The Role of Coupled Positive Feedback in the Expression of the SPI1 Type Three Secretion System in Salmonella

Abstract

Salmonella enterica serovar Typhimurium is a common food-borne pathogen that induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). The genes encoding the SPI1 T3SS are tightly regulated by a network of interacting transcriptional regulators involving three coupled positive feedback loops. While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown. To determine the function of this circuit, we measured gene expression dynamics at both population and single-cell resolution in a number of SPI1 regulatory mutants. Using these data, we constructed a mathematical model of the SPI1 gene circuit. Analysis of the model predicted that the circuit serves two functions. The first is to place a threshold on SPI1 activation, ensuring that the genes encoding the T3SS are expressed only in response to the appropriate combination of environmental and cellular cues. The second is to amplify SPI1 gene expression. To experimentally test these predictions, we rewired the SPI1 genetic circuit by changing its regulatory architecture. This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal. Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics.

HilD Induces Expression of Salmonella Pathogenicity Island 2 Genes by Displacing the Global Negative Regulator H-NS from ssrAB

“…HilD, HilC, and RtsA are homologous AraC-like regulators that bind to very similar DNA sites; therefore, when these regulators are overexpressed, they are able to self-activate their own expression and to activate the expression of one another, as well as to induce the expression of most genes belonging to the HilD regulon; however, HilD is considered to be dominant, as deletion of hilD, but not of hilC or rtsA, drastically reduces the expression of the targets of HilD (23,24,(26)(27)(28)(29)(30)(61)(62)(63)(64). Our results indicate that the expression of ssrAB is not affected in a ⌬hilC (21) or ⌬rtsA mutant, but a plasmid expressing HilC can restore the expression of ssrAB in the ⌬hilD mutant (unpublished data).…”

The Salmonella Spi1 Virulence Regulatory Protein HilD Directly Activates Transcription of the Flagellar Master OperonflhDC

“…In a feed-forward loop, each of those regulators can activate the hilD, hilC, and rtsA genes, as well as the gene encoding the transcriptional Spi1 activator HilA (12). HilD is a dominant regulator of hilA transcription, while HilC and RtsA amplify hilA gene expression (12,13).…”

Identification of HilD-Regulated Genes in Salmonella enterica Serovar Typhimurium

“…The OmpR/ToxR family transcription factor HilA is considered to be the master regulator of SPI-1, as it is capable of activating expression of all SPI-1 genes required for the assembly of a functional T3SS (6). However, HilA activation is dependent upon a coupled, positive-feedback loop comprising the AraC/XylS family transcription factors HilD, HilC, and RtsA, which drive HilA expression above the SPI-1 activation threshold (7,8). HilD, HilC, and RtsA are capable of self-activating expression in addition to activating expression of one another and of hilA.…”