An understanding of the expression profiles of Salmonella SPI-1 factors and other proteins in the presence of reactive oxygen species such as H2O2 should provide insight into the identification of virulent determinants important for Salmonella to survive in macrophages and cause systemic infection in the spleen in vivo. The expression of Salmonella genes (including those encoding SPI-1 factors) in vitro under various conditions

has been extensively studied [17–21]. However, most of these studies were performed #Selleckchem ALK inhibitor randurls[1|1|,|CHEM1|]# by examining the transcription levels of Salmonella genes either using microarray or a reporter system [17, 19–23]. Recently, proteomic analysis of Salmonella protein expression in the spleen of infected animals has been reported [24]. Furthermore, Smith and co-workers have reported global protein profiles of Salmonella enterica serovars Typhimurium and Typhi cultured at the stationary phase, logarithmic click here (log) phase, or phagosome-mimicking culture

conditions, and the expression profiles of proteins in infected macrophages [25–28]. However, to our knowledge, global expression profiling of Salmonella proteins upon exposure to reactive oxygen species such as H2O2 has not been reported, and efforts to identify proteins whose expression levels are affected by oxidative stress have been limited mostly to a few proteins at a time [9, 29, 30]. In addition,

expression of Salmonella proteins including those of SPI-1 in vivo during the established phase of infection has not been extensively studied. In this study, we have modified the procedure Clomifene of Stable Isotope Labeling by Amino acids in Cell culture (SILAC) [31, 32] to develop a mass spectrometry (MS)-based approach to carry out quantitative proteomic analysis of Salmonella. Using this procedure, we have identified 76 proteins from a strain of Salmonella enterica serovar Enteritidis that are differentially regulated upon exposure to H2O2. The results on selected SPI-1 proteins were confirmed by Western blot analyses, validating the accuracy and reproducibility of our approach for quantitative analyses of protein expression. The expression of several SPI-1 proteins was further analyzed in infected macrophages and in the spleen of infected mice. These results suggest a possible role for SPI-1 proteins in Salmonella infection in the presence of oxidative stress and in systemic infection in an animal host. Results Stable isotope labeling of Salmonella with 15N-containing growth media We used a virulent clinical isolate of Salmonella enterica serovar Enteritidis SE2472 for this analysis. Our previous studies have shown that almost all clinical strains analyzed, including SE2472, exhibited similar levels of resistance to H2O2 [33].