either TIGR4 or Didtr at 3, 6, 12, 24, 36 and 48 h after infection. Bacterial density was determined by plating serially diluted blood samples on BAP and incubating 18?4 hr.
