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Implication of Quorum Sensing in Salmonella enterica Serovar Typhimurium Virulence: the luxS Gene Is Necessary for Expression of Genes in Pathogenicity Island 1

Jeongjoon Choi, Dongwoo Shin, and Sangryeol Ryu^*

Department of Food and Animal Biotechnology, School of Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea

Received 11 December 2006/ Returned for modification 5 April 2007/ Accepted 29 June 2007

	ABSTRACT

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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.

	INTRODUCTION

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Bacterial cells are able to modify gene expression patterns in the response to a variety of extracellular signals. In contrast, some bacterial species release and detect signaling molecules and consequently control gene expression as their populations are growing. This type of bacterial signal transduction, termed quorum sensing, was initially reported in marine bacterium Vibrio fischeri, for which the signaling molecule and its sensing mechanism were identified (29). The quorum-sensing system in V. fischeri consists of two proteins: LuxI, which is responsible for the synthesis of the acylhomoserine lactone autoinducer, and LuxR, which is activated by this autoinducer and thus regulates expression of the luciferase operon (15).

Although both gram-negative and gram-positive bacteria use species-specific autoinducer 1 (AI-1) for activation of the quorum-sensing system, signaling molecules differ: the former use acetylated homoserine lactones (AHLs) (35), whereas peptide pheromones are used in the latter (12). In addition, certain bacterial species, including Vibrio harveyi, also harbor quorum-sensing systems activated by autoinducer 2 (AI-2) in which the signaling molecules may be used as a universal language for communication with other bacteria rather than exclusively used for intrabacterial communication (41). In both V. harveyi and Salmonella enterica serovar Typhimurium, the luxS gene encoding the AI-2 synthase (i.e., LuxS protein) is directly involved in production of the AI-2 molecules from S-adenyl-methionine (35). The LuxS protein catalyzes the final step in biosynthesis of AI-2, generating the signaling molecule 4,5-dihydroxy-2,3-pentanedione (DPD) (26). V. harveyi and Salmonella have two distinct AI-2 molecules derived from DPD. It has been found that the V. harveyi AI-2 is (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate and the Salmonella AI-2 is (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (26).

It has been reported that a variety of bacterial phenotypes, such as bioluminescence, sporulation, biofilm formation, conjugation, motility, competence, and antibiotic production, are regulated in response to signaling molecules of quorum-sensing systems (11, 24, 28, 30, 33). The potential role of quorum sensing in virulence has been studied in several different pathogens. In Escherichia coli O157:H7, the expression of the type III secretion system and other virulence factors is mediated by another autoinducer, AI-3 (6, 7, 33). In Streptococcus pyogenes, inactivation of the luxS gene resulted in the increased hemolytic activity but reduced secretion of cysteine protease, a putative virulence factor (24). In addition, S. pyogenes luxS mutant was internalized with greater efficiency by HEp-2 epithelial cells than by the wild-type parent (25). In Porphyromonas gingivalis, mutation of the luxS gene decreased the expression of two cysteine proteases even though the mutant strain displayed the normal virulence phenotype in mice (3). In Neisseria meningitidis, inactivation of the luxS gene reduced the ability to cause bacteremia in the infant rat (40).

A number of bacterial virulence proteins are translocated into host cells during Salmonella infection via type III secretion systems (16, 20). In particular, a type III secretion system encoded by genes clustered in Salmonella pathogenicity island 1 (SPI-1) is required for invasion of Salmonella into epithelial cells (17) and is activated by various environmental cues reflecting complex conditions present in the intestinal lumen before host cell invasion (18). A series of studies in Salmonella enterica indicated that a quorum-sensing signal molecule, AI-2, is imported during transition from the mid-exponential phase to the early stationary phase, and the lsr operon encoding the Lsr transport apparatus and proteins for processing of phospho-AI-2 is also maximally expressed in the mid-exponential phase (36). The coincidence between the fact that the expression of SPI-1 is upregulated at the moment from mid-exponential to stationary growth of Salmonella cells (23) and the induction kinetics of the Salmonella quorum-sensing system prompted us to investigate whether the quorum-sensing system is implicated in the regulation of SPI-1. We provide here evidence that the luxS gene is required for the induction of InvF regulator and, consequently, its target genes in SPI-1. Our findings may provide a link between quorum sensing and regulatory cascades of virulence genes in SPI-1.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in the present study are listed in Table 1. The serovar Typhimurium strains used in the present study are derived from strain SL1344. Phage P22-mediated transductions were performed as described previously (4). The V. harveyi strains were kindly provided by J.-H. Rhee (21). All Salmonella strains were grown aerobically or anaerobically at 37°C in Luria-Bertani (LB) broth. V. harveyi strains were grown aerobically in autoinducer bioassay (AB) medium (13) at 30°C. Antibiotics were used in the following concentrations: ampicillin at 50 µg/ml, chloramphenicol at 25 µg/ml, kanamycin at 50 µg/ml, and streptomycin at 50 µg/ml.

Construction of plasmids. Plasmid pJJ2 was constructed by cloning of the luxS gene from serovar Typhimurium strain SL1344 into the pACYC184 plasmid vector (5). The luxS gene was amplified by PCR using the primers luxS-F4 (5'-GATAATCCTGAACTAAGCTTCTCCGC-3') and luxS-R4 (5'-GGTTATGAGAAAAGCATGCACCGATCA-3'), and the resulting product was cloned between the HindIII and SphI sites of pACYC184.

ß-Galactosidase assay. ß-Galactosidase assays were carried out in duplicate, and the activity was determined as described previously (27).

RNA isolation and analysis of gene expression using real-time PCR. Salmonella strains were grown in LB broth anaerobically to stationary phase, and total RNA was isolated by using an RNeasy minikit (QIAGEN). After DNase treatment of the RNA solution, cDNA was synthesized by using Omnitranscript reverse transcription reagents (QIAGEN) and random hexamers. Quantification of cDNA was carried out using 2xiQ SYBR green Supermix (Bio-Rad Laboratories), and real-time amplification of PCR product was analyzed by using iCycler iQ real-time detection system (Bio-Rad Laboratories). The relative amount of cDNA was calculated by using a standard curve obtained from PCR on serially diluted genomic DNA as templates. The mRNA expression level of the target gene was normalized to 16S rRNA expression level. The sequences of the primers used are presented in Table 2.

Invasion assay. HEp-2 cells were grown in Dulbecco modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 U/ml). Confluent monolayers for infection with bacteria were prepared in 24-well tissue culture plates. Each well was seeded with 2 x 10⁵ cells suspended in DMEM-10% fetal bovine serum without antibiotics and incubated for 1 h at 37°C under 5% CO₂. The wells were washed three times with phosphate-buffered saline (PBS) before bacterial cells were added. Bacterial cells were washed with PBS, suspended in prewarmed DMEM, and then added onto the cell monolayer at a multiplicity of infection of 10:1. After 1 h of incubation, the wells were washed three times with prewarmed PBS to remove extracellular bacteria and then incubated for 1 h with prewarmed medium supplemented with 100 µg of gentamicin/ml to kill the extracellular bacteria. Afterward, the wells were washed three times with PBS, lysed in 1% Triton X-100 for 30 min, and then diluted with PBS. Dilution of the suspension was plated on LB agar medium to enumerate CFU. To show the relative invasion abilities of wild type and mutants, each number of colonies was divided by the mean value of the colonies in wild-type Salmonella.

Animal experiments. Six-week-old female BALB/c mice were purchased from Institute of Laboratory Animal Resources in Seoul National University. Mice in each group were infected by oral gavage with 10⁷ Salmonella cells in 100 µl of PBS. Water and food were withdrawn 4 h before infection and were provided again at 2 h postinfection (8). For analysis of bacterial colonization in organs, all mice were euthanized with mix of ketamine and xylamine at designated time points, and the spleen and liver were removed aseptically and placed in 1.5-ml tubes. The organs were then homogenized in 1 ml of ice-cold PBS and serially diluted. Bacterial loads were determined by plating these samples on MacConkey agar plates containing 50 µg of streptomycin per ml.

	RESULTS

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Induction of the invF gene expression is dependent on the AI-2 activity. A quorum-sensing system that is activated by AI-2 has been identified in S. enterica (37). It has also been reported that the AI-2 activity of Salmonella begins to significantly increase during the entry of Salmonella cells into exponential growth, peaks at late exponential phase, and then decreases to 25% of the maximum level, reaching the new steady state (Fig. 1A) (36). In serovar Typhimurium, the luxS gene is required for a burst of AI-2 activity because the luxS gene encodes an enzyme (i.e., AI-2 synthase) catalyzing the biosynthesis of the AI-2 molecule (Fig. 1A) (36).

View larger version (31K): [in this window] [in a new window]   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 ß-galactosidase activities (in Miller units) were determined (27). The AI-2 activities determined above are also shown as dashed lines.

To verify further that quorum sensing is necessary for the induction of invF transcription, we carried out complementation experiments. Real-time PCR analysis with total RNA isolated from isogenic strains reproduced the result that the transcription level of invF gene was decreased in the mutant strain with the luxS gene deleted (Fig. 2A). This result was indeed due to the LuxS function because the phenotype of luxS deletion was restored by either introduction of the plasmid-linked luxS gene or by the addition of DPD, a synthetic AI-2 molecule (26), into the luxS mutant (Fig. 2A). In the case of the hilA gene, the moderately decreased level of mRNA expression (i.e., <2-fold) was also observed in the absence of the luxS gene (Fig. 2B). However, given that the introduction of the LuxS-expressing plasmid rescued the hilA gene transcription but the addition of DPD did not (Fig. 2B), the effect of LuxS on the expression of hilA might result from one of multiple functions of this protein other than quorum sensing (39).

View larger version (43K): [in this window] [in a new window]   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.

The luxS gene is necessary for the expression of a subset of virulence genes in SPI-1. The InvF regulator positively controls the expression of the sicA gene encoding a chaperone; these two proteins together then activate the transcription of sigD and sopE that encode secreted effector proteins that promote invasion (22). Thus, we hypothesized that the reduced expression of the InvF regulator in luxS deletion mutant could consequently affect expression of its target genes. To test this idea, we directly measured the mRNA levels of the sicA, sigD, and sopE genes by using real-time PCR analysis. The results in Fig. 2A showed that the decreased expression of invF gene in a luxS mutant strain caused approximately 40 to 50% reduction of the sicA, sigD, and sopE transcription levels. Consistent with the complementation experiment for the invF gene, the decreased transcription of the InvF-regulated genes were restored by either the addition of DPD or the introduction of a plasmid copy of the luxS gene to the luxS deletion mutant (Fig. 2A).

To investigate further whether the LuxS-mediated quorum-sensing effect is limited to InvF regulatory events, we also measured the transcription of prgH, the first gene of the prg operon, which is directly regulated by the HilA regulatory protein (2). We observed that transcription of the prgH gene was reduced in the Salmonella strain lacking the luxS gene and that this phenotype was not complemented by the addition of DPD, as observed for the hilA transcription (Fig. 2B). Therefore, these data suggest that quorum sensing is implicated in the expression of the InvF regulator and its regulated gene targets.

The luxS gene is required for Salmonella invasion into eukaryotic cells. The entry of Salmonella organisms into mucosal tissues of animal hosts is mediated by various gene products expressed from SPI-1 (16). In particular, a Salmonella strain with the invF gene deleted is significantly impaired in its ability to enter host cells as a result of decreased expression of the InvF-controlled type III secreted proteins (14). Therefore, we tested the possibility that the reduced expression of SPI-1 genes by luxS deletion might affect the invasiveness of Salmonella into eukaryotic cells. Murine epithelial HEp-2 cells were infected with isogenic strains of Salmonella grown in SPI-1-inducing growth conditions (i.e., oxygen-limited conditions) (2, 31). We found that invasion of Salmonella luxS gene mutant occurred significantly less often (i.e., 33%) than that of the wild-type strain, and this phenotypic defect was recovered in a mutant strain grown with either extracellular DPD or a plasmid copy of the luxS gene (Fig. 3). This result is consistent with the transcription profiles of the invF gene and its targets that were changed by the quorum-sensing activity (Fig. 2A) and suggests that LuxS-mediated quorum sensing is required for the efficient invasion of Salmonella into host tissue.

View larger version (47K): [in this window] [in a new window]   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).

View larger version (15K): [in this window] [in a new window]   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 x 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.

	DISCUSSION

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Salmonella must express a series of proteins from SPI-1 to invade epithelial cells and move into deeper tissue of animal hosts during the course of infection (22). We initially observed that transcription of the invF gene began to increase in Salmonella cells at about the same time as the initial burst of AI-2 activity occurred, and this cell-density-dependent induction of the invF transcription was abrogated in the Salmonella mutant with the luxS gene deleted (Fig. 1B). This was due to the lack of quorum-sensing activity because the reduction of the invF transcription was restored either by the addition of DPD or by the introduction of the LuxS-expressing plasmid into the luxS deletion mutant (Fig. 2). The subsequent transcription assay also showed that the mRNA levels of other SPI-1 components such as sicA, sigD, and sopE were decreased to ca. 50 to 60% compared to the wild-type levels (Fig. 2), a finding consistent with the report that the InvF protein is a transcriptional activator of these genes (14, 22). Thus, the absence of quorum-sensing activity resulted in decreased invasiveness of Salmonella into epithelial cells (Fig. 3), which in turn attenuated its virulence in orally infected mice (Fig. 4).

We should also mention that the LuxS protein was necessary for the full expression of other SPI-1 genes such as HilA and PrgH (Fig. 2B). However, it does not seem that quorum sensing via the production of AI-2 is solely responsible for this phenomenon, because there was a complementary effect of the luxS expression from the plasmid but no recovery effect of DPD addition on the transcription of these two genes (Fig. 2B). Based on the report that the E. coli LuxS protein plays a role in metabolic pathways, which are unrelated with quorum sensing (39), we assume that one of multiple functions of LuxS is involved in the transcriptional regulation of hilA, where the controlled expression of the LuxS protein (i.e., at a specific timing with appropriate amounts) from the original locus on chromosome might be critical for this regulation.

In the Salmonella quorum-sensing system, a regulatory protein, LsrR, negatively controls the expression of the lsr operon encoding the Lsr transport apparatus, and this operon is derepressed by the processing of phospho-AI-2 through LsrK (36). However, it is unlikely that expression of the invF gene and its regulated targets is regulated by the LsrR protein since a mutant Salmonella strain with the lsrR gene deleted does not show any defect in growth-dependent SPI-1 gene expression (data not shown). In enterohemorrhagic E. coli, quorum sensing via the AI-3 molecules increased the levels of the QseA regulatory protein, which in turn activated type III secretion gene transcription and protein secretion in chromosomal pathogenicity islands named LEE (6, 32, 38). We found that Salmonella also harbors the yhcS gene encoding a putative regulatory protein that displays high amino acid identity to the QseA regulator (our unpublished data). Thus, it is tempting to test whether the YhcS protein of S. enterica would connect quorum sensing and the expression of SPI-1 mimicking a regulatory circuit found in E. coli O157:H7.

In response to environmental signals such as high osmolarity and low oxygen, the transcription of SPI-1 genes is upregulated in a highly complicated manner (2, 14). Among several transcriptional regulators, the HilA protein, a key player in SPI-1 expression, activates transcription of the prgH operon encoding the type III secretion apparatus plus another transcription factor, InvF (1), thereby the expression of a number of effector proteins (i.e., SigD and SopE) are promoted (14). Because of this regulatory hierarchy, it would appear that the effect of quorum sensing was limited to some of the genes (i.e., InvF-regulated genes) in SPI-1 (Fig. 2). Although we do not know the reason, this finding implies that Salmonella may require AI-2 signaling to produce and deliver enough effector proteins into host cells even after the HilA-mediated expression of type III secretion system. This might be a mechanism for Salmonella to secure the initial infection process by delivering virulence factors for modification of host response only when its population size attains a certain level.

	ACKNOWLEDGMENTS

 
We thank Michael M. Meijler for kindly providing DPD and Joon-Heang Rhee for V. harveyi strains BB170, BB152 and BB120. We also thank V. L. Miller for the Salmonella strains with invF- and hilA-lacZ fusions in chromosome.

J.C. was the recipient of a graduate fellowship provided by the Ministry of Education through the Brain Korea 21 Project. This study was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Science and Technology, Seoul, Republic of Korea (MG05-0201-04-0).

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Food and Animal Biotechnology, School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. Phone: 82-2-880-4856. Fax: 82-2-873-5095. E-mail: sangryu{at}snu.ac.kr

Published ahead of print on 9 July 2007.

Editor: V. J. DiRita

Present address: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.

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