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Infect Immun. Jun 2005; 73(6): 3219–3227.
PMCID: PMC1111876

Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Is Necessary for Complete Virulence in a Mouse Model of Infectious Enterocolitis

Abstract

Salmonella species cause a wide range of disease in multiple hosts. Salmonella enterica serovar Typhimurium causes self-limited intestinal disease in humans and systemic typhoid-like illness in susceptible mice. The prevailing dogma in murine S. enterica serovar Typhimurium pathogenesis is that distinct virulence mechanisms—Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2)—perform distinct roles in pathogenesis, the former being important for invasion and intestinal disease and the latter important for intracellular survival and systemic persistence and disease. Although evidence from bovine infections has suggested that SPI2 has a role in ileal disease, there is no evidence that SPI2 is important for inflammation in a disease that more closely recapitulates human colitis. Using S. enterica serovar Typhimurium strains that lack functional type III secretion systems, we demonstrate that SPI2 is essential for complete virulence in murine infectious enterocolitis. Using a recently characterized murine model (M. Barthel,S. Hapfelmeier, L. Quintanilla-Martinez, M. Kremer, M. Rohde, M. Hogardt, K. Pfeffer, H. Russmann, and W. D. Hardt, Infect. Immun. 71:2839-2858, 2003), we demonstrate that SPI1 mutants are unable to cause intestinal disease 48 h after infection and that SPI2-deficient bacteria also cause significantly attenuated typhlitis. We show that at the peak of inflammation in the cecum, SPI2 mutants induce diminished intercellular adhesion molecule 1 expression and neutrophil recruitment but that wild-type and mutant Salmonella are similarly distributed in the lumen of the infected organ. Finally, we demonstrate that attenuation of intestinal inflammation is accompanied by resolution of typhlitis in the mutant, but not wild-type, infections. Collectively, these results indicate that SPI2 is needed for enterocolitis, as well as for systemic disease.

Salmonella species are facultative intracellular gram-negative bacteria that cause a wide array of disease including systemic disease and enterocolitis in a multitude of hosts (reviewed in [2]). Murine infection with Salmonella enterica serovar typhimurium has been used predominantly to model human typhoid (caused by Salmonella enterica serovar Typhi), while bovine infection with Salmonella enterica serovar Dublin or S. enterica serovar Typhimurium has been a prevailing model of intestinal disease. These models have been exploited to gain critical insight into the pathogenesis of disease cause by salmonellae, including (for example) that invasion-associated genes are required for intestinal secretory and inflammatory disease, that intracellular survival in both the intestinal epithelium and macrophages is essential for systemic pathogenesis, and that M cells of the ileal Peyer's patches are the site of invasion for systemic infection in murine typhoid prior to dissemination to liver and spleen via the reticuloendothelial system (8, 14, 15, 28). A central hypothetical theme that has emerged as a result of these discoveries is the distinct role of different virulence systems—Salmonella pathogenicity island 1 (SPI1) and SPI2—in the pathogenesis of intestinal and systemic disease.

Both of these horizontally acquired genomic islands encode a type III secretion system (TTSS), capable of secreting bacterial proteins into the host cell or extracellular milieu. The prevailing view is that SPI1 is necessary for cell invasion and essential for intestinal disease, while SPI2 is required for intracellular survival and persistence in target organs such as the spleen and liver (reviewed in reference 22). Soon after the identification of these virulence systems, it was demonstrated that in mice, SPI1-deficient S. enterica serovar Typhimurium is incapable of causing systemic disease following oral infection but is not attenuated when introduced intraperitoneally (10, 28), while SPI2 mutants inoculated by the latter route are attenuated for systemic infection (23) but maintain intestinal virulence in cows and rabbits after oral infection (7, 25).

Whether SPI2 plays a role in intestinal inflammatory disease caused by S. enterica serovar Typhimurium is unclear. It has been shown with cattle that diarrhea caused by S. enterica serovar Typhimurium is SPI1 but not SPI2 dependent (25) and that Salmonella strains lacking SPI2 regulatory genes and wild-type (WT) Salmonella are equally pathogenic in a rabbit model of gastroenteritis (7). However, it has also been reported that SPI2 plays at least some role in bovine inflammatory disease (3, 25). Bispham et al. (3) demonstrated that bacteria lacking the SPI2 translocon component sseD or the SPI2 TTSS apparatus protein encoded by the ssaT gene induced less fluid secretion into bovine ileal loops inoculated with mutant bacteria than did WT and that there was diminished neutrophilic influx into infected tissue.

There are significant practical and theoretical limitations of these models, however. Due to the technically demanding nature of bovine experiments, the number of experimental animals used in these experiments is limited. Furthermore, cattle used are typically outbred and show significant variation in disease severity, depending on age. In addition, although investigations have demonstrated disease in the ilea of these animals, the role of SPI2 in tissues more commonly affected in human S. enterica serovar Typhimurium infection, e.g., the cecum and colon, have not been investigated. Consequently, it is difficult to extrapolate the findings of these studies to human disease.

A recently characterized model of infectious cecal inflammation (typhlitis) in mice provides an alternate model for the study of human S. enterica serovar Typhimurium-induced intestinal disease with significant advantages over the bovine model (1). Following oral administration of streptomycin, mice challenged with S. enterica serovar Typhimurium display signs of intestinal inflammatory pathology with many histopathological similarities to human disease, including severe inflammation in the large bowel with little or no inflammatory pathology in the ileum (1, 4, 17). This model has been utilized to demonstrate that Salmonella-induced intestinal inflammation requires bacterial activity dependent on the SPI1 effector sipA, as well as functional flagella and chemotaxis (1, 11, 24). Antibiotic treatment is a risk factor for acquiring S. enterica serovar Typhimurium colitis (6), and the dependence of inflammation on streptomycin treatment is inferred to be due to alterations in the host microflora, resulting in environmental changes and decreased colonization resistance (20, 21).

In this study, we exploited the streptomycin pretreatment model to show that SPI2 is required for complete intestinal virulence in the cecum and colon in vivo in a disease model that shares clinical and histopathological features with human disease.

MATERIALS AND METHODS

Bacterial culture.

Salmonella enterica serovar Typhimurium SL1344 (wild type [WT]), invA mutant (invA::kan SB103; SPI1), and ssaR mutant (ΔssaR; SPI2) (5) were grown with overnight shaking (200 rpm) in 3 ml Luria-Bertani (LB) broth with 50 μg/ml streptomycin ± 50 μg/ml kanamycin at 37°C for 18 h.

Mouse experiments.

Inbred 10-week-old C57BL/6 mice (Jackson Laboratories) were deprived of food and water for 4 h prior to administration of 20 mg of streptomycin/mouse by oral gavage. Two hours subsequently, food and water were provided ad libitum. Twenty hours after oral streptomycin treatment, food and water were once again withdrawn for 4 h, after which 3 × 106 or 3 × 108 bacteria in 100 μl LB broth were administered by oral gavage. Control mice were given 100 μl sterile LB broth. Water and food were provided ad libitum. Mice were euthanized with CO2 at designated time points, and tissues were harvested aseptically for bacterial enumeration and histopathology. All animal experiments were conducted in a manner consistent with the ethical requirements of the Animal Care Committee at the University of British Columbia.

Bacterial enumeration.

Tissues were collected at various time points into 1.5 ml sterile PBS and homogenized with a tissue homogenizer (Polytron MR 21; Kinematica). Serial dilutions of the resulting mixture were plated on LB agar plates containing 100 μg/ml streptomycin. The threshold of detection was 50 CFU per organ.

Histopathology.

Colons, ceca, and ilea of experimental animals were fixed in 3% formalin for 18 h, followed by 18 h in 70% ethanol prior to being embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E), or they were fixed for 3 h in 3% paraformaldehyde, prior to being embedded and cryosectioned.

Pathological scores were determined by averaging six fields/sample as follows, based on a revision of previously published methods (quantitave and qualitative criteria were separated) and descriptions of human disease.

(i) Lumen.

Pathological scores were determined as follows (scores are given in parentheses after each category): empty (0), necrotic epithelial cells (scant, 1; moderate, 2; dense, 3), and polymorphonuclear leukocytes (PMNs) (scant, 2; moderate, 3; dense, 4).

(ii) Surface epithelium.

Pathological scores were determined as follows (scores are given in parentheses after each category): no pathological changes (0); mild, moderate, or severe regenerative changes (1, 2, or 3, respectively); patchy or diffuse desquamation (1 or 2); PMNs in epithelium (1); and ulceration (1).

(iii) Mucosa.

Pathological scores were determined as follows (scores are given in parentheses after each category): no pathological changes (0); rare (<15%), moderate (15 to 50%), or abundant (>50%) crypt abscesses (1, 2, or 3, respectively); presence of mucinous plugs (1); presence of granulation tissue (1).

(iv) Submucosa.

Pathological scores were determined as follows (scores are given in parentheses after each category): no pathological changes (0); mononuclear cell infiltrate (1 small aggregate, >1 aggregate, or large aggregates plus increased single cells) (0, 1, or 2, respectively); PMN infiltrate (no extravascular PMNs, single extravascular PMNs, or PMN aggregates) (0, 1, or 2, respectively); mild, moderate, or severe edema (0, 1, or 2, respectively).

Immunohistochemistry.

Paraffin-embedded tissues were deparaffinized in xylene (twice for 5 min each time); rehydrated in 100%, 95%, and 70% ethanol (each, 5 min); then washed in phosphate-buffered saline containing 0.1% bovine serum albumin (PBS-BSA; BSA from Sigma). Sections were outline with a wax pen and blocked for 30 min in 10% goat serum in PBS-BSA at room temperature. Sections were then washed three times in PBS-BSA, prior to incubation overnight at 4°C or 1 h room temperature with a primary antibody (anti-Salmonella lipopolysaccharide [LPS], 6.8 μg/ml) and anti-intercellular adhesion molecule 1 (anti-ICAM-1), 5μg/ml; BD Biosciences). Sections were then washed three times in PBS-BSA, prior to incubation with the appropriate fluorochrome- or streptavidin-conjugated secondary antibodies (0.1 mg/ml, 30 min at room temperature). Imaging was performed with a Zeiss Axioskop epifluorescence microscope and MetaMorph software for fluorescent images and with a Zeiss Axiostar microscope with a Nikon Powershot G5 camera. Light and fluorescent images were cropped and scaled in Adobe Photoshop, version 7.0.1.

Mast cell staining.

Paraffin-embedded sections were deparaffinized as above prior to treatment with the Naphthol AS-D Chloroacetate Esterase staining kit (Sigma). Sections were treated for 15 min to visualize mast cells or for 1 h to visualize neutrophils as previously described (29).

Quantitative measures of inflammation.

Mast cells from 100 sequential crypts were counted at a magnification of ×400. Goblet cells were enumerated from 10 random high-powered fields spanning muscularis mucosa to surface epithelium with H&E-stained sections. Mucosal and submucosal thicknesses were measured at six evenly spaced points per section for each experimental animal. Mucosal thickness was defined as the distance from the surface epithelium to the inner edge of the muscularis mucosa. Submucosa thickness was defined as the distance from muscularis mucosa to the muscularis externa. Averages for each mouse were compared.

Statistical analysis.

Total pathological scores were compared using Mann-Whitney U and Kruskall-Wallis nonparametric tests. Bacterial load was compared using analysis of variance and Tukey's multiple comparison posttests. All analyses were performed using Graphpad Prism version 3.0.

RESULTS

S. enterica serovar Typhimurium elicits intestinal inflammation that is most pronounced in the cecum of streptomycin-pretreated mice.

To assess induction of intestinal inflammation in response to S. enterica serovar Typhimurium, we infected Nramp1S C57BL/6 mice orally with 3 × 108 WT S. enterica serovar Typhimurium SL1344 24 h after oral gavage with 20 mg streptomycin. We observed extensive inflammatory changes in the large bowels of WT Salmonella-infected mice similar to those reported previously (1), as well as bacterial translocation to liver and spleen and ultimately fatal systemic disease (Fig. (Fig.1;1; data not shown). Common features in the intestine following Salmonella infection included neutrophilic infiltratation into the intestinal lumen, surface epithelial erosion and/or desquamation, inflammatory infiltratation into the lamina propria and submucosa, crypt abscesses, and submucosal edema. These features were absent from control mice given LB broth alone. Epithelial proliferation in the mucosa as assessed by immunohistochemistry with antiproliferating cell nuclear antigen (data not shown) was increased, resulting in increased mucosal thickness (Fig. (Fig.1F;1F; Table Table1).1). Submucosal edema was also evident (Fig. (Fig.1F;1F; Table Table1).1). Inflammatory features including mast cell recruitment and loss of goblet cells were common at 48 h in infected mice (Table (Table1)1) but absent from controls. Pathological changes were also uncommon in mice infected with WT S. enterica serovar Typhimurium but not pretreated with streptomycin (data not shown). Bacterial translocation to the liver and spleen occurred as early as 6 h postinfection, and bacterial colonization of these sites occurred consistently by 24 h.

FIG. 1.
Salmonella enterica serovar Typhimurium-elicited enterocolitis is most severe in the ceca of wild-type infected mice and is partially attenuated in the absence of a SPI2 TTSS. Streptomycin-treated C57Bl/6 mice were infected with 3 × 108 wild-type, ...
TABLE 1.
Inflammatory pathology in the ceca of WT, SPI1, and SPI2 mutant S. typhimurium serovar Typhimurium-infected mice

At 48 h postinfection, intestinal inflammation was maximal (Fig. (Fig.1;1; data not shown). Consistent with previous studies (1), we observed that WT S. enterica serovar Typhimurium-elicited intestinal inflammation at 48 h was most severe in the cecum, with less severe inflammation in the colon and little or no inflammatory change in the ileum (Fig. 1A to C). We focused subsequent comparisons of intestinal pathology on the ceca at this time point, due to the consistent severity of histopathology.

SPI2 contributes to cecal inflammation in S. enterica serovar Typhimurium infection.

To assess the role of SPI2 in intestinal disease, we compared intestinal pathology at 48 h in mice infected with WT S. enterica serovar Typhimurium or bacterial strains lacking a functional SPI1 or SPI2 TTSS. In agreement with previous studies, we observed that a functional SPI1 TTSS is essential to elicit intestinal inflammation (1, 11). Significant histopathological changes were absent from all SPI1-infected mice in all three tissues examined (Fig. 1 A to C), and histopathology scores were statistically indistinguishable from uninfected mice (data not shown). Mucosal hypertrophy and submucosal edema were absent from SPI1-infected mice, and mast cell and goblet cell numbers were not different than uninfected controls (Table (Table1;1; data not shown).

Intestinal inflammation in the absence of SPI2 type III secretion was also significantly attenuated compared to WT (P < 0.0001; Fig. Fig.11 and Table Table1).1). In all three tissues assessed, intestinal pathology was intermediate between SPI1- and WT-infected mice (Fig. (Fig.1;1; Table Table1).1). The intermediate phenotype associated with SPI2 mutant infection was not due to the absence of one specific pathological feature but rather reflected the diffuse attenuation of the inflammatory phenotype in all measured parameters (Fig. (Fig.1A;1A; Table Table1).1). This intermediate pathology was not due to differences in bacterial content of the tissues examined, as bacterial loads of all strains were comparable at the time point assessed (Table (Table1),1), suggesting that SPI2 is actively involved in the induction of colitis and typhlitis.

S. enterica serovar Typhimurium in the ceca of infected mice is predominantly luminal, and bacterial presence in the mucosa coincides with severe mucosal inflammation.

To assess whether intestinal inflammation was attenuated in SPI2 mutants due to changes in the distribution of the bacteria within the gut, we assessed bacterial localization by immunohistochemistry. Twenty-four hours after streptomycin administration, mice were infected with WT, SPI1, or SPI2 S. enterica serovar Typhimurium as described above. Tissues were harvested 48 h after infection and stained for Salmonella LPS, actin, and nuclei. As observed previously, mice infected with SPI1, SPI2, and WT Salmonella displayed no, moderate, or severe inflammatory pathology, respectively (Fig. 2D, H, and L). Bacterial staining was predominantly confined to the lumen of infected tissues in mice infected with all bacterial strains (Fig. (Fig.2,2, insets). Bacteria that were in close association with the epithelium and mucosa were present in all infected mice. However, infiltration of bacteria into the deep mucosa was confined to areas in which there was moderate or severe inflammation (Fig. 2H and L). Severe inflammation was common in the absence of infiltrating bacteria, suggesting that bacterial penetration deep into the mucosa was a consequence rather than a cause of inflammation. The number of infiltrating bacteria of each strain tested correlated directly with the overall severity of intestinal pathology. Intracellular bacteria were not observed in any infected tissues at this time point.

FIG. 2.
S. enterica serovar Typhimurium in the ceca of infected mice is primarily extracellular and infiltrates the mucosa as inflammation progresses. Bacterial localization of SPI1 (A to D), SPI2 (E to H), and WT (I to L) S. enterica serovar Typhimurium 48 h ...

SPI2 mutants induce ICAM-1 expression and neutrophil recruitment less strongly than WT S. enterica serovar Typhimurium.

Since SPI2 bacteria were able to associate with the intestinal epithelium, we sought to determine whether the attenuation of typhlitis was due a decreased ability to induce leukocyte recruitment. To do this, mice were treated with streptomycin and infected as before; after 48 h, ceca were cryosectioned and stained for the CD18-β2 integrin receptor ICAM-1. In response to infection with WT S. enterica serovar Typhimurium, we noted significant ICAM-1 expression in the mucosa and submucosal vasculature, as was previously reported (1). Expression of ICAM-1 was absent from SPI1-infected tissues, confirming that SPI1 activity is essential to induce an inflammatory response in infected intestines. Epithelial cells maintained some ICAM-1 expression in response to SPI2-deficient Salmonella, although it was less intense and less extensive than that induced by WT bacteria (Fig. 3D to I). To determine if differences in ICAM-1 expression correlated with differences in neutrophil recruitment, we stained sections of infected ceca with a neutrophil detection kit that detects activity of the neutrophil-specific esterase.

FIG. 3.
SPI2 but not SPI1 mutant S. enterica serovar Typhimurium induces ICAM-1 and neutrophil recruitment in the intestines of streptomycin-treated mice 48 h after infection. Streptomycin-treated mice were infected with SPI1 (A to C), SPI2 (D to F), or WT (G ...

Rare neutrophils were present in all sections but were abundant only in WT-infected ceca (Fig. (Fig.4).4). Occasional small aggregates of neutrophils were present in SPI2-infected ceca, but large aggregates were not observed (Fig. (Fig.4B).4B). Infection with WT Salmonella, however, resulted in numerous large aggregates and greatly increased single cells in the mucosa, lamina propria, and submucosa (Fig. (Fig.4C4C).

FIG. 4.
Neutrophil infiltration is markedly reduced in S. enterica serovar Typhimurium-induced typhlitis in the absence of SPI2. Forty-eight hours after infection with SPI1 mutant (A), SPI2 mutant (B), or WT (C) S. enterica serovar Typhimurium, ceca were retrieved, ...

SPI2 mutant S. enterica serovar Typhimurium causes transient moderate infectious colitis in streptomycin-treated mice.

To test whether the absence of SPI2 type III secretion influences the course of S. enterica serovar Typhimurium-induced disease, we orally infected susceptible mice and examined bacterial loads and histopathological changes at 2 and 5 days after infection. A smaller dose (3 × 106) of bacteria was used in these experiments, as WT infection with 108 bacteria was fatal before 5 days. Unlike mice infected with WT bacteria, mice infected with SPI2 mutants showed no infection-associated morbidity such as ruffling of fur, wasting, or splenomegaly (Table (Table2;2; data not shown). We also observed a dramatic attenuation of intestinal pathology in mice infected with SPI2 mutants compared to WT bacteria. Mice infected with WT S. enterica serovar Typhimurium continued to have significant and extensive typhlitis, whereas mice infected with SPI2 mutant bacteria had almost completely recovered from the infection and showed no gross abnormalities and little or no histopathological evidence of active disease (Fig. (Fig.5;5; Table Table22).

FIG. 5.
Cecal inflammation caused by S. enterica serovar Typhimurium is controlled by day 5 in the absence of a functional SPI2 TTSS in streptomycin-treated mice. (A to C) Histopathology formalin-fixed, paraffin-embedded sections of ceca were stained with hemotoxylin ...
TABLE 2.
Systemic and intestinal pathology at 5 days in WT and SPI2 mutant in serovar Typhimurium-infected mice

In addition to significant differences in bacterial load by day 5, WT and SPI2 mutant bacteria were distributed differently by day 5 of infection. While WT bacteria were seen invading tissues extensively by day 5 (Fig. (Fig.5D),5D), SPI2 mutant bacteria were exclusively luminal and no mucosal invasion was observed (Fig. (Fig.5E5E).

At 2 days postinfection, bacterial burden in the colon after WT or SPI2 mutant infection was similar. By 5 days, the number of SPI2 mutant Salmonella organisms was significantly decreased while WT Salmonella persisted (Fig. (Fig.6A).6A). Bacterial burden in the liver and spleen had also decreased significantly by this time as expected (Fig. 6B and C). These data demonstrate that SPI2 is essential for persistent intestinal disease in S. enterica serovar Typhimurium-infected mice.

FIG. 6.
Bacterial persistence in the intestines, liver, and spleen is significantly diminished between 2 and 5 days after oral SPI2 mutant, but not WT S. enterica serovar Typhimurium infection. Mice were infected with 2 × 106 bacteria orally 24 h after ...

DISCUSSION

The data presented here are the first demonstration that SPI2 is necessary for complete virulence in a model that recapitulates S. enterica serovar Typhimurium-elicited human colitis. Furthermore, our data suggest that SPI2 is necessary for complete intestinal virulence during the bacterium-epithelium interaction and demonstrates that intestinal inflammation is resolved in SPI2 mutant but not WT S. enterica serovar Typhimurium-infected mice.

Although there are differences in the overall presentation of murine and human S. enterica serovar Typhimurium-elicited intestinal disease, including the absence of diarrhea and the development of systemic typhoid-like infection in mice, this model has numerous advantages over existing models of Salmonella enterocolitis, including low cost, availability of mice, and reproducibility. Like human intestinal disease induced by S. enterica serovar Typhimurium, murine inflammation is predominantly colitic with little or no ileal inflammation (4, 17). In contrast, although colitis is observed in cows upon S. enterica serovar Typhimurium infection, previously published models of S. enterica serovar Typhimurium pathogenesis in cows have focused primarily on inflammatory disease of the ileum. This focus may have arisen from substantial evidence that bacterial invasion in S. enterica serovar Typhimurium infection occurs in the M cells of ileal Peyer's patches, a behavior required for systemic virulence (14, 19). Although this may represent an important pathogenic mechanism in murine typhoid, it has not been demonstrated that intestinal invasion per se is essential for intestinal disease. Rather, it has been demonstrated that ileal inflammation requires invasion-associated virulence genes (27, 28) and that intestinal invasion in inoculated ilea occurs in an SPI1-dependent manner (3, 9). It is in this paradigm that conflicting evidence for the involvement of SPI2 in intestinal disease has arisen (3, 7). By exploiting the murine S. enterica serovar Typhimurium colitis model, we have demonstrated for the first time that SPI2 is necessary for complete virulence in a model representative of human disease and that SPI2 is necessary for the persistence of infection in murine large intestines.

The SPI2 virulence system is important in a range of bacterial adaptations to the host, including vacuolar remodeling, intracellular survival, and resistance to the host immune response. Although survival of many host defenses is SPI2 dependent, it is not clear whether this role of SPI2 is important in inflammation of the large bowel. The SPI2 dependence of intracellular survival in epithelial cells has been demonstrated in vitro (18), and this may be important in intestinal disease; however, we observed inflammation in this model at a time point at which the vast majority of bacteria were luminal, suggesting that extracellular bacteria may be responsible for intestinal inflammation. Others have shown that the attenuation of intestinal virulence in SPI2 mutant-infected bovine ilea was not accompanied by a decrease in the number of intracellular bacteria (3), and only a small proportion of total intestinal bacterial burden in infected human intestines were intracellular (12).

While it is not clear what role SPI2 plays in intestinal inflammation, our data imply that SPI2 is important for the induction of inflammation at the epithelial interface, rather than in deeper tissues, as ICAM-1 expression, a marker of epithelial inflammation and an inducer of neutrophil recruitment, was diminished in SPI2 mutant infections. The uniform intermediacy of the inflammatory phenotype associated with SPI2 mutant infection suggests a decrease in the dose of inflammatory stimulus in the gut rather than the complete loss of a single virulence strategy. Although bacterial numbers are similar in the entire gut, proinflammatory stimuli such as flagellin may be compartmentalized differently within mice infected with various mutants. Flagellin is an important S. enterica serovar Typhimurium inflammatory stimulus (30). Salmonella isolates lacking intact flagella are attenuated in this murine colitis model (24), and it has recently been demonstrated that SPI2-mediated vesicular transport is necessary for the transcytosis of flagellin across polarized epithelia (16). The attenuation of cecal inflammation in the absence of SPI2 may be a result of the decreased SPI2-dependent delivery of proinflammatory flagellin to the basolateral epithelium.

In addition to the attenuation of acute cecal inflammation, we have demonstrated that SPI2 mutant S. enterica serovar Typhimurium infection is resolved in mice. This is dramatically different from the course of WT infection, in which bacterial colonization of intestinal and systemic sites is maintained over time and intestinal disease continues unresolved, ultimately resulting in fatal infection. Resistance to antimicrobial defenses such as complement and evasion of the phagocyte NADPH oxidase complex within phagocytes is SPI2 dependent (13, 26). While loss of the resistance to soluble antimicrobial defenses may decrease the ability of SPI2-deficient bacteria within the gut to persist while extracellular, failure to survive phagocytic killing may diminish the survival of bacteria within phagocytic cells that have migrated into the intestinal lumen. It is possible, therefore, that SPI2 is necessary for persistence within the intestinal environment itself, without the need for extensive cellular invasion.

We have clearly demonstrated that SPI2 is essential for complete virulence in the large intestines of infected mice. These results parallel the recently published findings of Hapfelmeier et al. (11a). It is important now to carefully consider the dichotomous roles of SPI1 and SPI2 in the intestinal and/or systemic paradigm of S. enterica serovar Typhimurium infection.

Acknowledgments

We thank Wanyin Deng, Claudia Lupp, Brian Coombes, and other members of the Finlay lab for helpful discussions and comments. Microscopy facilities were provided with the assistance of Elizabeth Slow at the Centre for Molecular Medicine and Therapeutics in Vancouver. Histology services were provided by WAXIT, Inc., Vancouver.

B.A.C. is supported by the Canadian Institutes for Health Research and the Michael Smith Foundation for Health Research. Funding for this work was provided by the CIHR and the Howard Hughes Medical Institute. B.B.F. is a CIHR Distinguished Investigator, an HHMI International Research Scholar, and the University of British Columbia Peter Wall Distinguished Professor.

Notes

Editor: D. L. Burns

REFERENCES

1. Barthel, M., S. Hapfelmeier, L. Quintanilla-Martinez, M. Kremer, M. Rohde, M. Hogardt, K. Pfeffer, H. Russmann, and W. D. Hardt. 2003. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Infect. Immun. 71:2839-2858. [PMC free article] [PubMed]
2. Baumler, A. J., R. M. Tsolis, T. A. Ficht, and L. G. Adams 1998. Evolution of host adaptation in Salmonella enterica. Infect. Immun. 66:4579-4587. [PMC free article] [PubMed]
3. Bispham, J., B. N. Tripathi, P. R. Watson, and T. S. Wallis. 2001. Salmonella pathogenicity island 2 influences both systemic salmonellosis and Salmonella-induced enteritis in calves. Infect. Immun. 69:367-377. [PMC free article] [PubMed]
4. Boyd, J. F. 1985. Pathology of the alimentary tract in Salmonella typhimurium food poisoning. Gut 26:935-944. [PMC free article] [PubMed]
5. Brumell, J. H., C. M. Rosenberger, G. T. Gotto, S. L. Marcus, and B. B. Finlay. 2001. SifA permits survival and replication of Salmonella typhimurium in murine macrophages. Cell. Microbiol. 3:75-84. [PubMed]
6. Dore, K., J. Buxton, B. Henry, F. Pollari, D. Middleton, M. Fyfe, R. Ahmed, P. Michel, A. King, C. Tinga, and J. B. Wilson. 2004. Risk factors for Salmonella typhimurium DT104 and non-DT104 infection: a Canadian multi-provincial case-control study. Epidemiol. Infect. 132:485-493. [PMC free article] [PubMed]
7. Everest, P., J. Ketley, S. Hardy, G. Douce, S. Khan, J. Shea, D. Holden, D. Maskell, and G. Dougan. 1999. Evaluation of Salmonella typhimurium mutants in a model of experimental gastroenteritis. Infect. Immun. 67:2815-2821. [PMC free article] [PubMed]
8. Fields, P. I., R. V. Swanson, C. G. Haidaris, and F. Heffron. 1986. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc. Natl. Acad. Sci. USA 83:5189-5193. [PMC free article] [PubMed]
9. Frost, A. J., A. P. Bland, and T. S. Wallis. 1997. The early dynamic response of the calf ileal epithelium to Salmonella typhimurium. Vet. Pathol. 34:369-386. [PubMed]
10. Galan, J. E. 1996. Molecular genetic bases of Salmonella entry into host cells. Mol. Microbiol. 20:263-271. [PubMed]
11. Hapfelmeier, S., K. Ehrbar, B. Stecher, M. Barthel, M. Kremer, and W.-D. Hardt. 2004. 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. Infect. Immun. 72:795-809. [PMC free article] [PubMed]
11a. Hapfelmeier, S., B. Stecher, M. Barthel, M. Kremer, A. J. Muller, M. Heikenwalder, T. Stallmach, M. Hensel, K. Pfeffer, S. Akira, and W. D. Hardt. 2005. The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms. J. Immunol. 174:1675-1685. [PubMed]
12. Haque, A., F. Bowe, R. J. Fitzhenry, G. Frankel, M. Thomson, R. Heuschkel, S. Murch, M. P. Stevens, T. S. Wallis, A. D. Phillips, and G. Dougan. 2004. Early interactions of Salmonella enterica serovar typhimurium with human small intestinal epithelial explants. Gut 53:1424-1430. [PMC free article] [PubMed]
13. Hensel, M., J. E. Shea, B. Raupach, D. Monack, S. Falkow, C. Gleeson, T. Kubo, and D. W. Holden. 1997. Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella pathogenicity island 2. Mol. Microbiol. 24:155-167. [PubMed]
14. Jones, B. D., N. Ghori, and S. Falkow. 1994. Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer's patches. J. Exp. Med. 180:15-23. [PMC free article] [PubMed]
15. Leung, K. Y., and B. B. Finlay. 1991. Intracellular replication is essential for the virulence of Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 88:11470-11474. [PMC free article] [PubMed]
16. Lyons, S., L. Wang, J. E. Casanova, S. V. Sitaraman, D. Merlin, and A. T. Gewirtz. 2004. Salmonella typhimurium transcytoses flagellin via an SPI2-mediated vesicular transport pathway. J. Cell Sci. 117:5771-5780. [PubMed]
17. Mcgovern, V. J., and L. J. Slavutin. 1979. Pathology of Salmonella colitis. Am. J. Surg. Pathol. 3:483-490. [PubMed]
18. Ochman, H., F. C. Soncini, F. Solomon, and E. A. Groisman. 1996. Identification of a pathogenicity island required for Salmonella survival in host cells. Proc. Natl. Acad. Sci. USA 93:7800-7804. [PMC free article] [PubMed]
19. Penheiter, K. L., N. Mathur, D. Giles, T. Fahlen, and B. D. Jones. 1997. Non-invasive Salmonella typhimurium mutants are avirulent because of an inability to enter and destroy M cells of ileal Peyer's patches. Mol. Microbiol. 24:697-709. [PubMed]
20. Que, J. U., S. W. Casey, and D. J. Hentges. 1986. Factors responsible for increased susceptibility of mice to intestinal colonization after treatment with streptomycin. Infect. Immun. 53:116-123. [PMC free article] [PubMed]
21. Que, J. U., and D. J. Hentges. 1985. Effect of streptomycin administration on colonization resistance to Salmonella typhimurium in mice. Infect. Immun. 48:169-174. [PMC free article] [PubMed]
22. Santos, R. L., S. Zhang, R. M. Tsolis, R. A. Kingsley, L. G. Adams, and A. J. Baumler. 2001. Animal models of Salmonella infections: enteritis versus typhoid fever. Microbes Infect. 3:1335-1344. [PubMed]
23. Shea, J. E., M. Hensel, C. Gleeson, and D. W. Holden. 1996. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 93:2593-2597. [PMC free article] [PubMed]
24. Stecher, B., S. Hapfelmeier, C. Muller, M. Kremer, T. Stallmach, and W. D. Hardt. 2004. Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice. Infect. Immun. 72:4138-4150. [PMC free article] [PubMed]
25. Tsolis, R. M., L. G. Adams, T. A. Ficht, and A. J. Baumler. 1999. Contribution of Salmonella typhimurium virulence factors to diarrheal disease in calves. Infect. Immun. 67:4879-4885. [PMC free article] [PubMed]
26. Vazquez-Torres, A., Xu, Y. S., J. Jones-Carson, D. W. Holden, S. M. Lucia, M. C. Dinauer, P. Mastroeni, and F. C. Fang. 2000. Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase. Science 287:1655-1658. [PubMed]
27. Wallis, T. S., and E. E. Galyov. 2000. Molecular basis of Salmonella-induced enteritis. Mol. Microbiol. 36:997-1005. [PubMed]
28. Watson, P. R., E. E. Galyov, S. M. Paulin, P. W. Jones, and T. S. Wallis. 1998. Mutation of invH, but not stn, reduces Salmonella-induced enteritis in cattle. Infect. Immun. 66:1432-1438. [PMC free article] [PubMed]
29. Wyllie, S., P. Seu, F. Q. Gao, P. Gros, and J. A. Goss. 2002. Disruption of the Nramp1 (also known as Slc11a1) gene in Kupffer cells attenuates early-phase, warm ischemia-reperfusion injury in the mouse liver. J. Leukoc. Biol. 72:885-897. [PubMed]
30. Zeng, H., A. Q. Carlson, Y. W. Guo, Yu, Y. M., L. S. Collier-Hyams, J. L. Madara, A. T. Gewirtz, and A. S. Neish. 2003. Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella. J. Immunology 171:3668-3674. [PubMed]

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