At different time points postinfection, mice were sacrificed and the spleen, stomach, and cecum were harvested. The numbers of bacteria in these three organs were determined. No bacteria were found in the stomach at 12–24 hours postinfection, consistent with the fact that the systemicSalmonellainfection does not spread to the organ or is cleared at this early time point (data not shown). The expression of the tagged proteins in the bacterial strains isolated from the spleen and cecum of infected mice was detected using Western analysis
with an anti-FLAG antibody and normalized Emricasan cost using the expression of bacterial protein DnaK as the internal control (Figure6A–B). Normalization of samples was also carried out by loading total protein extracted from the same CFU (e.g. 5 × 107CFU) of bacteria in each lane. The protein level of DnaK did not appear to be significantly different in bacteria from the spleen and cecum as similar amount of the DnaK protein was
detected from 5 × 107CFU of each bacterial strain regardless of infection route (intraperitoneally or intragastrically) or time point postinfection (12–24 hours or 5–7 days) (data not shown). Figure 6 Western analyses of the expression of the tagged proteins from the internalized bacterial strains T-prgI (lane 1), T-sipA (lane 2), T-sptP (lane 3), T-spaO (lane 4), T-sopE2 (lane 5), and T-sipB (lane 6) recovered from spleens. BALB/c mice were intraperitoneally heptaminol infected with 1 × Doramapimod concentration 105CFU of the tagged strains, and internalized bacteria were Selleck MK-8931 recovered from the spleens at 5 days
post inoculation. The expression of bacterial DnaK was used as the internal control (B). Protein samples were reacted with antibodies against the FLAG sequence (A) and DnaK (B). Each lane was loaded with material from 5 × 107CFU bacteria. Salmonellastrains isolated from both the spleen and cecum at 18 hours postinfection continued to express PrgI, SpoE2, SipB, and SipA. In contrast, a substantial level of SpaO was detected inSalmonellaisolated from the cecum but not the spleen, while that of SptP was observed inSalmonellarecovered from the spleen but not the cecum (Figure7A–B). These results suggest that SpaO and SptP are differentially expressed bySalmonellawhen they colonize specific organs and tissues. Figure 7 Level of the tagged proteins from the internalized bacterial strains T-prgI, T-sipA, T-sptP, T-spaO, T-sopE2, and T-sipB recovered from the spleen (A) and cecum (B). BALB/c mice were intraperitoneally infected with 1 × 107or 1 × 105CFU of the tagged strains, and internalized bacteria were recovered from the spleen at 18 hours or 5 days post inoculation, respectively.