Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD

Abstract

The invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is mediated by a type III secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). Expression of the SPI1 T3SS is tightly regulated by the combined action of HilC, HilD, and RtsA, three AraC family members that can independently activate hilA, which encodes the direct regulator of the SPI1 structural genes. Expression of hilC, hilD, and rtsA is controlled by a number of regulators that respond to a variety of environmental signals. In this work, we show that one such signal is iron mediated by Fur (ferric uptake regulator). Fur activates hilA transcription in a HilD-dependent manner. Fur regulation of HilD does not appear to be simply at the transcriptional or translational level but rather requires the presence of the HilD protein. Fur activation of SPI1 is not mediated through the Fur-regulated small RNAs RfrA and RfrB, which are the Salmonella ortholog and paralog of RyhB that control expression of sodB. Fur regulation of HilD is also not mediated through the known SPI1 repressor HilE or the CsrABC system. Although understanding the direct mechanism of Fur action on HilD requires further analysis, this work is an important step toward elucidating how various global regulatory systems control SPI1.

FIG. 1.

Model for Fur regulation of SPI1. The arrows indicate direct activation of gene expression. HilD, HilC, and RtsA are each capable of activating hilA transcription. HilE acts by directly inhibiting HilD function. Fur presumably acts through an intermediate (indicated by X) to control HilD function. For clarity, the genes encoding HilD, HilC, RtsA, and HilA are not shown. See reference for a more complete model of SPI1 regulation.

FIG. 2.

Fur activates expression of hilA. (A) Regulation of hilA and sitA by Fur and metal chelation. The strains contained transcriptional lacZ fusions to hilA or sitA promoters, and either the strains were fur⁺ or the fur gene was deleted, as indicated. The strains were grown in both SPI1-inducing conditions and SPI1-inducing conditions with 200 μM dipyridyl. The strains used were JS404, JS412, JS575, and JS583. (B) Fur overexpression induces hilA expression when HilD is present. The strains contained a transcriptional lacZ fusion to hilA, and hilC, hilD, or rtsA was deleted, as indicated. The strains also contained either pWKS30 (vector control) or pFur. All strains were grown in SPI1-inducing conditions with ampicillin. The strains used were JS591 through JS598.

FIG. 3.

Fur regulates expression of hilA via HilD. The strains contained a transcriptional lacZ fusion to hilA, and hilC, hilD, rtsA, or fur was deleted, as indicated. The strains were grown in SPI1-inducing conditions. The strains used were JS575 through JS590.

FIG. 4.

Fur does not directly regulate hilD transcription. The strains contained a transcriptional lacZ fusion to hilD, and either fur was deleted or the strain contained pFur or pHilC, as indicated. The strains were grown in SPI1-inducing conditions. The strains used were JS659 through JS662. Wt, wild type.

FIG. 5.

Fur regulation of hilD requires the HilD protein. The strains contained a transcriptional (A) or translational (B) lacZ fusion to hilD inserted into yjeT. The strains were either hilD⁺ or ΔhilD and contained pWKS30 (vector control), pFur, pBAD30 (vector control), or pHilC, as indicated. The strains were grown in SPI1-inducing conditions. The strains used were JS702 through JS731.

FIG. 6.

Fur regulates expression of rtsA via HilD. The strains contained a transcriptional lacZ fusion to either hilC or rtsA, and hilC, hilD, or rtsA was deleted, as indicated. The strains were grown in SPI1-inducing conditions. The strains used were JS641 through JS656.

FIG. 7.

Fur does not regulate hilA expression by controlling HilE. The strains contained a transcriptional lacZ fusion to hilA, and hilC, hilD, rtsA, fur, or hilE was deleted, as indicated. The strains were grown in SPI1-inducing conditions. The strains used were JS575 through JS579 and JS633 through JS640.

FIG. 8.

Fur regulation of sodB and Fur regulation of hilA are mechanistically different. (A) Fur controls expression of sodB, but not SPI1, via RfrA and RfrB. The strains contained a translational lacZ fusion to sodB or a transcriptional fusion to hilA, and fur, rfrA, or rfrB was deleted, as indicated. The strains were grown in SPI1-inducing conditions. The strains used were JS575, JS583, and JS613 through 626. (B) Fur regulation of SPI1 is not dependent on Hfq. The strains contained translational lacZ fusions to sodB or hilD or a transcriptional fusion to hilA, and fur or hfq was deleted, as indicated. The strains were grown in SPI1-inducing conditions. The strains used were JS575, JS583, JS619, JS620, and JS629 through JS632. Wt, wild type.