Salmonella enterica serovar Typhimurium colonizes and invades host intestinal epithelial cells using the type three secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The level of SPI1 T3SS gene expression is controlled by the transcriptional activator HilA, encoded on SPI1. Expression of hilA is positively regulated by three homologous transcriptional regulators, HilD, HilC, and RtsA, belonging to the AraC/XylS family. These regulators also activate the hilD, hilC, and rtsA genes by binding to the same DNA sequences upstream of these promoters, forming a complex feed-forward loop to control SPI1 expression. Despite the apparent redundancy in function, HilD has a unique role in SPI1 regulation because the majority of external regulatory inputs act exclusively through HilD. To better understand SPI1 regulation, the nature of interaction between HilD, HilC, and RtsA has been characterized using biochemical and genetic techniques. Our results showed that HilD, HilC, and RtsA can form heterodimers as well as homodimers in solution. Comparison with other AraC family members identified a putative α-helix in the N-terminal domain, which acts as the dimerization domain. Alanine substitution in this region results in reduced dimerization of HilD and HilC and also affects their ability to activate hilA expression. The dimer interactions of HilD, HilC, and RtsA add another layer of complexity to the SPI1 regulatory circuit, providing a more comprehensive understanding of SPI1 T3SS regulation and Salmonella pathogenesis. IMPORTANCE The SPI1 type three secretion system is a key virulence factor required for Salmonella to both cause gastroenteritis and initiate serious systemic disease. The system responds to numerous environmental signals in the intestine, integrating this information via a complex regulatory network. Here, we show that the primary regulatory proteins in the network function as both homodimers and heterodimers, providing information regarding both regulation of virulence in this important pathogen and general signal integration to control gene expression.

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.

The light chain of botulinum neurotoxin A (BoNT/A-LC) is a Zn-dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C-terminus of SNAP25 (residues 141-206) retains full activity for BoNT/A-LC-catalyzed cleavage at P1-P1' (Gln197-Arg198). Using the structure of a complex between the C-terminus of SNAP25 and BoNT/A-LC as a model to design SNAP25-derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala-Asn-Gln-Arg (residues 195-198) at the substrate cleavage site, with the intent to identify possible side-chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1-P1' residues to explore the spatial tolerance within the active-site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A-LC. Among the SNAPIs tested, several known cleavage-resistant, single-point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly-insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A-LC. Two of the Gly-insertion mutants exhibited 10-fold lower IC 50 values than the wildtype 66-mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.