PhoP-Mediated Repression of the SPI1 Type 3 Secretion System in Salmonella enterica Serovar Typhimurium

Abstract

Salmonella must rapidly adapt to various niches in the host during infection. Relevant virulence factors must be appropriately induced, and systems that are detrimental in a particular environment must be turned off. Salmonella infects intestinal epithelial cells using a type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The system is controlled by three AraC-like regulators, HilD, HilC, and RtsA, which form a complex feed-forward loop to activate expression of hilA, encoding the main transcriptional regulator of T3SS structural genes. This system is tightly regulated, with many of the activating signals acting at the level of hilD translation or HilD protein activity. Once inside the phagosomes of epithelial cells, or in macrophages during systemic stages of disease, the SPI1 T3SS is no longer required or expressed. Here, we show that the PhoPQ two-component system, critical for intracellular survival, appears to be the primary mechanism by which Salmonella shuts down the SPI1 T3SS. PhoP negatively regulates hilA through multiple distinct mechanisms: direct transcriptional repression of the hilA promoter, indirect transcriptional repression of both the hilD and rtsA promoters, and activation of the small RNA (sRNA) PinT. Genetic analyses and electrophoretic mobility shift assays suggest that PhoP specifically binds the hilA promoter to block binding of activators HilD, HilC, and RtsA as a mechanism of repression. IMPORTANCE Salmonella is one of the most common foodborne pathogens, causing an estimated 1.2 million illnesses per year in the United States. A key step in infection is the activation of the bacterial invasion machinery, which induces uptake of the bacterium into epithelial cells and leads to induction of inflammatory diarrhea. Upon entering the vacuolar compartments of host cells, Salmonella senses an environmental transition and represses the invasion machinery with a two-component system relevant for survival within the vacuole. This adaptation to specific host niches is an important example of how signals are integrated for survival of the pathogen.

The Small RNA PinT Contributes to PhoP-Mediated Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System in Salmonella enterica Serovar Typhimurium

“…5, deletion of pinT in the wild-type background slightly increased hilA expression, but had no effect on rtsA expression when these strains were cultured under SPI1-inducing conditions. As expected, constitutive activation of PhoP in the phoQ24 background led to significant repression of both hilA and rtsA (and hilD) (60). Deletion of pinT in the phoQ24 background conferred significant increases in both hilA (ϳ4-fold) and rtsA (ϳ2-fold) expression.…”

“…PinT, a PhoPQ-activated sRNA, regulates hilA and rtsA translation. The PhoPQ two-component system regulates expression of the SPI1 genes primarily by repressing hilA and hilD transcription (11,60) (Fig. 1A).…”

“…PhoPQ represses SPI1 expression primarily by blocking activation of the hilA promoter and indirectly repressing hilD and rtsA transcription (60). Given the effects of PinT on hilA and rtsA translation, we tested how PinT contributes to the PhoPQ-mediated regulation of the SPI1 T3SS.…”

“…Deletion of pinT in the phoQ24 background conferred significant increases in both hilA (ϳ4-fold) and rtsA (ϳ2-fold) expression. However, the major effect of phoQ24 on the expression of SPI1 is via transcriptional regulation of hilD, rtsA, and hilA (60). Thus, regulation of hilA and rtsA by PinT is an additional layer on top of the transcriptional repression of the SPI1 T3SS mediated by the PhoPQ two-component system to effect complete and rapid shutoff of the system.…”

“…The PhoPQ two-component system regulates SPI1 gene expression by repressing hilA transcription (11), but the mechanism of action was not clear. We have shown that the PhoP represses hilA transcription by blocking activation of the promoter and also indirectly affects hilD and rtsA transcription, providing mechanistic insight into how the SPI1 system is shut off after invasion (60). PhoPQ also induces two sRNAs: MgrR and PinT (33,61,62).…”