The intestinal fatty acid propionate inhibits Salmonella invasion through the post-translational control of HilD

Abstract

To cause disease, Salmonella must invade the intestinal epithelium employing genes encoded within Salmonella Pathogenicity Island 1 (SPI1). We show here that propionate, a fatty acid abundant in the intestine of animals, repressed SPI1 at physiologically relevant concentration and pH, reducing expression of SPI1 transcriptional regulators and consequently decreasing expression and secretion of effector proteins, leading to reduced bacterial penetration of cultured epithelial cells. Essential to repression was hilD, which occupies the apex of the regulatory cascade within SPI1, as loss of only this gene among those of the regulon prevented repression of SPI1 transcription by propionate. Regulation through hilD, however, was achieved through the control of neither transcription nor translation. Instead, growth of Salmonella in propionate significantly reduced the stability of HilD. Extending protein half-life using a Lon protease mutant demonstrated that protein stability itself did not dictate the effects of propionate and suggested modification of HilD with subsequent degradation as the means of action. Furthermore, repression was significantly lessened in a mutant unable to produce propionyl-CoA, while further metabolism of propionyl-CoA appeared not to be required. These results suggest a mechanism of control of Salmonella virulence in which HilD is post-translationally modified using the high-energy intermediate propionyl-CoA.

© 2013 Blackwell Publishing Ltd.

Figures

Fig. 1. A model for the control of SPI1 by propionate and genetic regulators

Arrows indicate positive control while T-shaped bars indicate negative control.

Fig 2. Propionate represses Salmonella invasion gene expression

A) Propionate represses sipC expression. Cultures of a sipC::lacZY reporter fusion strain were grown as standing cultures without additive (black bars) or with 10 mM propionic acid (grey bars), with β-galactosidase assays used to assess sipC expression. B) Propionate represses regulators of Salmonella SPI1. Cultures of strains with the hilA::lacZY or invF::lacZY reporter fusion were grown without additive (black bars) or with 10 mM propionic acid (grey bars). β-galactosidase assays were used to assess hilA and invF expression. C) Propionate represses sipC expression under anaerobic conditions. Cultures of a sipC::lacZY reporter fusion strain were grown anaerobically without additive (black bars) or with 10 mM propionic acid (grey bars), with β-galactosidase assays used to assess sipC expression. Error bars indicate standard deviation, and an asterisk (*) indicates a statistically significant difference due to the presence of propionic acid at p < 0.05.

Fig 3. Propionate represses Salmonella invasion

A) Propionate decreases Salmonella invasion of HEp-2 cells. The wild type strain was grown either with no additive or with 10 mM propionic acid. Invasion of HEp-2 cells was assessed using a gentamicin protection assay. Invasion is shown relative to the wild type without additive, which was set to 100%. Error bars indicate standard deviation, and an asterisk (*) indicates a statistically significant difference due to the presence of propionic acid at p < 0.05. B) Propionate diminishes production of SPI1 secreted effector proteins. The wild type strain was grown with no additive (lane 1) or with 10 mM propionic acid (lane 2). Secreted proteins were then isolated and analyzed by SDS-PAGE. Proteins with apparent molecular weights of 89, 67, 42, and 38 kDa that were reduced in cultures grown with propionic acid are designated with arrowheads. Molecular weights are shown on the right.

Fig. 4. Production of the intermediate propionyl-CoA is important for the repressive effect of propionate

A) Wild type and mutant strains with the sipC::lacZY reporter fusion were grown under as standing cultures in media containing either no additive (black bars), 10 mM propionic acid (grey bars), or 10 mM butyric acid (white bars). β-galactosidase assays were used to assess sipC expression. An asterisk (*) indicates a statistically significant difference for each respective strain grown with propionate or butyrate compared to no additive at p < 0.05. Two asterisks (**) indicates a statistically significant difference for a mutant strain grown with propionate compared to wild type grown with propionate at p < 0.05. B) Wild type and mutant strains with the sipC::lacZY reporter fusion were grown under anaerobic conditions in media containing these same additives. An asterisk (*) indicates a statistically significant difference for mutants compared to the wild type. Error bars indicate standard deviation.

Fig. 5. Propionate represses SPI1 through the central regulator hilD

A) Propionate reduces expression of SPI1 regulators. The wild type strain was grown with either no additive (black bars) or with 10 mM propionic acid (grey bars), total RNA was extracted and cDNA synthesis was performed. cDNA was then used as template for real-time PCR to measure relative expression of hilC, hilD, and rtsA, with expression of these invasion genes normalized to that of 16S rRNA. B) Activators of SPI1 other than hilD do not mediate the repressive effect of propionate. Wild type and mutant strains with the sipC::lacZY reporter fusion were grown in media containing either no additive (black bars) or 10 mM propionic acid (grey bars), and β-galactosidase assays were used to assess sipC expression. The wild type is shown using a separate axis to better discern the differences among mutant strains. C) Inhibitors of SPI1 do not mediate the repressive effect of propionate. Wild type and mutant strains with the sipC::lacZY reporter fusion were grown in media containing either no additive (black bars) or 10 mM propionic acid (grey bars), and β-galactosidase assays were used to assess sipC expression. For all panels, error bars indicate standard deviation. An asterisk (*) indicates a statistically significant difference for a given strain with the addition of propionate as compared to no additive at p < 0.05.

Fig. 6. hilD, but not hilC, is required for the repressive effect of propionate

A) The wild type; B) ΔhilD mutant; C)ΔhilC mutant, and; D) ΔhilC,ΔhilD mutant with the sopB::luxCDABE reporter fusion plasmid were grown in media containing either no additive (open symbols) or 10 mM propionic acid (closed symbols). Light production was used to assess sopB expression, and optical density at 600 nm (OD600) was used to measure growth. The measure of luminescence/OD600, in arbitrary units, was used to normalize for variation in growth rate. Error bars indicate standard deviation.

Fig. 7. Functional HilD is required for the repressive effect of propionate on hilD expression

Wild type (circles) and the ΔhilD mutant (squares) with the hilD::luxCDABE reporter fusion plasmid were grown in media containing either no additive (open symbols) or 10mM propionic acid (closed symbols). Light production was used to assess hilD expression, and optical density at 600 nm (OD600) was used to measure growth. The measure of luminescence/OD600, in arbitrary units, was used to normalize for variation in growth rate. Error bars indicate standard deviation.

Fig. 8. Propionate affects hilD through neither transcription nor translation

A) Propionate regulates hilA through hilD. Strains carrying a single-copy chromosomal transcriptional hilA-lac fusion and rtsA under the control of a tetracycline-inducible tetRA promoter were grown with or without propionic acid and with the concentrations of tetracycline shown. β-galactosidase assays were used to assess hilA expression. B) Propionate does not function through the regulation of hilD transcription. Strains carrying a single-copy chromosomal transcriptional hilA-lac fusion and hilD under the control of a tetracycline-inducible tetRA promoter were grown with or without propionic acid and with the concentrations of tetracycline shown. β-galactosidase assays were used to assess hilA expression. C) Propionate does not affect the translation of hilD. Strains carrying a single-copy chromosomal translational hilD’-‘lac fusion and of the genotype shown were grown with 10 mM propionic acid or without additive and were assessed for β-galactosidase production. For all panels, error bars indicate standard deviation. An asterisk (*) indicates a statistically significant difference for a given strain with the addition of propionate as compared to no additive at p < 0.05. Two asterisks (**) indicates a statistically significant difference with mutation of csrBC at p < 0.05.

Fig. 9. Propionate controls SPI1 through the post-translational regulation of HilD

A) Propionate reduces the stability of HilD. A wild type or Δlon mutant strain carrying the hilA-lac transcriptional fusion and a chromosomal HilD-3xFLAG construct under the control of the tetRA promoter was grown in the presence of 0.8 μg ml-1 of tetracycline, with or without propionic acid. Transcription and translation were halted at time zero by the addition of an antibiotic cocktail, total protein was extracted, and HilD was quantified by western blotting using an anti-FLAG antibody. Densitometric analysis of the blot was performed using ImageJ software, with all samples normalized to the wild type strain grown without propionic acid at time zero. ND = Not Determined. B) Propionate exhibits regulation of hilA through the post-translational regulation of HilD. β-galactosidase assays were used to assess hilA expression at time zero using the assay conditions described above.