Identification of a new Salmonella enterica serovar Enteritidis locus involved in cell invasion and in the colonisation of chicks

Poultry products contaminated with Salmonella enterica serovar Enteritidis are a major cause of foodborne disease in industrialized countries. Knowledge of how poultry is colonised is essential for reducing contamination of these products. We have characterized the bacterial yfg-eng locus involved in chicken colonisation. Its sequencing revealed four open reading frames (ORF), yfgM, yfgL, engA and yfgJ, all transcribed in the same orientation. An yfgL mutant of S. Enteritidis colonised the caeca (P < 0.05) and the spleens (P < 0.01) of one-day-old chicks subnormally 2 and 5 days after oral inoculation. This lower virulence was correlated with reduced secretion of the SPI-1 and flagellar proteins in the yfgL mutant compared to the wild-type strain. Consistent with this, the S. Enteritidis yfgL mutant was less motile than the wild type and fewer invaded enterocytes (P < 0.05) and avian HD11 macrophages (P < 0.001). All these defects could be partially overcome by inserting the yfg-eng locus into the mutant on a recombinant plasmid.     

In Vivo Genetic Analysis Indicates That PhoP-PhoQ and the Salmonella Pathogenicity Island 2 Type III Secretion System Contribute Independently to Salmonella enterica Serovar Typhimurium Virulence

Many virulence factors are required for Salmonella enterica serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection by S. enterica serovar Typhimurium: the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently to S. typhimurium virulence. Our results also suggest that mutation of phoP may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.     

Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants

Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA' reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.     

A Novel Phage Element of Salmonella enterica Serovar Enteritidis P125109 Contributes to Accelerated Type III Secretion System 2-Dependent Early Inflammation Kinetics in a Mouse Colitis Model

Salmonella enterica subsp. I serovar Enteritidis exhibits type III secretion system 2 (TTSS2)-dependent early colonization and inflammation kinetics faster than those of closely related S. enterica serovar Typhimurium. To investigate the accelerated TTSS-2-dependent pathogenic potential of S. Enteritidis, we focused on its genome. Results of a previously published comparative genomic study revealed the presence of mutually exclusive genes in both serovars. In this study, we investigated the roles of six S. Enteritidis-specific genes in vivo by using differential fluorescence induction (DFI) through putative gene-specific promoters. The promoter construct associated with the gene locus SEN1140 induced green fluorescent protein (GFP) expression in the gut lumen, lamina propria, mesenteric lymph nodes, and related systemic organs. To further investigate the potential role of SEN1140, we compared a SEN1140 deletion mutant with S. Typhimurium in a TTSS1-deficient background. Interestingly, the S. Enteritidis mutant lacking SEN1140 did not show the unique TTSS-2-dependent early colonization and inflammation kinetic phenotype of S. Typhimurium. Consistent with this result, complementation of SEN1140 restored the TTSS-2-dependent accelerated inflammatory potential of S. Enteritidis. This report presents a suitable screening strategy that uses a combination of DFI, fluorescence-activated cell sorting, quantitative PCR, and wild-type isogenic tagged-strain techniques to explore the unique roles of S. Enteritidis-specific genes in bacterial pathogenesis.     

Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens

Salmonella enterica serovar Gallinarum is a host-specific serotype that causes the severe systemic disease fowl typhoid in domestic poultry and a narrow range of other avian species but rarely causes disease in mammalian hosts. Specificity of the disease is primarily at the level of the reticuloendothelial system, but few virulence factors have been described other than the requirement for an 85-kb virulence plasmid. In this work, by making functional mutations in the type III secretion systems (TTSS) encoded by Salmonella pathogenicity island 1 (SPI-1) and SPI-2, we investigated the role of these pathogenicity islands in interactions between Salmonella serovar Gallinarum and avian cells in vitro and the role of these pathogenicity islands in virulence in chickens. The SPI-1 mutant showed decreased invasiveness into avian cells in vitro but was unaffected in its ability to persist within chicken macrophages. In contrast the SPI-2 mutant was fully invasive in nonphagocytic cells but failed to persist in macrophages. In chicken infections the SPI-2 mutant was attenuated while the SPI-1 mutant showed full virulence. In oral infections the SPI-2 mutant was not observed in the spleen or liver, and following intravenous inoculation it was cleared rapidly from these sites. SPI-2 function is required by Salmonella serovar Gallinarum for virulence, primarily through promoting survival within macrophages allowing multiplication within the reticuloendothelial system, but this does not preclude the involvement of SPI-2 in uptake from the gut to the spleen and liver. SPI-1 appears to have little effect on virulence and survival of Salmonella serovar Gallinarum in the host.     

Virulent Salmonella enterica serovar typhimurium evades adaptive immunity by preventing dendritic cells from activating T cells

Dendritic cells (DCs) constitute the link between innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) in bacteria and by presenting bacterial antigens to T cells. Recognition of PAMPs renders DCs as professional antigen-presenting cells able to prime na?ve T cells and initiate adaptive immunity against bacteria. Therefore, interfering with DC function would promote bacterial survival and dissemination. Understanding the molecular mechanisms that have evolved in virulent bacteria to evade activation of adaptive immunity requires the characterization of virulence factors that interfere with DC function. Salmonella enterica serovar Typhimurium, the causative agent of typhoid-like disease in the mouse, can prevent antigen presentation to T cells by avoiding lysosomal degradation in DCs. Here, we show that this feature of virulent Salmonella applies in vivo to prevent activation of adaptive immunity. In addition, this attribute of virulent Salmonella requires functional expression of a type three secretion system (TTSS) and effector proteins encoded within the Salmonella pathogenicity island 2 (SPI-2). In contrast to wild-type virulent Salmonella, mutant strains carrying specific deletions of SPI-2 genes encoding TTSS components or effectors proteins are targeted to lysosomes and are no longer able to prevent DCs from activating T cells in vitro or in vivo. SPI-2 mutant strains are attenuated in vivo, showing reduced tissue colonization and enhanced T-cell activation, which confers protection against a challenge with wild-type virulent Salmonella. Our data suggest that impairment of DC function by the activity of SPI-2 gene products is crucial for Salmonella pathogenesis.     

The Salmonella enterica serovar typhimurium translocated effectors SseJ and SifB are targeted to the Salmonella-containing vacuole

The Salmonella enterica serovar Typhimurium type III secretion system (TTSS) encoded in Salmonella pathogenicity island 2 (SPI-2) promotes replication within host cells and systemic infection of mice. The SPI-2 TTSS is expressed following Salmonella internalization into host cells and translocates effectors across the membrane of the Salmonella-containing vacuole (SCV). Two effectors with similar amino-terminal domains, SseJ and SifB, localize to the SCV membrane in infected HEp-2 cells and subsequently traffic away from the SCV along Salmonella-induced-filaments (Sifs). Following infection of RAW cells, SseJ and SifB localize to the SCV as well as LAMP-1-positive, vesicular-appearing structures distant from the SCV. Trafficking of SseJ and SifB away from the SCV requires the SPI-2 effector SifA. Deletion of sseJ, but not sifB, leads to attenuation of Salmonella replication in mice following intraperitoneal inoculation. The contribution of SseJ to in vivo replication is identical in wild-type and sifA deletion backgrounds, suggesting that SseJ trafficking away from the SCV along Sifs is unnecessary for its virulence function.     

Role of receptor proteins for enterobactin and 2,3-dihydroxybenzoylserine in virulence of Salmonella enterica

Single, double, and triple mutants of an enterobactin-deficient mutant strain of Salmonella enterica serovar Typhimurium were constructed that were defective in the expression of the iron-regulated outer membrane proteins (IROMPs) FepA, IroN, and Cir, which are proposed to function as catecholate receptors. Uptake of naturally occurring and chemically synthesized catecholate molecules by these mutants was assessed in standard growth promotion assays. Unique patterns of uptake were identified for each IROMP; specifically, FepA and IroN were confirmed to be required for transport of enterobactin, and all three proteins were shown to function as receptors for the enterobactin breakdown product 2,3-dihydroxybenzoylserine. The fepA, iroN, and cir alleles were transduced to enterobactin-proficient strains of S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, and the resulting phenotypes were confirmed by analysis of outer membrane protein profiles, by sensitivity to KP-736, a catecholate-cephalosporin conjugate, and by growth promotion tests on egg white agar. Intragastric infections of mice with the S. enterica serovar Typhimurium strains indicated that the parental strain and the fepA iroN double mutant were similarly virulent but that the fepA iroN cir triple mutant was significantly attenuated. Moreover, in mixed infections, the fepA iroN mutant showed similar cecal colonization and invasion of the liver to the parental strain, while the triple mutant showed significantly reduced cecal colonization and no measurable spread to the liver. Infections of 4-day-old chicks with S. enterica serovar Enteritidis strains also indicated that mutation of the fepA iroN genes did not significantly reduce cecal colonization and systemic spread compared with those of the parental strain. The results indicate that, while enterobactin uptake is not essential for the virulence of S. enterica serovars in mouse and chicken infection models, the ability to take up 2,3-dihydroxybenzoylserine via any of the three catecholate siderophore receptors appears to play an important role, since the S. enterica serovar Typhimurium triple mutant was significantly attenuated in the mouse model. Salmochelins appear not to be involved in the virulence of S. enterica.     

SspA is required for lethal Salmonella enterica serovar Typhimurium infections in calves but is not essential for diarrhea

Salmonella pathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether the Salmonella enterica serovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with a sptP mutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with an sspA mutant developed diarrhea, which resolved within 10 days but did not result in mortality. The sspA mutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.     

Role of the Salmonella pathogenicity island 1 effector proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium colitis in streptomycin-pretreated mice

Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) induces enterocolitis in humans and cattle. The mechanisms of enteric salmonellosis have been studied most extensively in calf infection models. The previous studies established that effector protein translocation into host cells via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is of central importance in serovar Typhimurium enterocolitis. We recently found that orally streptomycin-pretreated mice provide an alternative model for serovar Typhimurium colitis. In this model the SPI-1 TTSS also plays a key role in the elicitation of intestinal inflammation. However, whether intestinal inflammation in calves and intestinal inflammation in streptomycin-pretreated mice are induced by the same SPI-1 effector proteins is still unclear. Therefore, we analyzed the role of the SPI-1 effector proteins SopB/SigD, SopE, SopE2, and SipA/SspA in elicitation of intestinal inflammation in the murine model. We found that sipA, sopE, and, to a lesser degree, sopE2 contribute to murine colitis, but we could not assign an inflammation phenotype to sopB. These findings are in line with previous studies performed with orally infected calves. Extending these observations, we demonstrated that in addition to SipA, SopE and SopE2 can induce intestinal inflammation independent of each other and in the absence of SopB. In conclusion, our data corroborate the finding that streptomycin-pretreated mice provide a useful model for studying the molecular mechanisms of serovar Typhimurium colitis and are an important starting point for analysis of the molecular events triggered by SopE, SopE2, and SipA in vivo.     

Salmonella pathogenicity island 2-mediated overexpression of chimeric SspH2 proteins for simultaneous induction of antigen-specific CD4 and CD8 T cells

Salmonella enterica serovar Typhimurium employs two different type III secretion systems (TTSS) encoded within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) for targeting of effector proteins into the cytosol of eukaryotic cells during different stages of the infection cycle. The SPI1 TTSS translocates virulence factors across the plasma membrane when the bacterium initially contacts the host cell. In contrast, the SPI2 TTSS functions to translocate proteins across the membrane of the Salmonella-containing vacuole and promotes intracellular survival and replication. The aim of the present study was to directly compare the potentials of SPI1 and SPI2 type III effector proteins to act as carrier molecules for a heterologous antigen. The p60 protein of Listeria monocytogenes was used as a model antigen to construct chimeric SopE2 (SPI1), SifA (SPI2), and SspH2 (SPI2) proteins. SPI1- and SPI2-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of p60 fusion proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with recombinant Salmonella strains expressing these hybrid proteins revealed comparable numbers of p60-specific CD8 T cells. However, only overexpression of translocated SspH2/p60 from a medium-copy-number vector induced simultaneous antigen-specific CD4 and CD8 T-cell responses, suggesting that SspH2 is an attractive carrier molecule for foreign-protein delivery.     

Infection of mice by Salmonella enterica serovar Enteritidis involves additional genes that are absent in the genome of serovar Typhimurium

Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro, rfa, rfb, phoP, and phoQ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system (SEN4290 to SEN4292), the peg fimbrial operon (SEN2144A to SEN2145B), a putative pathogenicity island (SEN1970 to SEN1999), and a type VI secretion system remnant SEN1001, encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A ΔSEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro. Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro. The genes identified here may perform novel virulence functions not characterized in previous Salmonella models.     

SseJ deacylase activity by Salmonella enterica serovar Typhimurium promotes virulence in mice

Salmonella enterica serovar Typhimurium utilizes a type III secretion system (TTSS) encoded on Salmonella pathogenicity island-2 (SPI2) to promote intracellular replication during infection, but little is known about the molecular function of SPI2-translocated effectors and how they contribute to this process. SseJ is a SPI2 TTSS effector protein that is homologous to enzymes called glycerophospholipid-cholesterol acyltransferases and, following translocation, localizes to the Salmonella-containing vacuole and Salmonella-induced filaments. Full virulence requires SseJ, as sseJ null mutants exhibit decreased replication in cultured cells and host tissues. This work demonstrates that SseJ is an enzyme with deacylase activity in vitro and identifies three active-site residues. Catalytic SseJ mutants display wild-type translocation and subcellular localization but fail to complement the virulence defect of an sseJ null mutant. In contrast to the wild type, SseJ catalytic mutants fail to down regulate Salmonella-induced filament formation and fail to restore the sifA null mutant phenotype of loss of phagosomal membrane to sifA sseJ null double mutants, suggesting that wild-type SseJ modifies the vacuolar membrane. This is the first demonstration of an enzymatic activity for a SPI2 effector protein and provides support for the hypothesis that the deacylation of lipids on the Salmonella-containing vacuole membrane is important to bacterial pathogenesis.     

Discovery of Salmonella virulence factors translocated via outer membrane vesicles to murine macrophages

Salmonella enterica serovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors. Salmonella virulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ～70 amino acids and have high sequence homology to each other (>85% identity). Salmonella lacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all three Salmonella gene-encoded type III secretion systems (T3SSs)-Salmonella pathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novel Salmonella virulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.     

SseK1 and SseK2 are novel translocated proteins of Salmonella enterica serovar typhimurium

Salmonella enterica is a gram-negative, facultative intracellular pathogen that causes disease symptoms ranging from gastroenteritis to typhoid fever. A key virulence strategy is the translocation of bacterial effector proteins into the host cell, mediated by the type III secretion systems (TTSSs) encoded in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. In S. enterica serovar Typhimurium LT2, we identified the protein products of STM4157 and STM2137 as novel candidate secreted proteins by comparison to known secreted proteins from enterohemorrhagic Escherichia coli and Citrobacter rodentium. The STM4157 and STM2137 proteins, which we have designated SseK1 and SseK2, respectively, are 61% identical at the amino acid level and differ mainly in their N termini. Western analysis showed that in vitro accumulation and secretion of these proteins in serovar Typhimurium were affected by mutations in the two-component systems SsrA/B and PhoP/Q, which are key mediators of intracellular growth and survival. SPI-2 TTSS-dependent translocation of recombinant SseK1::Cya was evident at 9 h postinfection of epithelial cells, while translocation of SseK2::Cya was not detected until 21 h. Remarkably, the translocation signal for SseK1 was contained within the N-terminal 32 amino acids. Fractionation of infected epithelial cells revealed that following translocation SseK1 localizes to the host cytosol, which is unusual among the currently known Salmonella effectors. Phenotypic analysis of DeltasseK1, DeltasseK2, and DeltasseK1/DeltasseK2 mutants provided evidence for a role that was not critical during systemic infection. In summary, this work demonstrates that SseK1 and SseK2 are novel translocated proteins of serovar Typhimurium.     

Characterization of Salmonella pathogenicity island 1 type III secretion-dependent hemolytic activity in Salmonella enterica serovar Typhimurium

A number of bacteria that are pathogenic for animals and plants possess a type III secretion system (TTSS) to translocate virulence-associated proteins into host cells. In several bacteria, it has been reported that the TTSS is correlated with an ability to cause contact-dependent hemolysis in vitro. Here, we showed that the Salmonella enterica serovar Typhimurium strain SL1344 exhibited Salmonella pathogenicity island 1 type III secretion-dependent, contact-mediated, hemolytic activity. Mutations with a single deletion in genes encoding putative pore-forming proteins, SipB and SipC, secreted by the TTSS abolished hemolytic activity. In addition, the osmoprotection studies revealed that molecules larger than PEG2000 conferred significant protection against lysis induced by Salmonella. These results indicate that the hemolysis generated by Salmonella is due to the formation of pores within the erythrocyte membrane.     

Salmonella pathogenicity island 2-encoded proteins SseC and SseD are essential for virulence and are substrates of the type III secretion system

Survival of Salmonella enterica serovar Typhimurium within host phagocytic cells is a critical step in establishing systemic infection in mice. Genes within Salmonella pathogenicity island 2 (SPI-2) encode a type III secretion system that is required for establishment of systemic infection. Several proteins encoded by SPI-2 have homology to type III secreted proteins from enteropathogenic Escherichia coli and Yersinia and, based on that homology, are predicted to be secreted through the SPI-2 type III secretion system. We have investigated the roles of two of these proteins, SseC and SseD. We demonstrate here that the SseD protein is required for systemic Salmonella infection of the mouse, and we confirmed the virulence requirement for the SseC protein. Experiments were performed, using cellular fractionation and immunoblotting, to identify the subcellular location of the SseC and SseD proteins. Both proteins were found to localize predominantly to the bacterial cell membrane. In addition, our work revealed that SseC and SseD are exposed to the extracellular environment and are loosely associated with the bacterial membrane. Furthermore, localization of SseC and SseD to the bacterial membrane was found to require a functional SPI-2 type III secretion system. Collectively, these results indicate that the SseC and SseD proteins are secreted by the SPI-2 type III secretion system to the bacterial membrane in order to perform their virulence functions.     

Salmonella enterica serovar Senftenberg human clinical isolates lacking SPI-1

Nontyphoidal Salmonella species cause gastrointestinal disease worldwide. The prevailing theory of Salmonella enteropathogenesis is that bacterial invasion of the intestinal epithelium is essential for virulence and that this requires the virulence-associated genomic region Salmonella pathogenicity island 1 (SPI-1). Recent studies of Salmonella enterica infection models have demonstrated that enterocolitis and diarrhea in mice and cows can occur independently of SPI-1. In this study, we sought to confirm whether two S. enterica serovar Senftenberg clinical isolates lacked genes essential for SPI-1 function. Two clinical strains were isolated and identified as being S. enterica serovar Senftenberg from four stool samples from a food-borne disease outbreak affecting seven individuals in Shenzhen, Guangdong Province, China, using conventional methods, pulsed-field gel electrophoresis and multilocus sequence typing. The possibility of coinfection with other potential bacteria or usual viruses was excluded. Two isolates were analyzed for the presence of invA, sipA, ssaR, sifA, and sopE2 by PCR and Southern blotting and were then assayed for the presence of SPI-1 by PCR and long-range PCR for fhlA-hilA, hilA-spaP, and spaP-invH and Southern blot analysis. A long-range PCR fragment from fhlA to mutS covering the 5' and 3' flanks of SPI-1 was also amplified from the two clinical isolates and sequenced. In addition, the two clinical isolates were assayed for enteroinvasiveness in vitro. Murine infection models were also examined. Biochemical tests and serotyping confirmed that the two clinical isolates are S. enterica serovar Senftenberg. However, they lacked genes critical for SPI-1 function but contained SPI-2 genes and were attenuated for the invasion of cultured intestinal epithelial cells. In conclusion, clinical S. enterica serovar Senftenberg strains isolated from a food-borne disease outbreak lack the invasion-associated locus SPI-1, indicating that SPI-1 is not essential for human gastroenteritis.