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.

Author Summary

Salmonella is a causative agent for a wide range of diseases in humans, including gastroenteritis and enteric fever. A key step in the infection process occurs when Salmonella invades intestinal epithelial cells using a molecular hypodermic needle. Salmonella uses these needles to inject proteins into host cells that enable the bacterium to enter and replicate within them. The production of these needles, and the corollary decision to invade the host, is tightly controlled by a complex network of interacting regulatory proteins that, when studied individually, seemingly have either redundant or antagonizing effects. To understand how this ensemble of regulators dynamically controls the expression of these invasion genes, we systematically deconstructed the network and then used this information to analyze their composite behavior by computer simulation. Our analysis demonstrates that this regulatory network ensures that the invasion genes are expressed only when the invasion signals, a combination of environmental and cellular cues, exceed a defined threshold. Once induced, this network further amplifies and accelerates the expression of the invasion genes. These results further our understanding of this important pathogen by unraveling a key mechanism during infection, namely the decision to invade.