Implication of quorum sensing in Salmonella enterica serovar typhimurium virulence: the luxS gene is necessary for expression of genes in pathogenicity island 1

Abstract

Despite the fact that the regulatory system sensing density of cell population and its signaling molecule have been identified in Salmonella enterica, the biological significance of this phenomenon termed as quorum sensing remains unknown. In this report, we provide evidence that the luxS gene is necessary for Salmonella virulence phenotypes. Transcription assays showed that the cell-density-dependent induction of the invF gene was abolished in a Salmonella strain with the luxS gene deleted. The effect of the luxS deletion was also investigated in other InvF-regulated genes expressed from Salmonella pathogenicity island 1 (SPI-1). The decreased expression of SPI-1 genes in the strain with luxS deleted could be restored by either the addition of a synthetic signal molecule or the introduction of a plasmid copy of the luxS gene. Thus, the reduced expression of invF and its regulated genes in Salmonella cells lacking quorum sensing resulted in the attenuation of virulence phenotypes both in vitro and in vivo.

FIG. 1.

Induction kinetics of the invF gene show a correlation with AI-2 activity. (A) Extracellular AI-2 activities were determined during the growth of Salmonella strains. Aliquots of wild-type (•) and its isogenic luxS deletion mutant (○) that were grown anaerobically in LB broth were removed at specific time intervals, and the AI-2 activity in the cell culture fluids was measured by using a V. harveyi BB170 bioassay (34). Identical growth of two Salmonella strains is indicated as dashed lines. (B) Time course transcription of the invF and the hilA genes. Aliquots of wild-type (closed symbols) and luxS deletion mutant (open symbols) strains harboring a lacZ transcriptional fusion to either the invF (squares) or the hilA (triangles) promoter were obtained as described above, and the β-galactosidase activities (in Miller units) were determined (27). The AI-2 activities determined above are also shown as dashed lines.

FIG. 2.

The quorum-sensing activity is required for the expression of a subset of SPI-1 genes. Total RNA was isolated from wild-type (WT), luxS mutant (luxS), luxS mutant supplemented with 24 μM DPD (luxS + DPD), and luxS mutant harboring pluxS plasmid (luxS + pluxS) that were grown anaerobically to the stationary phase. (A) The mRNA levels of the invF gene and its regulated targets—sicA, sigD, and sopE—were determined with three different samples by using real-time PCR analysis. (B) The transcription of the hilA gene and the HilA-regulated prgH was also examined. Expression levels of the target genes were normalized to that of 16S rRNA gene. Shown is the average of three independent experiments, and error bars indicate the standard deviation from the mean.

FIG. 3.

The Salmonella luxS mutant displays an invasion defect for epithelial cells. Wild-type (WT), luxS mutant (luxS), luxS mutant supplemented with 24 μM DPD (luxS + DPD), and luxS mutant harboring pluxS plasmid (luxS + pluxS) were grown anaerobically for 3 h, and ∼10⁶ CFU of bacterial cells were used for infection of HEp-2 cells in 24-well plates. Note that DPD was added before infection into DMEM without antibiotics. Invasion of Salmonella was allowed for 1 h, and extracellular bacterial cells were eliminated by a wash with PBS and treatment with 100 μg of gentamicin/ml. The invasion ability of the strain was determined by plating the intracellular bacteria on LB agar after cell lysis with 1% Triton X-100. The relative Salmonella invasion was calculated by dividing the numbers of intracellular bacteria by those of bacteria used for the initial infection, and the resulting values were normalized further to the wild-type level. *, P < 0.01 (Student t test).

FIG. 4.

The luxS deletion attenuates Salmonella virulence in mice. Isogenic Salmonella strains grown aerobically were used to infect BALB/c mice. A group of five mice were orally inoculated with 1.3 × 10⁷ CFU of Salmonella. To analyze bacterial colonization in organs, mice were sacrificed at 48 and 120 h after infection. The spleens and livers were removed, homogenized, and then plated on MacConkey agar plates containing 50 μg of streptomycin per ml.