For BALB/c mice infected intragastrically with 1 × 106 CFU of the tagged or the wild type strains, Protein Tyrosine Kinase inhibitor all infected mice died within 7 days post infection and no significant
difference was observed among the wild type and the tagged strains (Figure 5A). No significant difference in the colonization of the internal organs such as spleen, liver, and ileum, was observed between the parental (wild type) SE2472 strain and the tagged strains regardless of the route of inoculation (Table 4). These results suggest that tagging of the target ORF does not impair the invasiveness, growth, and virulence of the bacteria, and that the tagged strains can be used as model strains to study infection of Salmonella in check details vitro and in vivo, including the expression of the SPI-1 proteins. Table 4 The numbers of bacteria (CFU) in different organs from animals. Salmonella strains Colonization (i.p.) Colonization (i.g.) log CFU per organ log CFU per organ Liver Spleen Liver Ileum SE2472 9.0 ± 0.5 8.3 ± 0.5 9.1 ± 0.5 8.2 ± 0.5 SipA(HF) 9.1 ± 0.5 8.2 ± 0.5 8.9 ± 0.5 8.3 ± 0.5 SipC(HF) 9.2 ± 0.5 8.4 ± 0.5 9.0 ± 0.5 8.2 ± 0.5 SopB(HF) 9.0 ± 0.5 8.4 ± 0.5 9.2 ± 0.5 8.1 ± 0.5 * BALB/c mice were either infected intraperitoneally (i.p.) with 1 × 104 CFU or intragastrically (i.g.) with 1 × 106 CFU bacteria. A group of 5 mice was infected and the organs were
harvested at 4 (for i.p. infection) or 6 days (for i.g. inoculation) post infection. Each sample was analyzed in triplicate and the analysis was repeated at least three times. The CFU of the sample was expressed as the average of the values obtained. The concentrations of bacteria were recorded as CFU/ml of organ homogenate. The limit of bacteria detection in the organ homogenates
was 10 CFU/ml. Figure 5 (A) Mortality of BALB/c mice infected with Salmonella strains, (B) Western blot analyses of the synthesis of the tagged proteins from SE2472 (lane 1), SipC(HF) (lanes 2-3), SipA(HF) (lanes 4-5), and SopB(HF) (lanes 6-7), and (C) Effect of the STA-9090 ic50 treatment of hydrogen peroxide on the expression Farnesyltransferase of the tagged SPI-1 proteins. (A) Mice (5 animals per group) were infected intragastrically with 1 × 106 CFU of each bacterial strain. Mortality of mice was monitored for at least 10 days postinfection. (B) The expression of bacterial FliC was used as the internal control. The bacterial strains were grown in LB broth in the absence (-, lanes 2, 4, and 6) and presence of 5 mM H2O2 (H2O2, lanes 3, 5, and 7) at 37°C for 2 hours. SE2472 was grown in the absence of H2O2 (lane 1). Protein samples were separated in SDS-polyacrylamide gels and reacted with antibodies against the FLAG sequence (top panel) and FliC (low panel). Each lane was loaded with material from 5 × 107 CFU bacteria.