HilD is an Arac-like transcriptional regulator encoded in the Salmonella pathogenicity island 1 (SPI-1), which actives transcription of many genes within and outside SPI-1 that are mainly required for invasion of Salmonella into host cells. HilD controls expression of target genes directly or by acting through distinct regulators; three different regulatory cascades headed by HilD have been described to date. Here, by analyzing the effect of HilD on the yobH gene in Salmonella enterica serovar typhimurium (S. Typhimurium), we further define an additional regulatory cascade mediated by HilD, which was revealed by previous genome-wide analyses. in this regulatory cascade, HilD acts through SprB, a LuxR-like regulator encoded in SPI-1, to induce expression of virulence genes. Our data show that HilD induces expression of sprB by directly counteracting H-nS-mediated repression on the promoter region upstream of this gene. then, SprB directly activates expression of several genes including yobH, slrP and ugtL. Interestingly, we found that YobH, a protein of only 79 amino acids, is required for invasion of S. typhimurium into HeLa cells and mouse macrophages. thus, our results reveal a novel S. typhimurium invasion factor and provide more evidence supporting the HilD-SprB regulatory cascade.The genus Salmonella groups facultative anaerobic Gram-negative bacteria and is divided into two species, S. enterica and S. bongori. The former is responsible for diseases ranging from gastroenteritis to severe systemic infections in a wide range of hosts, and it comprises 6 subspecies that are further divided into serovars 1,2 . The broad-host-range S. enterica serovar Typhimurium (S. Typhimurium) is a common cause of gastroenteritis in humans and many animals worldwide; furthermore, it can also cause systemic infection in humans and some animals, including laboratory mice 1,3,4 . For this reason, S. Typhimurium is frequently used as a model for studying the host-pathogen interactions during infection with Salmonella.

When Salmonella is grown in the nutrient-rich lysogeny broth (LB), the AraC-like transcriptional regulator HilD positively controls the expression of genes required for Salmonella invasion of host cells, such as the Salmonella pathogenicity island 1 (SPI-1) genes. However, in minimal media, the two-component system PhoP/Q activates the expression of genes necessary for Salmonella replication inside host cells, such as the SPI-2 genes. Recently, we found that the SL1344_1872 hypothetical gene, located in a S. Typhimurium genomic island, is co-expressed with the SPI-1 genes. In this study we demonstrate that HilD induces indirectly the expression of SL1344_1872 when S. Typhimurium is grown in LB; therefore, we named SL1344_1872 as grhD1 for gene regulated by HilD. Furthermore, we found that PhoP positively controls the expression of grhD1, independently of HilD, when S. Typhimurium is grown in LB or N-minimal medium. Moreover, we demonstrate that the grhD1 gene is required for the invasion of S. Typhimurium into epithelial cells, macrophages and fibroblasts, as well as for the intestinal inflammatory response caused by S. Typhimurium in mice. Thus, our results reveal a novel virulence factor of Salmonella, whose expression is positively and independently controlled by the HilD and PhoP transcriptional regulators.

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