Salmonella enterica serovar Typhimurium injects a set of effector proteins into the cytoplasm of host cells via Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS) and induces bacterial invasion into host cells. Cyclic-di-GMP, a circular RNA dinucleotide synthesized by diguanylate cyclases, functions as a second messenger in a variety of physiological processes, including motility, biofilm formation, and virulence factor production. In this study, in order to address how cyclic-di-GMP regulates virulence factor production in S. Typhimurium, the role of cyclic-di-GMP in SPI-1 regulation was investigated. Overexpression of a diguanylate cyclase AdrA significantly decreased the mRNA expression and protein production of HilD, a master regulator of SPI-1, thereby down-regulating the expression of cognate SPI-1 genes. However, the negative regulation of SPI-1 by cyclic-di-GMP was abolished in the absence of H-NS or Lon, which represses hilD expression and controls HilD turnover, respectively. Considering that AdrA overexpression did not influence hns and lon expression, cyclic-di-GMP might exert negative regulation on SPI-1 by modulating the activity of H-NS and Lon. With regard to a pivotal role of cyclic-di-GMP in controlling motile and sessile lifestyles in diverse bacterial pathogens, this study suggests a new role of cyclic-di-GMP in Salmonella lifestyle conversion between colonization in intestinal lumen and invasion into host cells
