g. inoculation) post infection. Each sample was analyzed in triplicate and the analysis was repeated at least twice. 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. In vitrosynthesis and secretion of the tagged SPI-1 proteins under different cultured conditions After being ingested from contaminated food or polluted water,Salmonellawill encounter a series of extreme environmental changes such as acidity in the

stomach, hypoxia, hyperosmolarity, and other conditions BTSA1 research buy such as fermentation in the gut [22–24]. The expression of bacterial genes including those of SPI-1 is expected to be regulated to allow bacteria to adapt to new environments and to prepare for the invasion of the intestinal epithelium. To investigate the synthesis and secretion of the SPI-1 proteins, each of the tagged strains was grown under five different conditions that resembled the early stages of its natural infection, and the expression of the tagged proteins was

studied. (A) Expression in rich media LB broth Western analyses were carried out to detect the expression of the Cilengitide tagged proteins with an anti-FLAG antibody (Figure2Aand2B), using the expression of bacterial DnaK protein as the internal control (Figure2C). KPT-8602 purchase 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. SPI-1 proteins PrgI, SopE2, SpaO, SptP, SipB, and SipA were detected inSalmonellacultured in LB broth (Figure2B). Furthermore, SpoE2, SptP, SipB, and SipA but not PrgI or SpaO Acetophenone were detected in the culture supernatant (Figure2A, data not

shown). These results are consistent with the previous observations that SpaO and PrgI are the structural components of the needle complex [5]. Figure 2 Western analyses of the synthesis (B) and secretion (A) of the tagged proteins from bacterial strains T-prgI (13 KD)(lane 1), T-spoE2 (29 KD) (lane 2), T-spaO (36 KD) (lane 3), T-sptP (62 KD) (lane 4), T-sipB (65 KD) (lane 5), and T-sipA (76 KD) (lane 6). The expression of bacterial DnaK was used as the internal control (C). Protein samples were separated in SDS-polyacrylamide gels and reacted with antibodies against the FLAG sequence (A-B) and DnaK (C). Each lane was loaded with material from 5 × 107CFU bacteria. The molecular masses of some of the proteins in the PageRuler protein size markers (Fermentas) are shown and given in kiloDaltons (KD). (B) Expression under different pH conditions Acidity in the stomach is the first stressSalmonellameets after being ingested orally. Because the environment in the intestine is relatively basic,Salmonellawill encounter an increase in pH after it reaches the intestine.