One of these T3SSs is encoded by a cluster of virulence genes termedSalmonellaPathogenicity Island 1 (SPI-1). The second T3SS is encoded by another cluster of genes in a separate pathogenicity island termedSalmonellaPathogenicity this website Island 2 (SPI-2). Each of the T3SSs is constituted by a secretome (secretion apparatus), its substrates (effector proteins) and chaperone proteins [7,9]. These two

T3SSs perform quite different functions inSalmonellainfection. It is generally believed that SPI-1 T3SS is responsible for invasion of non-phagocytic cells, while SPI-2 T3SS is essential for the intracellular replication and systemic infection [7,9]. In addition to the well-characterized SPI-1 and SPI-2, many other SPIs have been described inSalmonellabut their roles have not yet been fully investigated [10–12]. Chracterization of the expression patterns of the genes of SPI-1 and other SPIs should provide insight into the functional roles of these factors inSalmonellainfection. The modulation of expression of genes in SPI-1 is remarkably complex and needs further characterization [13,14]. For example, in contrast to the current model of SPI-mediated pathogenesis, several studies have shown that the expression of some SPI-1 genes is induced upon invasion of both macrophages and epithelial cells and that

several SPI-1 factors GDC0449 are essential for intracellular replication [15–17]. Furthermore, SPI-1 proteins, SipA, SopA, SopB, SopD, and SopE2 were found to be expressed bySalmonellain infected animals at the late stages of infection [17]. These results suggest that in addition to its generally recognized role in invasion, the SPI-1 factors may play an important role post-invasion. Hence, the role

of the SPI-1 factors in bacterial pathogenesis, especially during the late stages of salmonellosis, needs further characterization and their expressionin vivoneeds to be studied. Extensive studies have been carried out to investigate the expression of SPI-1 under different conditionsin vitro[13,18]. Bay 11-7085 However, most of these studies were performed by examining the transcription levels of these genes either using microarray or a reporter system [18–20], and protein expression under the native promoter for these T3SS factors has not been extensively investigated. In addition, little is known about the expression of these factorsin vivo, especially during the MAPK inhibitor established phase of infection. In this study, we constructedSalmonellastrains that contained a FLAG epitope sequence inserted in frame into the carboxyl terminus of SPI-1 genesprgI,sipA,sipB,sopE2,spaO, andsptP, and characterized the expression of the tagged proteinsin vitroandin vivoduring murine salmonellosis. The FLAG epitope is an octapeptide protein tag that has been widely used for tagging a protein, which in turn can be detected and studied using the anti-FLAG antibody [21].