HilD, HilC, and RtsA Form Homodimers and Heterodimers To Regulate Expression of the Salmonella Pathogenicity Island I Type III Secretion System.

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.