Role of cross talk in regulating the dynamic expression of the flagellar Salmonella pathogenicity island 1 and type 1 fimbrial genes

Abstract

Salmonella enterica, a common food-borne pathogen, differentially regulates the expression of multiple genes during the infection cycle. These genes encode systems related to motility, adhesion, invasion, and intestinal persistence. Key among them is a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). In addition to the SPI1 T3SS, other systems, including flagella and type 1 fimbriae, have been implicated in Salmonella pathogenesis. In this study, we investigated the dynamic expression of the flagellar, SPI1, and type 1 fimbrial genes. We demonstrate that these genes are expressed in a temporal hierarchy, beginning with the flagellar genes, followed by the SPI1 genes, and ending with the type 1 fimbrial genes. This hierarchy could mirror the roles of these three systems during the infection cycle. As multiple studies have shown that extensive regulatory cross talk exists between these three systems, we also tested how removing different regulatory links between them affects gene expression dynamics. These results indicate that cross talk is critical for regulating gene expression during transitional phases in the gene expression hierarchy. In addition, we identified a novel regulatory link between flagellar and type 1 fimbrial gene expression dynamics, where we found that the flagellar regulator, FliZ, represses type 1 fimbrial gene expression through the posttranscriptional regulation of FimZ. The significance of these results is that they provide the first systematic study of the effect of regulatory cross talk on the expression dynamics of flagellar, SPI1, and type 1 fimbrial genes.

FIG. 1.

Coordinate regulation of the flagellar, SPI1, and type 1 fimbrial genes. The master regulator for flagellar gene expression is FlhD₄C₂ (9). The FlhD₄C₂ complex, in turn, activates two additional regulators, FliA and FliZ, encoded within the fliAZ operon. FliA is a flagellum-specific alternate sigma factor essential for the expression of the motor, filament, and chemotaxis genes. FliZ is a posttranslational activator of FlhD₄C₂ (71). FliZ also activates HilD (38, 41, 53, 57, 82) and represses FimZ posttranscriptionally (this study). In the SPI1 T3SS, HilD, HilC, and RtsA form three interlocking positive-feedback loops where all three activate each other's and their own expression (23). In addition, they can independently activate HilA expression. HilA is required for the expression of the genes encoding the SPI1 T3SS. SPI1 gene expression is negatively regulated by HilE, which binds to HilD and prevents it from activating the SPI1 promoters. RtsB, encoded within the same operon as RtsA, binds to the P_flhDC promoter and represses motility (22). In type 1 fimbriae, FimW and FimZ form a coupled feedback loop where they can activate their own and each other's expression (72). They can also independently activate the expression of the P_fimA promoter, which controls the expression of genes encoding type 1 fimbriae. FimY and FimW also participate in a negative-feedback loop, where FimY activates FimW expression and FimW represses FimY expression. FimZ also binds to the P_flhDC promoter and represses the expression of the flagellar genes (10) and induces the expression of HilE to repress SPI1 gene expression (5, 72).

FIG. 2.

Dynamic expression of the flagellar, SPI1, and type 1 fimbrial genes. Time course dynamics of the P_flgA (flagellar), P_hilA (SPI1), and P_fimA (type 1 fimbrial) promoter activities in wild-type cells as determined using luciferase transcriptional reporters. For reference purposes, the optical density (OD600) was plotted to illustrate how each system is activated during different phases of growth. In these experiments, cells were first grown overnight at 37°C in LB without salt and then subcultured 1:500 in fresh LB-1% salt. Cells were then grown statically with luminescence, and optical density readings were taken every 20 min. Average promoter activities from three independent experiments on separate days are reported. For each experiment, six samples were tested. Error bars denote standard deviations. A.U., arbitrary units.

FIG. 3.

Effect of transcriptional cross talk on flagellar, SPI1, and type 1 fimbrial gene expression dynamics. (A to C) The flagellar genes amplify SPI1 gene expression and delay type 1 fimbrial gene expression. P_flgA (A), P_hilA (B), and P_fimA (C) promoter activities in the wild type and the ΔflhDC mutant are shown. (D to F) The SPI1 genes reduce the duration of flagellar gene expression and accelerate the induction of type 1 fimbrial gene expression. P_flgA (D), P_hilA (E), and P_fimA (F) promoter activities in the wild type and the ΔSPI1 mutant are shown. (G to I) Type 1 fimbrial genes do not affect flagellar gene expression and reduce the duration of SPI1 gene expression. P_flgA (G), P_hilA (H), and P_fimA (I) promoter activities in the wild type and the ΔfimYZW mutant are shown. Experiments were performed as described for Fig. 2. The mutants were also tested to see whether they affected growth. However, no change in optical density as a function of time was observed (data not shown).

FIG. 4.

FliZ controls the magnitude of SPI1 gene expression and the dynamics of type 1 fimbrial gene expression. (A to C) Deleting FliZ represses flagellar and SPI1 gene expression and accelerates the induction of type 1 fimbrial genes. P_flgA (A), P_hilA (B), and P_fimA (C) promoter activities in the wild type and the ΔfliZ mutant are shown. (D to F) Overexpressing FliZ increases the magnitude of flagellar and SPI1 gene expression and delays the induction of type 1 fimbrial genes. P_flgA (D), P_hilA (E), and P_fimA (F) promoter activities in the wild type and the ΔfliZ mutant constitutively expressing FliZ from a P_LtetO-1 promoter on a plasmid (pFliZ) are shown. Experiments were performed as described for Fig. 2.

FIG. 5.

FliZ regulates type 1 fimbrial gene expression though FimZ. (A) FliZ is unable to regulate P_fimA promoter activity in the absence of FimZ. P_fimA promoter activities in the wild type, the ΔfimZ mutant, the ΔfliZ mutant, the ΔfimZ ΔfliZ mutant, the ΔfliZ mutant expressing FliZ from the constitutive P_LtetO-1 promoter on a plasmid (pFliZ), and the ΔfimZ ΔfliZ mutant harboring pFliZ are shown. (B) FliZ regulates FimZ posttranscriptionally. P_fimA promoter activities in the P_fimZ::tetRA ΔfimY mutant, the P_fimZ::tetRA ΔfimY ΔfliZ mutant, and the P_fimZ::tetRA ΔfimY ΔfliZ mutant expressing FliZ from the constitutive P_LtetO-1 promoter on a plasmid are shown. In the genetic background P_fimZ::tetRA, FimZ is under the control of a tetracycline-inducible promoter. Overnight cultures were subcultured 1:1,000 in fresh LB and then grown statically at 37°C for 24 h. FimZ expression was induced by addition of 15 μg/ml tetracycline. Fluorescence and optical density (OD600) values were then measured for each sample. Average promoter activities from three independent experiments on separate days are reported. For each experiment, six samples were tested. Error bars denote standard deviations.

FIG. 6.

RtsB controls the dynamics of flagellar and type 1 fimbrial gene expression. (A to C) Deleting RtsB increases the duration of flagellar gene expression and slows the induction of type 1 fimbrial genes. P_flgA (A), P_hilA (B), and P_fimA (C) promoter activities in the wild type and the ΔrtsB mutant are shown. (D to F) Overexpressing RtsB inactivates flagellar gene expression and accelerates the induction of type 1 fimbrial genes. P_flgA (D), P_hilA (E), and P_fimA (F) promoter activities in the wild type and the ΔrtsB mutant constitutively expressing RtsB from the constitutive P_LtetO-1 promoter on a plasmid (pRtsB) are shown. Dynamic luminescence experiments were performed as described for Fig. 2. (G) RtsB regulates type 1 fimbrial gene expression through FliZ. P_fimA promoter activities in the wild type, the ΔrtsB mutant, the ΔfliZ mutant, the ΔfliZ ΔrtsB mutant, and the ΔrtsB and ΔfliZ ΔrtsB mutants constitutively expressing RtsB are shown. Endpoint fluorescence experiments were performed as described for Fig. 5.

FIG. 7.

FimZ controls the dynamics of SPI1 gene expression. (A to C) Deleting FimZ increases the duration of SPI1 gene expression. P_flgA (A), P_hilA (B), and P_fimA (C) promoter activities in the wild type and the ΔfimZ mutant are shown. (D to F) Overexpressing FimZ represses both flagellar and SPI1 gene expression. P_flgA (D), P_hilA (E), and P_fimA (F) promoter activities in the wild type and the P_fimZ::tetRA mutant, where FimZ is under the control of a tetracycline-inducible promoter, are shown. FimZ expression was induced by addition of 15 μg/ml tetracycline. Experiments were performed as described for Fig. 2.

FIG. 8.

Salmonella invasion program. (A) Diagram of transcriptional cross talk between the flagellar, SPI1, and type 1 fimbrial gene systems. (B) Inferred logic of transcriptional cross talk, where the decision to “move” results from flagellar gene expression, the decision to “invade” results from SPI1 gene expression, and the decision to “persist” results from type 1 fimbrial gene expression.