Salmonella Pathogenicity Island 1 Genes Research Articles

Salmonella Pathogenicity Island 1 (SPI-1) and Its Complex Regulatory Network.

Salmonella species can infect a diverse range of birds, reptiles, and mammals, including humans. The type III protein secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) delivers effector proteins required for intestinal invasion and the production of enteritis. The T3SS is regarded as the most important virulence factor of Salmonella. SPI-1 encodes transcription factors that regulate the expression of some virulence factors of Salmonella, while other transcription factors encoded outside SPI-1 participate in the expression of SPI-1-encoded genes. SPI-1 genes are responsible for the invasion of host cells, regulation of the host immune response, e.g., the host inflammatory response, immune cell recruitment and apoptosis, and biofilm formation. The regulatory network of SPI-1 is very complex and crucial. Here, we review the function, effectors, and regulation of SPI-1 genes and their contribution to the pathogenicity of Salmonella.

S. Enteritidis and S. Typhimurium Harboring SPI-1 and SPI-2 Are the Predominant Serotypes Associated With Human Salmonellosis in Saudi Arabia.

Non-typhoidal Salmonella (NTS) strains are Gram negative bacterial pathogens that are associated with foodborne illness worldwide. During the process of infection, Salmonella uses two molecular injectisomes known as Type 3 Secretion Systems (T3SS) to secrete virulence factors that are encoded by Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 into host cells. These secretion systems play a major role in virulence, as shown in various animal models, but little is known about their role in human infections. In Saudi Arabia, NTS strains frequently cause human infections but data regarding these pathogenic strains is fairly limited. The aim of this study was to characterize Salmonella human clinical isolates in Riyadh, Saudi Arabia, by determining their serotype, testing for the presence of SPI-1 and SPI-2 genes and to determine the antibiotic resistance profiles of these strains. Using the rapid Check and Trace Salmonella™ (CTS) system our results demonstrate that S. Enteritidis and S. Typhimurium were the predominant serovars, followed by S. Livingstone, S. Kentucky and S. Poona among a list of 36 serovars reported for the first time in the country. In addition, SPI-1 genes were detected in 99% of the isolates, while the sifA gene (SPI-2) was not detected in 13.5% of the isolates. These results suggest that both the SPI-1 and SPI-2 virulence determinants are important for human infection. Moreover, we report the presence of a Multi-Drug (MDR) carbapenem resistant S. Kentucky isolate harboring the blaOXA−48 gene not reported previously in Saudi Arabia.

Optimal translational fidelity is critical for Salmonella virulence and host interactions.

Translational fidelity is required for accurate flow of genetic information, but is frequently altered by genetic changes and environmental stresses. To date, little is known about how translational fidelity affects the virulence and host interactions of bacterial pathogens. Here we show that surprisingly, either decreasing or increasing translational fidelity impairs the interactions of the enteric pathogen Salmonella Typhimurium with host cells and its fitness in zebrafish. Host interactions are mediated by Salmonella pathogenicity island 1 (SPI-1). Our RNA sequencing and quantitative RT-PCR results demonstrate that SPI-1 genes are among the most down-regulated when translational fidelity is either increased or decreased. Further, this down-regulation of SPI-1 genes depends on the master regulator HilD, and altering translational fidelity destabilizes HilD protein via enhanced degradation by Lon protease. Our work thus reveals that optimal translational fidelity is pivotal for adaptation of Salmonella to the host environment, and provides important mechanistic insights into this process.

SlyA and HilD Counteract H-NS-Mediated Repression on the ssrAB Virulence Operon of Salmonella enterica Serovar Typhimurium and Thus Promote Its Activation by OmpR.

H-NS-mediated repression of acquired genes and the subsequent adaptation of regulatory mechanisms that counteract this repression have played a central role in the Salmonella pathogenicity evolution. The Salmonella pathogenicity island 2 (SPI-2) is an acquired chromosomal region containing genes necessary for Salmonella enterica to colonize and replicate in different niches of hosts. The ssrAB operon, located in SPI-2, encodes the two-component system SsrA-SsrB, which positively controls the expression of the SPI-2 genes but also other many genes located outside SPI-2. Several regulators have been involved in the expression of ssrAB, such as the ancestral regulators SlyA and OmpR, and the acquired regulator HilD. In this study, we show how SlyA, HilD, and OmpR coordinate to induce the expression of ssrAB under different growth conditions. We found that when Salmonella enterica serovar Typhimurium is grown in nutrient-rich lysogeny broth (LB), SlyA and HilD additively counteract H-NS-mediated repression on ssrAB, whereas in N-minimal medium (N-MM), SlyA antagonizes H-NS-mediated repression on ssrAB independently of HilD. Interestingly, our results indicate that OmpR is required for the expression of ssrAB independently of the growth conditions, even in the absence of repression by H-NS. Therefore, our data support two mechanisms adapted for the expression of ssrAB under different growth conditions. One involves the additive action of SlyA and HilD, whereas the other involves SlyA, but not HilD, to counteract H-NS-mediated repression on ssrAB, thus favoring in both cases the activation of ssrAB by OmpR.IMPORTANCE The global regulator H-NS represses the expression of acquired genes and thus avoids possible detrimental effects on bacterial fitness. Regulatory mechanisms are adapted to induce expression of the acquired genes in particular niches to obtain a benefit from the information encoded in the foreign DNA, as for pathogenesis. Here, we show two mechanisms that were integrated for the expression of virulence genes in Salmonella Typhimurium. One involves the additive action of the regulators SlyA and HilD, whereas the other involves SlyA, but not HilD, to counteract H-NS-mediated repression on the ssrAB operon, thus favoring its activation by the OmpR regulator. To our knowledge, this is the first report involving the coordinated action of two regulators to counteract H-NS-mediated repression.

LoiA directly represses lon gene expression to activate the expression of Salmonella pathogenicity island-1 genes

Salmonella enterica serovar Typhimurium uses virulence factors encoded by Salmonella pathogenicity island 1 (SPI-1) to invade the intestinal epithelium. The low oxygen-induced transcriptional regulator LoiA is established to be a positive regulator of SPI-1 genes and can also repress the expression of the ATP-dependent Lon protease, a negative regulator of SPI-1 genes. Whether the repression of lon by LoiA is associated with its regulation of SPI-1 genes, as well as the regulatory mechanism involved, is unknown. In this study, we showed that LoiA directly represses the expression of the lon gene during invasion by binding to the promoter region of lon. The activation of S. Typhimurium SPI-1 gene expression by LoiA is associated with its repression of lon. Moreover, through invasion and mouse infection assays, we found that the repression of lon by LoiA contributes to S. Typhimurium invasion of intestinal epithelial cells and virulence in mice. However, LoiA does not influence lon expression during S. Typhimurium growth in macrophages, which may be associated with the low expression level of loiA in macrophages. Collectively, these findings establish the direct regulation of lon by LoiA and describe a novel mechanism by which LoiA regulates SPI-1 gene expression.

Guanosine tetraphosphate relieves the negative regulation of Salmonella pathogenicity island-2 gene transcription exerted by the AT-rich ssrA discriminator region

The repressive activity of ancestral histone-like proteins helps integrate transcription of foreign genes with discrepant AT content into existing regulatory networks. Our investigations indicate that the AT-rich discriminator region located between the −10 promoter element and the transcription start site of the regulatory gene ssrA plays a distinct role in the balanced expression of the Salmonella pathogenicity island-2 (SPI2) type III secretion system. The RNA polymerase-binding protein DksA activates the ssrAB regulon post-transcriptionally, whereas the alarmone guanosine tetraphosphate (ppGpp) relieves the negative regulation imposed by the AT-rich ssrA discriminator region. An increase in the GC-content of the ssrA discriminator region enhances ssrAB transcription and SsrB translation, thus activating the expression of downstream SPI2 genes. A Salmonella strain expressing a GC-rich ssrA discriminator region is attenuated in mice and grows poorly intracellularly. The combined actions of ppGpp and DksA on SPI2 expression enable Salmonella to grow intracellularly, and cause disease in a murine model of infection. Collectively, these findings indicate that (p)ppGpp relieves the negative regulation associated with the AT-rich discriminator region in the promoter of the horizontally-acquired ssrA gene, whereas DksA activates ssrB gene expression post-transcriptionally. The combined effects of (p)ppGpp and DksA on the ssrAB locus facilitate a balanced SPI2 virulence gene transcription that is essential for Salmonella pathogenesis.

The transcriptional regulator VarN contributes to Salmonella Typhimurium growth in macrophages and virulence in mice

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major intracellular pathogen of humans and animals and its survival and growth in macrophages is essential for its pathogenicity. The S. Typhimurium genome encodes more than 50 putative regulatory proteins, but their involvement in pathogenicity and their regulatory roles are largely unknown. In this study, we investigated the biological function of the S. Typhimurium STM4320 gene (named varN), which encodes a putative MerR family transcriptional regulator. We found that varN is upregulated 2.6- to 6.8-fold after S. Typhimurium enters murine macrophages. A varN mutant reduced S. Typhimurium growth in murine macrophages and attenuated virulence in mice. Moreover, we showed that deletion of varN decreased the transcription of Salmonella pathogenicity island-2 (SPI-2) genes, which are required for S. Typhimurium growth in macrophages, indicative of the positive regulation of SPI-2 by VarN. We confirmed that the virulence defects of the varN mutant are entirely dependent on its regulation of SPI-2. Thus, our results revealed that VarN is a novel activator of the expression of SPI-2 genes and contributes to S. Typhimurium growth in macrophages and virulence in mice. Our findings provide a significant example of how a putative regulatory protein facilitates S. Typhimurium pathogenicity.

The Hcp-like protein HilE inhibits homodimerization and DNA binding of the virulence-associated transcriptional regulator HilD in Salmonella

HilD is an AraC-like transcriptional regulator that plays a central role in Salmonella virulence. HilD controls the expression of the genes within the Salmonella pathogenicity island 1 (SPI-1) and of several genes located outside SPI-1, which are mainly required for Salmonella invasion of host cells. The expression, amount, and activity of HilD are tightly controlled by the activities of several factors. The HilE protein represses the expression of the SPI-1 genes through its interaction with HilD; however, the mechanism by which HilE affects HilD is unknown. In this study, we used genetic and biochemical assays revealing how HilE controls the transcriptional activity of HilD. We found that HilD needs to assemble in homodimers to induce expression of its target genes. Our results further indicated that HilE individually interacts with each the central and the C-terminal HilD regions, mediating dimerization and DNA binding, respectively. We also observed that these interactions consistently inhibit HilD dimerization and DNA binding. Interestingly, a computational analysis revealed that HilE shares sequence and structural similarities with Hcp proteins, which act as structural components of type 6 secretion systems in Gram-negative bacteria. In conclusion, our results uncover the molecular mechanism by which the Hcp-like protein HilE controls dimerization and DNA binding of the virulence-promoting transcriptional regulator HilD. Our findings may indicate that HilE's activity represents a functional adaptation during the evolution of Salmonella pathogenicity.

HilD and PhoP independently regulate the expression of grhD1, a novel gene required for Salmonella Typhimurium invasion of host cells

When Salmonella is grown in the nutrient-rich lysogeny broth (LB), the AraC-like transcriptional regulator HilD positively controls the expression of genes required for Salmonella invasion of host cells, such as the Salmonella pathogenicity island 1 (SPI-1) genes. However, in minimal media, the two-component system PhoP/Q activates the expression of genes necessary for Salmonella replication inside host cells, such as the SPI-2 genes. Recently, we found that the SL1344_1872 hypothetical gene, located in a S. Typhimurium genomic island, is co-expressed with the SPI-1 genes. In this study we demonstrate that HilD induces indirectly the expression of SL1344_1872 when S. Typhimurium is grown in LB; therefore, we named SL1344_1872 as grhD1 for gene regulated by HilD. Furthermore, we found that PhoP positively controls the expression of grhD1, independently of HilD, when S. Typhimurium is grown in LB or N-minimal medium. Moreover, we demonstrate that the grhD1 gene is required for the invasion of S. Typhimurium into epithelial cells, macrophages and fibroblasts, as well as for the intestinal inflammatory response caused by S. Typhimurium in mice. Thus, our results reveal a novel virulence factor of Salmonella, whose expression is positively and independently controlled by the HilD and PhoP transcriptional regulators.

The microbiota metabolite indole inhibits Salmonella virulence: Involvement of the PhoPQ two-component system.

The microbial community present in the gastrointestinal tract is an important component of the host defense against pathogen infections. We previously demonstrated that indole, a microbial metabolite of tryptophan, reduces enterohemorrhagic Escherichia coli O157:H7 attachment to intestinal epithelial cells and biofilm formation, suggesting that indole may be an effector/attenuator of colonization for a number of enteric pathogens. Here, we report that indole attenuates Salmonella Typhimurium (Salmonella) virulence and invasion as well as increases resistance to colonization in host cells. Indole-exposed Salmonella colonized mice less effectively compared to solvent-treated controls, as evident by competitive index values less than 1 in multiple organs. Indole-exposed Salmonella demonstrated 160-fold less invasion of HeLa epithelial cells and 2-fold less invasion of J774A.1 macrophages compared to solvent-treated controls. However, indole did not affect Salmonella intracellular survival in J774A.1 macrophages suggesting that indole primarily affects Salmonella invasion. The decrease in invasion was corroborated by a decrease in expression of multiple Salmonella Pathogenicity Island-1 (SPI-1) genes. We also identified that the effect of indole was mediated by both PhoPQ-dependent and independent mechanisms. Indole also synergistically enhanced the inhibitory effect of a short chain fatty acid cocktail on SPI-1 gene expression. Lastly, indole-treated HeLa cells were 70% more resistant to Salmonella invasion suggesting that indole also increases resistance of epithelial cells to colonization. Our results demonstrate that indole is an important microbiota metabolite that has direct anti-infective effects on Salmonella and host cells, revealing novel mechanisms of pathogen colonization resistance.

Signal transduction pathway mediated by the novel regulator LoiA for low oxygen tension induced Salmonella Typhimurium invasion.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major intestinal pathogen of both humans and animals. Salmonella pathogenicity island 1 (SPI-1)-encoded virulence genes are required for S. Typhimurium invasion. While oxygen (O2) limitation is an important signal for SPI-1 induction under host conditions, how the signal is received and integrated to the central SPI-1 regulatory system in S. Typhimurium is not clear. Here, we report a signal transduction pathway that activates SPI-1 expression in response to low O2. A novel regulator encoded within SPI-14 (STM14_1008), named LoiA (low oxygen induced factor A), directly binds to the promoter and activates transcription of hilD, leading to the activation of hilA (the master activator of SPI-1). Deletion of loiA significantly decreased the transcription of hilA, hilD and other representative SPI-1 genes (sipB, spaO, invH, prgH and invF) under low O2 conditions. The response of LoiA to the low O2 signal is mediated by the ArcB/ArcA two-component system. Deletion of either arcA or arcB significantly decreased transcription of loiA under low O2 conditions. We also confirmed that SPI-14 contributes to S. Typhimurium virulence by affecting invasion, and that loiA is the virulence determinant of SPI-14. Mice infection assays showed that S. Typhimurium virulence was severely attenuated by deletion of either the entire SPI-14 region or the single loiA gene after oral infection, while the virulence was not affected by either deletion after intraperitoneal infection. The signal transduction pathway described represents an important mechanism for S. Typhimurium to sense and respond to low O2 conditions of the host intestinal tract for invasion. SPI-14-encoded loiA is an essential element of this pathway that integrates the low O2 signal into the SPI-1 regulatory system. Acquisition of SPI-14 is therefore crucial for the evolution of S. Typhimurium as an intestinal pathogen.

The protein acyltransferase Pat post-transcriptionally controls HilD to repress Salmonella invasion.

N-Lysine acylation is a post-translational modification important for both prokaryotic and eukaryotic cells to control a wide array of cellular functions. Here we demonstrate that the protein acyltransferase Pat regulates genes on Salmonella Pathogenicity Island 1 (SPI1) that are required for the invasion of the intestinal epithelium. Mutation of pat slightly increased spleen colonization by Salmonella in streptomycin-treated mice, with more of the pat mutant reaching the spleen than the wild type strain. Growth of Salmonella under specific conditions selectively induced expression of Pat, and deletion of pat increased SPI1 gene expression under the same growth conditions. In addition, over-expression of Pat repressed SPI1 expression and bacterial entry into epithelial cells. These results demonstrate that Salmonella invasion is negatively controlled by Pat. Regulation of the SPI1 central regulator HilD was essential for Pat to exert its effects. The control of HilD by Pat was through post-transcriptional mechanisms, moderately repressing hilD translation while significantly reducing HilD stability. Additionally, growth of Salmonella in the presence of histone deacetylases inhibitors reduced expression of SPI1 by affecting HilD stability, supporting the concept that altering the stability of this regulator is required for Pat to control Salmonella invasion.

Inherent Variability of Growth Media Impacts the Ability of Salmonella Typhimurium to Interact with Host Cells

Efficient invasion of non-phagocytic cells, such as intestinal epithelial cells, by Salmonella Typhimurium is dependent on the Salmonella Pathogenicity Island 1 (SPI-1)-encoded Type Three Secretion System. The environmental cues involved in SPI-1 induction are not well understood. In vitro, various conditions are used to induce SPI-1 and the invasive phenotype. Although lysogeny broth (LB) is widely used, multiple formulations exist, and variation can arise due to intrinsic differences in complex components. Minimal media are also susceptible to variation. Still, the impact of these inconsistencies on Salmonella virulence gene expression has not been well studied. The goal of this project is to identify growth conditions in LB and minimal medium that affect SPI-1 induction in vitro using both whole population and single cell analysis. Here we show, using a fluorescent reporter of the SPI-1 gene prgH, that growth of Salmonella in LB yields variable induction. Deliberate modification of media components can influence the invasive profile. Finally, we demonstrate that changes in SPI-1 inducing conditions can affect the ability of Salmonella to replicate intracellularly. These data indicate that the specific media growth conditions impact how the bacteria interact with host cells.

Virulence Gene Regulation by L-Arabinose in Salmonella enterica.

Invasion of the intestinal epithelium is a critical step in Salmonella enterica infection and requires functions encoded in the gene cluster known as Salmonella Pathogenicity Island 1 (SPI-1). Expression of SPI-1 genes is repressed by L-arabinose, and not by other pentoses. Transport of L-arabinose is necessary to repress SPI-1; however, repression is independent of L-arabinose metabolism and of the L-arabinose-responsive regulator AraC. SPI-1 repression by L-arabinose is exerted at a single target, HilD, and the mechanism appears to be post-translational. As a consequence of SPI-1 repression, l-arabinose reduces translocation of SPI-1 effectors to epithelial cells and decreases Salmonella invasion in vitro. These observations reveal a hitherto unknown role of L-arabinose in gene expression control and raise the possibility that Salmonella may use L-arabinose as an environmental signal.

Temporal regulation of Salmonella pathogenicity Island 1 (SPI-1) hilA by Hfq in Salmonella enterica serovar typhimurium

Hfq is a global regulatory RNA-binding protein abundant in many bacteria. In terms of virulence regulation in Salmonella, Hfq binds to and stabilizes the mRNA of hilD, a master regulator of Salmonella pathogenicity island 1 (SPI-1), and Hfq activates SPI-1 gene transcription during the late exponential-growth phase. In contrast to the positive roles of Hfq in SPI-1 regulation, we observed that the absence of Hfq increased the expression of SPI-1 regulators, including HilA and InvF, during the late stationary phase. This result suggests that Hfq has pleiotropic functions, controlling SPI-1 positively or negatively depending on the growth conditions.

LeuO, a dormant sentinel for SPI‐1?

A new mechanism for the turning-off of gene expression in Salmonella Pathogenicity Island 1 (SPI-1) is proposed by Espinosa and Casadesús, which involves the action of the LeuO quiescent regulator, by two different pathways. A major one through the activation of the hilE gene, where the HilE protein would in turn inactivate HilD, a major positive transcriptional regulator of SPI-1; and a minor HilE-HilD-independent pathway. This could constitute a back-up or an aid for the turn-off of SPI-1 genes mediated by the nucleoid protein H-NS.

Hfq and ArcA Are Involved in the Stationary Phase-Dependent Activation of Salmonella Pathogenicity Island 1 (SPI1) Under Shaking Culture Conditions

In Salmonella enterica serovar Typhimurium, many genes encoded within Salmonella pathogenicity island 1 (SPI1) are required to induce intestinal/diarrheal disease. In this study, we compared the expression of four SPI1 genes (hilA, invF, prgH, and sipC) under shaking and standing culture conditions and found that the expression of these genes was highest during the transition from the exponential to stationary phase under shaking conditions. To identify regulators associated with the stationary phase-dependent activation of SPI1, the effects of selected regulatory genes, including relA/spoT (ppGpp), luxS, ihfB, hfq, and arcA, on the expression of hilA and invF were compared under shaking conditions. Mutations in the hfq and arcA genes caused a reduction in hilA and invF expression (more than 2-fold) in the early stationary phase only, whereas the lack of ppGpp and IHF decreased hilA and invF gene expression during the entire stationary phase. We also found that hfq and arcA mutations caused a reduction of hilD expression upon entry into the stationary phase under shaking culture conditions. Taken together, these results suggest that Hfq and ArcA regulate the hilD promoter, causing an accumulation of HilD, which can trigger a stationary phase-dependent activation of SPI1 genes under shaking culture conditions.

Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.

ClpP deletion causes attenuation of Salmonella Typhimurium virulence through mis-regulation of RpoS and indirect control of CsrA and the SPI genes

Salmonella enterica serovar Typhimurium requires the type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) and controlled by the master regulator, HilA, to penetrate the intestinal epithelium. Numerous regulators affect virulence through influence on this system, including the proteolytic component ClpP, the stationary phase regulator RpoS and the carbon-storage regulator CsrA. However, the mechanism behind the ClpP regulation is not fully understood. To elucidate this we examined differentially expressed genes in a ΔclpP mutant compared with WT using global transcriptomic analysis. SPI1 and SPI4 virulence genes were significantly downregulated in the ΔclpP mutant, whereas several RpoS-dependent genes and the fliC gene encoding flagellin were upregulated. While the ΔclpP mutant was attenuated in cell invasion, this attenuation was not present in a ΔclpP/rpoS : : amp double mutant, suggesting the repression of invasion was directed through RpoS. The expression of the csrA virulence regulator was increased in the ΔclpP mutant and decreased in the rpoS : : amp and ΔclpP/rpoS : : amp mutants, indicating that ClpP affects the csrA expression level as well. Thus, this study suggests that ClpP affects SPI1 expression and thereby virulence indirectly through its regulation of both RpoS and CsrA.

LsrR-Mediated Quorum Sensing Controls Invasiveness of Salmonella typhimurium by Regulating SPI-1 and Flagella Genes

Bacterial cell-to-cell communication, termed quorum sensing (QS), controls bacterial behavior by using various signal molecules. Despite the fact that the LuxS/autoinducer-2 (AI-2) QS system is necessary for normal expression of Salmonella pathogenicity island-1 (SPI-1), the mechanism remains unknown. Here, we report that the LsrR protein, a transcriptional regulator known to be involved in LuxS/AI-2-mediated QS, is also associated with the regulation of SPI-1-mediated Salmonella virulence. We determined that LsrR negatively controls SPI-1 and flagella gene expressions. As phosphorylated AI-2 binds to and inactivates LsrR, LsrR remains active and decreases expression of SPI-1 and flagella genes in the luxS mutant. The reduced expression of those genes resulted in impaired invasion of Salmonella into epithelial cells. Expression of SPI-1 and flagella genes was also reduced by overexpression of the LsrR regulator from a plasmid, but was relieved by exogenous AI-2, which binds to and inactivates LsrR. These results imply that LsrR plays an important role in selecting infectious niche of Salmonella in QS dependent mode.