HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium invades intestinal epithelial cells using a type three secretion system (TTSS) encoded on Salmonella Pathogenicity Island 1 (SPI1). The SPI1 TTSS injects effector proteins into the cytosol of host cells where they promote actin rearrangement and engulfment of the bacteria. We previously identified RtsA, an AraC-like protein similar to the known HilC and Hill) regulatory proteins. Like HilC and KID, RtsA activates expression of SPI1 genes by binding upstream of the master regulatory gene hilA to induce its expression. HilA activates the SPI1 TTSS structural genes. Here we present evidence that hilA expression, and hence the SPI1 TTSS, is controlled by a feedforward regulatory loop. We demonstrate that HilC, HilD and RtsA are each capable of independently inducing expression of the hilC, hilD and rtsA genes, and that each can independently activate hilA. Using competition assays in vivo, we show that each of the hilA regulators contribute to SPI1 induction in the intestine. Of the three, Hill) has a predominant role, but apparently does not act alone either in vivo or in vitro to sufficiently activate Spill. The two-component regulatory systems, SirA/BarA and OmpR/EnvZ, function through Hill), thus inducing hilC, rtsA and hilA. However, the two-component systems are not responsible for environmental regulation of SPI1. Rather, we show that 'SPI1 inducing conditions' cause independent activation of the rtsA, hilC and hilD genes in the absence of known regulators. Our model of SPI1 regulation provides a framework for future studies aimed at understanding this complicated regulatory network.