CsrA Positively and Directly Regulates the Expression of the pdu, pocR, and eut Genes Required for the Luminal Replication of Salmonella Typhimurium

Abstract

Enteric pathogens, such as Salmonella, have evolved to thrive in the inflamed gut. Genes located within the Salmonella pathogenicity island 1 (SPI-1) mediate the invasion of cells from the intestinal epithelium and the induction of an intestinal inflammatory response. Alternative electron acceptors become available in the inflamed gut and are utilized by Salmonella for luminal replication through the metabolism of propanediol and ethanolamine, using the enzymes encoded by the pdu and eut genes. The RNA-binding protein CsrA inhibits the expression of HilD, which is the central transcriptional regulator of the SPI-1 genes. Previous studies suggest that CsrA also regulates the expression of the pdu and eut genes, but the mechanism for this regulation is unknown. In this work, we show that CsrA positively regulates the pdu genes by binding to the pocR and pduA transcripts as well as the eut genes by binding to the eutS transcript. Furthermore, our results show that the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes mediated by PocR or EutR, which are the positive AraC-like transcriptional regulators for the pdu and eut genes, respectively. By oppositely regulating the expression of genes for invasion and for luminal replication, the SirA-CsrB/CsrC-CsrA regulatory cascade could be involved in the generation of two Salmonella populations that cooperate for intestinal colonization and transmission. Our study provides new insight into the regulatory mechanisms that govern Salmonella virulence. IMPORTANCE The regulatory mechanisms that control the expression of virulence genes are essential for bacteria to infect hosts. Salmonella has developed diverse regulatory mechanisms to colonize the host gut. For instance, the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the SPI-1 genes, which are required for this bacterium to invade intestinal epithelium cells and for the induction of an intestinal inflammatory response. In this study, we determine the mechanisms by which the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes, which are necessary for the replication of Salmonella in the intestinal lumen. Thus, our data, together with the results of previous reports, indicate that the SirA-CsrB/CsrC-CsrA regulatory cascade has an important role in the intestinal colonization by Salmonella.

Keywords: Csr; Salmonella; regulation.

FIG 1

Schematic representation of the pdu and eut operons. (A) The pdu and pocR genes. pduA is the first gene of the pdu operon. The PocR transcriptional regulator activates the expression of the pdu genes by acting on the promoters located upstream of pduA and pduF (adapted from [10]). (B) The eut genes. eutS is the first gene of the eut operon. The EutR transcriptional regulator activates the expression of the eut genes by acting on the promoter located upstream of eutS (adapted from [73]). The DNA fragments carried by the pduA′-′lacZ and pocR′-′lacZ fusions (A) as well as by the eutS′-′lacZ and P2-eutR′-′lacZ fusions (B) are shown; positions indicated are relative to the translational start codon.

FIG 2

The SirA-CsrB/C-CsrA cascade regulates the expression of the pdu and eut genes. The β-galactosidase activity of the pduA′-′lacZ (A) and eutS′-′lacZ (B) translational fusions was quantified in the indicated strains. β-galactosidase assays were performed with samples taken from bacterial cultures that were grown overnight in LB at 37°C. The data represent the average and the standard deviation of three independent experiments done in duplicate. The P values were calculated using one-way ANOVAs with Tukey’s post hoc tests (***, P < 0.001).

FIG 3

The SirA-CsrB/C-CsrA cascade requires PocR and EutR to regulate the expression of the pdu and eut genes. The β-galactosidase activity of the pduA′-′lacZ (A) and eutS′-′lacZ (B) translational fusions was quantified in the indicated strains. β-galactosidase assays were performed with samples taken from bacterial cultures that were grown overnight in LB at 37°C. The data represent the average and the standard deviation of three independent experiments done in duplicate. The P values were calculated using one-way ANOVAs with Tukey’s post hoc tests (***, P < 0.001).

FIG 4

The SirA-CsrB/C-CsrA cascade regulates the expression of PocR and EutR. The β-galactosidase activity of the pocR′-′lacZ (A) and P2-eutR′-′lacZ (C) translational fusions was quantified in the indicated strains. The data represent the average and the standard deviation of three independent experiments done in duplicate. The P values were calculated using one-way ANOVAs with Tukey’s post hoc tests (***, P < 0.001). Western blot analysis of PocR-FLAG (B) and EutR-FLAG (D) expression in the WT strain carrying a chromosomal 3xFLAG-tagged pocR or eutR gene, respectively, with the indicated plasmids. Monoclonal anti-FLAG antibodies were used to detect the FLAG-tagged proteins. As a loading control, the expression of GroEL was also detected using polyclonal anti-GroEL antibodies. The blots were performed three times in independent experiments. Representative images of the blots are shown. The fold change for the expression of FLAG-tagged proteins were calculated as the ratio of AU (arbitrary units) normalized with GroEL, using the ImageJ software package. The P values were obtained by using unpaired Student’s t tests (*, P < 0.05; **, P < 0.01). Western blots and β-galactosidase assays were performed with samples taken from bacterial cultures that were grown overnight in LB at 37°C.

FIG 5

The SirA-CsrB/C-CsrA cascade regulates the expression of pocR in the absence of PocR. (A) The β-galactosidase activity of the pocR′-′lacZ translational fusion was quantified in the indicated strains. β-galactosidase assays were performed with samples taken from bacterial cultures that were grown overnight in LB at 37°C. The data represent the average and the standard deviation of three independent experiments done in duplicate. The P values were calculated using one-way ANOVAs with Tukey’s post hoc tests (***, P < 0.001). Nonradioactive EMSAs using purified MBP-PocR and the DNA fragments contained in the pduA′-′lacZ (B) and pocR′-′lacZ (C) fusions. As a negative control, the DNA fragment contained in the eutR′-′lacZ fusion was included in each DNA-binding reaction. The immunodetection assays using anti-MBP monoclonal antibodies (the images below the EMSAs) show that the MBP-PocR protein forms overly large complexes with (pduA) or without (pocR and eutR) bound DNA, and these remained near the wells of the gels.

FIG 6

SirA and CsrB/C regulate the expression of the pdu and eut genes in the absence or presence of inducer molecules. The β-galactosidase activity of the pduA′-′lacZ (A) and eutS′-′lacZ (B) translational fusions was determined in the indicated strains that were grown in the absence or presence of either 12.5 mM 1,2-propanediol and 150 nM vitamin B₁₂ (A) or 10 mM ethanolamine and vitamin B₁₂ (B). β-galactosidase assays were performed with samples taken from bacterial cultures that were grown overnight in LB at 37°C. The data represent the average and the standard deviation of three independent experiments done in duplicate. The P values were calculated using one-way ANOVAs with Tukey’s post hoc tests (***, P < 0.001).

FIG 7

CsrA binds specifically to the pocR, pduA, and eutS transcripts. CsrA binding to the pocR (A), pduA (B), and eutS (C) RNAs was analyzed using EMSAs by incubating ³²P-labeled RNA fragments with increasing concentrations of purified CsrA-H6. The positions of bound and free RNA are marked. The simple binding curves for these data are shown. The K_d values of the CsrA interaction with the pocR, pduA, and eutS transcripts are shown. For the RNA competition experiments, labeled pocR (0.1 nM), pduA (0.2 nM), or eutS (0.2 nM) RNA was combined with 1, 30, or 100 nM unlabeled specific (pocR, pduA, and eutS) or nonspecific (phoB) competitor RNA and was incubated with purified CsrA.

FIG 8

The model for the regulation of the pdu/pocR/eut and hilD (SPI-1) genes by BarA/SirA-CsrB/C-CsrA. The expression of the pdu/pocR/eut and hilD (SPI-1) genes that are required for luminal replication and for the invasion of host cells, respectively, is oppositely controlled by the BarA/SirA-CsrB/C-CsrA regulatory cascade. See the text for details.