SirA, CsrBC and HilD form in vivo a regulatory cascade that controls the SP1-1 and SPI-2 gene expression when Salmonella Typhimurium is in the intestinal lumen and are required for cecal colonization and liver dissemination in the avian model.

When Salmonella Typhimurium grows in LB in vitro, BarA/SirA system induces the expression of CsrB/C, that sequester the regulator CsrA, thus derepressing HilD regulator. HilD activated induces HilA and SsrB expression, central regulators of SPI-1 and SPI-2, respectively. We analyze the in vivo contribution of these genes in 1-day- and 1-week-old chickens infected with a Wild Type strain of S. Typhimurium and the ΔsirA, ΔcsrB/C and ΔhilD mutants. CFUs determination in liver and cecum showed that the mutants colonized both organs in lower amounts compared with WT strain in both chicken models and they were affected in the ability to produce histological injuries in these organs. We analyzed whether these genes operate in cascade in vivo and prior to intestinal invasion, by analyzing hilA, ssrAB, hilD, csrB and sirA expression in the cecal contents of chickens inoculated with Wild Type and mutants 120 min after inoculation. Expression of hilA and ssrB, but not csrB and sirA, was decreased in ΔhilD strain. Expression of hilD, hilA and ssrB, but not sirA, was decreased in samples of ΔcsrB/C. In SirA absence, expression of all genes was decreased. Our findings demonstrate that SirA, CsrB/C and HilD conform a regulatory cascade in vivo, when Salmonella is in intestinal lumen and this cascade controls the expression of HilA and SsrB prior to intestinal invasion. We also demonstrate that these genes are necessary for the production of lesions during S. Typhimurium infection in chickens.