Integrating global regulatory input into the Salmonella pathogenicity island 1 type III secretion system

Abstract

Salmonella enterica serovar Typhimurium uses the Salmonella pathogenicity island 1 (SPI1) type III secretion system to induce inflammatory diarrhea and bacterial uptake into intestinal epithelial cells. The expression of hilA, encoding the transcriptional activator of the SPI1 structural genes, is directly controlled by three AraC-like regulators, HilD, HilC, and RtsA, each of which can activate the hilD, hilC, rtsA, and hilA genes, forming a complex feed-forward regulatory loop. A large number of factors and environmental signals have been implicated in SPI1 regulation. We have developed a series of genetic tests that allows us to determine where these factors feed into the SPI1 regulatory circuit. Using this approach, we have grouped 21 of the known SPI1 regulators and environmental signals into distinct classes on the basis of observed regulatory patterns, anchored by those few systems where the mechanism of regulation is best understood. Many of these factors are shown to work post-transcriptionally at the level of HilD, while others act at the hilA promoter or affect all SPI1 promoters. Analysis of the published transcriptomic data reveals apparent coregulation of the SPI1 and flagellar genes in various conditions. However, we show that in most cases, the factors that affect both systems control SPI1 independently of the flagellar protein FliZ, despite its role as an important SPI1 regulator and coordinator of the two systems. These results provide a comprehensive model for SPI1 regulation that serves as a framework for future molecular analyses of this complex regulatory network.

Figure 1

Working model for SPI1 regulation. Blue lines indicate transcriptional regulation. Red lines indicate post-transcriptional regulation. Green lines represent post-translational regulation. The effect of each regulator, positive (+) or negative (−) on hilA expression is indicated. For clarity, the genes encoding HilC, RtsA, and HilA are not shown.

Figure 2

Rationale for interpretation of panels A, B, and C of the bar graphs in Figures 3–6 and Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6, Figure S7, Figure S8, Figure S9, Figure S11, Figure S12, Figure S13, Figure S14, Figure S15, Figure S16. (A) The transcriptional hilA–lac fusion serves as a major readout for SPI1 expression. First, test the effects of a regulatory factor on hilA expression in both hilD⁺ and hilD⁻ backgrounds. (B) Second, test the effects of a regulatory factor on hilA expression in a background where the system can be induced via tetracycline control of rtsA with or without HilD. (C) Third, test the effects of a regulatory factor on hilD–lac transcriptional and hilD’-’lac translational locus fusions, which provide a readout of hilD transcription and translation, respectively, in the absence of HilD autoinduction. See detailed description of the experimental setup in Rationale and approach in Results.

Figure 3

Class I, HilE regulates hilA expression via the post-translational control of HilD. (A) β-Galactosidase activity in strains containing a hilA–lac transcriptional fusion and the indicated mutations after growth under SPI1 inducing conditions. (B) β-Galactosidase activity of strains containing a hilA–lac transcriptional fusion and indicated mutations with rtsA under the control of a tetracycline-regulated promoter. Strains were grown under SPI1-inducing conditions with the indicated tetracycline concentrations. (C) β-Galactosidase activity in strains containing a hilD–lac transcriptional or a hilD’-’lac translational fusion and the indicated mutations after growth under SPI1 inducing conditions. β-Galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± SD, where n = 4.

Figure 4

Class II, SirA activates hilA expression via the post-transcriptional control of hilD. See Figure 3 legend for details.

Figure 5

Class III, PhoPQ (PhoQ24) represses hilA expression independently of HilD. See Figure 3 legend for details.

Figure 6

Class IV, Hha represses SPI1 expression independently of HilD (affects all regulators in the feed-forward loop). See Figure 3 legend for details.

Figure 7

HilE and FliZ affect hilA expression independently of each other. β-Galactosidase activity in strains containing a hilA–lac transcriptional fusion and the indicated mutations after growth under SPI1 inducing conditions. β-Galactosidase activity units are defined as (μmol of ONP formed min⁻¹) × 10⁶/(OD₆₀₀ × ml of cell suspension) and are reported as mean ± SD, where n = 4.