Long Distance Effects of H-NS Binding in the Control of hilD Expression in the Salmonella SPI1 Locus.

Salmonella Typhimurium utilizes a type three secretion system (T3SS) encoded on the Salmonella pathogenicity island 1 (SPI1) to invade intestinal epithelial cells and induce inflammatory diarrhea. HilA activates expression of the T3SS structural genes. Expression of hilA is controlled by the transcription factors HilD, HilC and RtsA, which act in a complex feed-forward regulatory loop. The nucleoid-associated protein H-NS is a xenogenic silencer that has a major effect on SPI1 expression. In this work, we use genetic techniques to show that disruptions of the chromosomal region surrounding hilD have a cis-effect on H-NS-mediated repression of the hilD promoter; this effect occurs asymmetrically over ∼4 kb spanning the prgH-hilD intergenic region. CAT cassettes inserted at various positions in this region are also silenced in relation to the proximity to the hilD promoter. We identify a putative H-NS nucleation site, mutation of which results in de-repression of the locus. Furthermore, we genetically show that HilD abrogates H-NS-mediated silencing to activate the hilD promoter. In contrast, H-NS-mediated repression of the hilA promoter, downstream of hilD, is through its control of HilD, which directly activates hilA transcription. Likewise, activation of the prgH promoter, although in a region silenced by H-NS, is strictly dependent on HilA. In summary, we propose a model in which H-NS nucleates within the hilD promoter region to polymerize and exert its repressive effect. Thus, H-NS-mediated repression of SPI1 is primarily through control of hilD expression, with HilD capable of overcoming H-NS to autoactivate. IMPORTANCE The foodborne pathogen Salmonella relies on a type III secretion system to invade intestinal epithelial cells and initiate infection. This system was acquired through horizontal gene transfer, essentially creating the Salmonella genus. Expression of this critical virulence factor is controlled by a complex regulatory network. The nucleoid protein H-NS is a global repressor of horizontally acquired genomic loci. Here we identify the critical site of H-NS regulation in this system and show that alterations to the DNA over a surprisingly large region affect this regulation, providing important information regarding the mechanism of H-NS action.