A prerequisite for Salmonella enterica to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals through a variety of global regulatory systems. We have previously shown that three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control the expression of the hilA gene, which encodes the direct regulator of the SPI1 structural genes. In this work, we characterize a major positive regulator of this system, the flagellar protein FliZ. Through genetic and biochemical analyses, we show that FliZ posttranslationally controls HilD to positively regulate hilA expression. This mechanism is independent of other flagellar components and is not mediated through the negative regulator HilE or through FliZmediated RpoS regulation. We demonstrate that FliZ controls HilD protein activity and not stability. FliZ regulates HilD in the absence of Lon protease, previously shown to degrade HilD. Indeed, it appears that FliZ, rather than HilD, is the most relevant target of Lon as it relates to SPI1 expression. Mutants lacking FliZ are significantly attenuated in their ability to colonize the intestine but are unaffected during systemic infection. The intestinal attenuation is partially dependent on SPI1, but FliZ has additional pleiotropic effects.During the course of infection, Salmonella enterica serovar Typhimurium induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1) (20,51,53,55). The SPI1 locus is a 40-kb island and carries all of the genes for a functional T3SS apparatus, a number of secreted effectors, and the regulatory proteins HilA, HilC, and HilD (34). RtsA, homologous to HilD and HilC, is encoded on a 15-kb island inserted in the tRNA PheU gene (15). HilA is the master SPI1 regulator and directly binds to the promoters and activates expression of the prg-org and inv-spa operons, encoding the components of the apparatus (2,8,10,11,32). The expression of hilA is controlled by a complex feed-forward regulatory loop consisting of HilD, HilC, and RtsA, each of which can independently activate hilA expression (13) (Fig. 1). Of these three, HilD has the predominant role, but apparently, it alone cannot activate SPI1 sufficiently in vivo (13). HilC and RtsA act as amplifiers of the inducing signals. A number of additional regulatory systems also affect SPI1 regulation (1,17). Most of these systems seem to act through HilD (18), suggesting that HilD is the integration point for signal transduction into the SPI1 system. RtsA, HilD, and HilC directly activate dsbA, which encodes a periplasmic disulfide bond oxidoredu...

Upon contact with intestinal epithelial cells, Salmonella enterica serovar Typhimurium injects a set of effector proteins into the host cell cytoplasm via the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS) to induce inflammatory diarrhea and bacterial uptake. The master SPI1 regulatory gene hilA is controlled directly by three AraC-like regulators: HilD, HilC, and RtsA. Previous work suggested a role for DsbA, a periplasmic disulfide bond oxidase, in SPI1 T3SS function. RtsA directly activates dsbA, and deletion of dsbA leads to loss of SPI1-dependent secretion. We have studied the dsbA phenotypes by monitoring expression of SPI1 regulatory, structural, and effector genes. Here we present evidence that loss of DsbA independently affects SPI1 regulation and SPI1 function. The dsbA-mediated feedback inhibition of SPI1 transcription is not due to defects in the SPI1 T3SS apparatus. Rather, the transcriptional response is dependent on both the flagellar protein FliZ and the RcsCDB system, which also affects fliZ transcription. Thus, the status of disulfide bonds in the periplasm affects expression of the SPI1 system indirectly via the flagellar apparatus. RcsCDB can also affect SPI1 independently of FliZ. All regulation is through HilD, consistent with our current model for SPI1 regulation.

Disulfide bond formation in periplasmic proteins is catalysed by the DsbA/DsbB system in most Gram-negative bacteria. Salmonella enterica serovar Typhimurium also encodes a paralogous pair of proteins to DsbA and DsbB, DsbL and DsbI, respectively, downstream of a periplasmic arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI function as a redox pair contributing to periplasmic disulfide bond formation and, as such, affect transcription of the Salmonella pathogenicity island 1 (SPI1) type three secretion system genes and activation of the RcsCDB system, as well as ASST activity. In contrast to DsbA/DsbB, however, the DsbL/DsbI system cannot catalyse the disulfide bond formation required for flagellar assembly. Phylogenic analysis suggests that the assT dsbL dsbI genes are ancestral in the Enterobacteriaceae, but have been lost in many lineages. Deletion of assT confers no virulence defect during acute Salmonella infection of mice.