As an IVI antigen identified in a previous study using IVIAT method, the regulation of YncD expression usually can be induced in certain conditions encountered in vivo. In the genome of S. Typhi, the yncD gene is adjacent to the yncE gene but it has the opposite transcriptional orientation. The yncE gene is induced under iron restriction through the action of the global iron regulator Fur in E. coli; however, the regulator and the iron restriction did not affect the transcription of the yncD gene (McHugh et al., 2003). Upstream of the yncD gene, a possible PmrAB-box sequence, cattttcttaacttaat, was found, which indicated that the

expression of the yncD gene may be regulated by the PmrAB system (Marchal et al., Ivacaftor molecular weight 2004). In agreement with this anticipation, acidic pH, a main activation signal of the PmrAB system, was proved to induce the expression of yncD gene in the present study. The acid

condition is an ecological niche that pathogens usually encounter in vivo. Enteric pathogens share an oral route of infection (Gorden & Small, 1993; Maurer et al., 2005). During the initial infection, enteric bacteria PARP inhibition encounter low pH stresses in the human digestive tract (Drasar et al., 1969). Successful colonization requires survival through the stomach at pH 1–2 or the intestinal tract at pH 2–7 (Dressman et al., 1990). The bacteria respond to low pH stresses by regulating gene expression, which maintains internal pH homeostasis (Gorden & Small, 1993). Moreover, low pH is an important inducing factor of virulence genes as well. Low pH enhances the expression of numerous virulence factors, such as the ToxR-ToxT virulence regulon in Vibrio cholerae (Behari et al., 2001) and the phoP-phoQ regulon of Salmonella enterica (Bearson et al., 1998). It also enhances expression of genes for flagellar Epothilone B (EPO906, Patupilone) motility and catabolism (Maurer et al., 2005). Due to lack of information, the exact function of YncD remains unclear. However, our study showed that YncD plays a role

in the in vivo survival of S. Typhi. As the yncD gene knockout significantly reduces bacterial virulence and the attenuated strain shows an effective immunoprotection, the yncD gene is undoubtedly a good candidate gene for the construction of attenuated vaccine strains. This study was supported by the National Natural Science Foundation of China (Grant No. 30500435). We gratefully acknowledge Victor de Lorenzo of the Centro Nacional de Biotecnologia CSIC, Spain, for providing the Mini-Tn5 plasmid. K.X. and Z.C. contributed equally to this work. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.