Accelerated Type III Secretion System 2-Dependent Enteropathogenesis by a Salmonella enterica Serovar Enteritidis PT4/6 Strain

Bacterial strains and plasmids.The naturally streptomycin-resistant wild-type serovar Typhimurium strain SL1344 (19) and a streptomycin-resistant variant of serovar Enteritidis strain P125109 (32) have been described previously. An isogenic TTSS-1⁻ mutant of SL1344 (SB566; invC::aphT) has been described previously (14, 28). An isogenic TTSS-1⁻ mutant of P125109 (M1511; invC::aphT), isogenic TTSS-2⁻ mutants of SL1344 (M1516) and P125109 (M1512), and TTSS-1⁻ TTSS-2⁻ double mutants of SL1344 (M1517) and P125109 (M1513) were generated by P22 transduction of the invC::aphT and/or ssaV::cat allele of SB566 and HH110 (ATCC 14028; ssaV::cat) (14, 28). M1511 and SB566 were complemented by P22 transduction of the chromosomal wild-type invC region of M1300 (harboring a tetracycline resistance-tagged sipA_M45 allele [27]), yielding M2700 and M2701. The strains are listed in Table 1. All strains were verified by PCR (presence or absence of mutation) and serotyping (serovar Typhimurium versus serovar Enteritidis).

pM973 expressing green fluorescent protein (GFP) under the control of a TTSS-2 promoter (i.e., after host tissue invasion) was described recently (18). pDsRed expresses DsRed from the lac promoter. In the gut lumen, DsRed fluorescence is very weak. Once bacteria have entered the gut tissue, DsRed fluorescence is intense and allows enumeration of the bacteria by fluorescence microscopy, as described previously (17).

Bacteria were grown for 12 h at 37°C in Luria-Bertani broth (LB; with 0.3 M NaCl), diluted 1:20 in fresh medium, and subcultured for 4 h under mild aeration. Bacteria were washed in ice-cold phosphate-buffered saline (PBS). For mouse infections, we used 5 × 10⁷ CFU suspended in 50 μl of cold PBS, and for tissue culture infections, dilutions in PBS were used, as indicated.

Mice and infection experiments.Infection experiments were carried out in individually ventilated cages as described previously (29). All mice were specific pathogen free (SPF) and were bred at the Rodent Center HCI, ETH Zürich. This included C57BL/6 mice, B6.129P-CX3CR1^tm1Litt/J mice (20) (referred to as CX3CR1^gfp/+ mice; C57BL/6 background), MyD88^−/− mice (C57BL/6 background) (1), MyD88^+/+ littermate controls, and DTR^tg heterozygous transgenic mice [mouse line B6.FVB-Tg (Itgax-DTR/EGFP)57Lan/J] (21). The last group of mice and their nontransgenic littermate controls were bred by crossing DTR^tg × C57BL/6 mice. For depletion of DCs from DTR^tg mice, diphtheria toxin (DTX) was injected intraperitoneally (100 ng/25 g body weight) (21) 1 day before and 1 day after infection. Depletion efficiency was analyzed by fluorescence-activated cell sorting (CD45⁺ gate; CD11c⁺ CD11b⁺ cells) (17) and immunohistology (anti-CD11c staining) (see below) of the cecal mucosa and showed ≥80% depletion of CD11c⁺ cells (not shown).

Mice were pretreated intragastrically with 20 mg of streptomycin and infected 24 h later with 5 × 10⁷ CFU (by gavage) of the corresponding bacterial strain. Bacterial loads in the cecal lumen, mesenteric lymph nodes (MLNs), liver, and spleen were determined by plating homogenates on MacConkey agar plates as previously described (5). Animal experiments were approved by Swiss cantonal authorities and performed according to legal requirements.

Histopathological evaluation.Cryosections of cecal mucosa (5 μm) were stained with hematoxylin and eosin, and the degree of inflammation was quantified by a pathologist as described previously (29).

(i) Submucosal edema.Submucosal edema was deduced from the extension of the submucosa and scored by morphometric analysis according to the formula % SE = (b − a)/c, where % SE is the percentage of submucosal edema, a is the area enclosed by the mucosa (mucosa and intestinal lumen), b is the area enclosed by the borderline between the submucosa and the tunica muscularis (submucosa, mucosa, and intestinal lumen), and c is the area enclosed by the outer edge of the tunica muscularis (tunica muscularis, submucosa, mucosa, and lumen; area of the whole cecal cross section). Submucosal edema was scored as follows: 0, no pathological changes; 1, detectable edema (% SE < 10%); 2, moderate edema (% SE = 10 to 40%); and 3, profound edema (% SE ≥ 40%).

(ii) PMN infiltration into the lamina propria.Polymorphonuclear granulocytes (PMN) in the lamina propria were enumerated in 10 high-power fields (magnification, ×400; field diameter, 420 μm), and the average number of PMN/high-power field was calculated. The scores were as follows: 0, fewer than 5 PMN per high-power field; 1, 5 to 20 PMN per high-power field; 2, 21 to 60 PMN per high-power field; 3, 61 to 100 PMN per high-power field; and 4, more than 100 PMN per high-power field.

(iii) Goblet cells.The average number of goblet cells per high-power field (magnification, ×400) was calculated from 10 different regions of the cecal epithelium and scored as follows: 0, more than 28 goblet cells per high-power field (magnification, ×400) (in the ceca of normal SPF mice, we observed an average of 6.4 crypts per high-power field, and the average crypt consisted of 35 to 42 epithelial cells, 25 to 35% of which were differentiated into goblet cells); 1, 11 to 28 goblet cells per high-power field; 2, 1 to 10 goblet cells per high-power field; and 3, less than 1 goblet cell per high-power field.

(iv) Epithelial integrity.Epithelial integrity was scored as follows: 0, no pathological changes detectable in 10 high-power fields (magnification, ×400); 1, epithelial desquamation; 2, erosion of the epithelial surface (gaps of 1 to 10 epithelial cells per lesion); and 3, epithelial ulceration (gaps of >10 epithelial cells per lesion) (at this stage, there is generally granulation tissue below the epithelium).

The combined pathological score for each tissue sample was determined as the sum of these averaged scores, as follows: 0, intestine intact without any signs of inflammation; 1 or 2, minimal signs of inflammation (this was frequently found in the ceca of SPF mice); 3 or 4, slight inflammation; 5 to 8, moderate inflammation; and 9 to 13, profound inflammation.

Immunohistochemistry.Five-micrometer-thick acetone-fixed cryosections were stained as described previously (17), using rat anti-CD11b (M1/70; BMA Biomedicals AG Augst, Switzerland), rat anti-Ly6G (clone RB6-8C5; BD Biosciences Pharmingen), Armenian hamster anti-CD11c monoclonal antibody N418 (HB-224; American Type Culture Collection), or rat anti-CD8 (YTS169) (9). Goat anti-rat immunoglobulin (Ig; Caltag Laboratories, Burlingame, CA) or goat anti-Armenian hamster Ig (Jackson ImmunoResearch Europe Ltd., Soham, United Kingdom) served as a secondary antibody, and horseradish peroxidase-coupled donkey anti-goat Ig (with 2% normal mouse serum; Jackson ImmunoResearch Europe Ltd., Soham, United Kingdom) served as a tertiary reagent. Slides were developed with amino-ethylcarbazol and counterstained with hemalum.

Analysis of serovar Typhimurium colonization of the lamina propria.Cecal tissue was incubated overnight in PBS with 4% paraformaldehyde at 4°C and then for 8 h in PBS with 20% sucrose at 4°C and was snap frozen in O.C.T. compound (Sakura, Torrance, CA). Twenty-micrometer-thick cryosections were air dried and blocked (PBS with 10% goat serum). GFP-expressing Salmonella spp. harboring pM973 were enumerated in the mucosa as described previously (18). Briefly, sections were stained with Armenian hamster anti-CD54 antibody (lamina propria staining) (clone 3E2; Becton Dickinson), A647-phalloidin (staining of brush border and cortical actin of individual gut cells; Fluoprobes), and DAPI (4′,6-diamidino-2-phenylindole; Sigma Aldrich). Three to nine sections per cecum were analyzed. To analyze the localization of Salmonella spp. harboring pDsRed in CX3CR1^GFP/+-transgenic mice, six nonserial sections (stained with Cy3-conjugated goat anti-Armenian hamster Ig [Jackson ImmunoResearch Laboratories] and DAPI) per mouse were screened by fluorescence microscopy as described previously (17).

Modified gentamicin protection assay using automated image processing and analysis.HeLa cells were grown in clear-bottomed 96-well plates (Greiner). Salmonella spp. carried the plasmid pM975, encoding GFP under the control of a SPI-2 promoter (26). Strains were cultured for 12 h overnight at 37°C with mild aeration, and 150 μl was subcultured in 3 ml of LB medium with 0.3 M NaCl for an additional 4 h. Bacteria were serially diluted in ice-cold Dulbecco's modified Eagle's medium. Cells were infected for 20 min and incubated with gentamicin (400 μg/ml) to kill all bacteria that had remained outside host cells. After 4 h, cells were fixed with paraformaldehyde and stained with DAPI. Nine images per well (approximately 6,000 cells) were taken in the GFP and DAPI channels, using a CellWorx automated microscope (API). Image processing was done using CellProfiler (7) and customized Matlab scripts, as described previously (26). Any cell harboring GFP-expressing bacteria was considered “infected.”

Statistical analysis.The exact Mann-Whitney U test was used to perform group comparisons, using SPSS software, version 14.0.

To perform statistical analysis, minimal detectable bacterial colonization levels (CFU) were set to 10 CFU/MLN, 20 CFU/spleen, 60 CFU/liver, and 150 CFU/cecum in cases where no bacteria were detected by plating. P values of <0.05 (two-tailed test) were considered statistically significant.

Time course of acute colitis triggered by serovar Enteritidis and Typhimurium mutants lacking functional TTSS-1 and/or TTSS-2.Our previous work demonstrated that wild-type strains of serovars Typhimurium and Enteritidis elicit pronounced acute colitis in streptomycin-pretreated mice. For this study, we extended this analysis by comparing the virulence of isogenic strains of serovars Enteritidis (P125109) and Typhimurium (SL1344) lacking functional TTSS-1 and/or TTSS-2 (termed TTSS-1⁻ and TTSS-2⁻ strains hereafter) (Table 1).

Streptomycin-pretreated C57BL/6 mice were infected (5 × 10⁷ CFU by gavage) with the indicated bacterial strain and analyzed at 1, 2, 3, or 4 days p.i., as indicated (Fig. 2). Infections with the corresponding wild-type strains and TTSS-1⁻ TTSS-2⁻ double mutants served as positive and negative controls. We analyzed bacterial colonization of the cecal lumen, the MLNs, and the spleen and scored the inflammation of the cecal mucosa as described in Materials and Methods.

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FIG. 2.

Time course of acute colitis triggered by isogenic mutants of serovars Enteritidis and Typhimurium. Streptomycin-pretreated C57BL/6 mice (n = 5 to 14 animals per group) were infected with the indicated serovar Enteritidis (open circles) or serovar Typhimurium (filled circles) strains. Animals were sacrificed on days 1 to 4 p.i., and we determined the pathology (top row) and the bacterial loads in the cecal lumen (second row), the MLNs (third row), and the spleen (bottom row). The origin of the y axis in every graph represents the detection limit. *, statistically significant difference (P < 0.05). WT, wild type; re-compl., recomplemented strain.

In line with our previous data, both wild-type strains (P125109 and SL1344) efficiently colonized the intestinal lumen, MLNs, and spleen at days 1 to 2 p.i. In line with earlier data (30), the gut colonization density in wild-type-infected mice dropped at day 2 p.i. and increased again in the subsequent days. This phenomenon is not well understood. The reduced gut colonization levels were more pronounced for wild-type Salmonella spp. than for attenuated mutants (this work; also see reference 30) and might be related to higher gut passage rates or (unidentified) acute defenses. Significant mucosal inflammation was detected as early as day 1 p.i., and the intensity of inflammation increased in both groups until day 3 p.i. In the case of the wild-type serovar Enteritidis strain, we observed a slight drop in gut inflammation at day 4 p.i. This might be attributable to the advanced systemic infection (more spleen colonization). Reduced mucosal inflammation has been observed in mice suffering from severe sepsis (S. Hapfelmeier and W.-D. Hardt, unpublished observations) and might be linked to exhaustion of the immune system.

The TTSS-1⁻ TTSS-2⁻ double mutants of both strains failed to colonize the spleen and did not elicit cecal inflammation during the course of the experiment. This demonstrated that TTSS-1 and TTSS-2 are key virulence factors for eliciting enteric disease by both strains. Furthermore, we observed that the density of TTSS-1⁻ TTSS-2⁻ double mutants in the intestinal lumen dropped at days 3 and 4 p.i. This is in line with earlier observations (18, 30) and is attributable to competition with the normal murine gut flora: in the absence of mucosal inflammation, the pathogen can be outcompeted by the regrowing normal gut flora (30).

The TTSS-2 mutants of serovar Typhimurium SL1344 are known to elicit colitis via the “classical” pathway by day 1 p.i. (18). We did not observe significant differences between TTSS-2⁻ mutants of P125109 and SL1344. Thus, the “classical” pathway seems to operate in a similar fashion in both serovars.

Strikingly, the TTSS-1⁻ mutant of P125109 triggered gut inflammation significantly sooner than the TTSS-1⁻ mutant of SL1344 (see cecal pathology at day 1 and 2 p.i. [Fig. 2]). This phenotype could not be explained by different pathogen densities in the gut lumen and was alleviated by recomplementing the chromosomal TTSS-1 mutations in both strains (M2700 and M2701) (Fig. 2). Thus, P125109 may trigger the “alternative” pathway more efficiently than SL1344 does. This would be in line with the slightly increased kinetics of MLN and spleen colonization observed at days 1 and/or 2 p.i. (Fig. 2). Furthermore, we confirmed our findings by immunohistological analysis of cecal tissues from mice infected for 2 days with TTSS-1⁻ mutants of P125109 and SL1344 (Fig. 3A). At this time point, the P125109 TTSS-1⁻ mutant triggered massive infiltration of Ly6G⁺ (PMN marker), CD11b⁺ (macrophage marker), CD11c⁺ (DC marker), and to a lesser extent, CD8⁺ (cytotoxic T-cell marker) cells into the cecal mucosa, while the SL1344 TTSS-1⁻ mutant did not.

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FIG. 3.

Immunohistology and tissue loads verify that the alternative pathway is faster and/or more efficient in the case of serovar Enteritidis P125109. (A) Immunohistology of two typical C57BL/6 mice infected for 2 days with TTSS-1⁻ mutants of serovar Enteritidis P125109 and serovar Typhimurium SL1344. The animals were taken from the experiment shown in Fig. 2. Cryosections of cecal tissue were stained using antibodies directed against Ly6G, CD11b, CD11c, and CD8, as indicated. All stained cell types are typically located in the lamina propria. Bars, 200 μm. Lp, lamina propria; S, submucosa; L, lumen; If, inflammatory focus. (B) Streptomycin-pretreated C57BL/6 mice (n = 5 per group) were infected with serovar Enteritidis P125109 TTSS-1⁻ (pM973; open circles) or serovar Typhimurium SL1344 (pM973; filled circles) for 2 days. The number of bacteria in the lamina propria was analyzed by microscopy as described in Materials and Methods. *, statistically significant difference (P < 0.05).

In conclusion, TTSS-1 and TTSS-2 are key virulence factors for enteropathogenesis of serovar Enteritidis P125109 and serovar Typhimurium SL1344. TTSS-1-mediated effects were comparable between both strains (Fig. 2), while TTSS-2-mediated colitis was triggered more efficiently by serovar Enteritidis P125109 (Fig. 2 and 3A).

Serovar Enteritidis TTSS-1⁻ mutant reaches higher mucosal tissue loads at day 2 p.i.The serovar Typhimurium TTSS-1⁻ strain requires 3 days to reach sufficient gut tissue loads to trigger colitis via the alternative pathway (≈20 bacteria in the lamina propria per tissue section, i.e., ≈10⁶ CFU/g tissue [17]). The earlier onset of colitis suggested that serovar Enteritidis P125109 TTSS-1⁻ may colonize the gut tissue more efficiently. Thus, streptomycin-pretreated C57BL/6 mice were infected with TTSS-1⁻ mutants of serovar Enteritidis P125109 or serovar Typhimurium SL1344 harboring the reporter plasmid pM973 (5 × 10⁷ CFU by gavage) for 2 days. The lamina propria loads in the cecum tissue were analyzed by fluorescence microscopy (see Materials and Methods). Serovar Enteritidis P125109 TTSS-1⁻ had reached tissue densities of 20 to 120 CFU per tissue section, while serovar Typhimurium SL1344 TTSS-1⁻ densities were significantly lower (Fig. 3B). Thus, efficient lamina propria colonization can explain why serovar Enteritidis P125109 TTSS-1⁻ already elicits colitis via the alternative pathway by day 2 p.i.

Equivalent levels of TTSS-1-mediated host cell invasion by serovar Enteritidis P125109 and serovar Typhimurium SL1344.TTSS-1 mediates host cell invasion (25). Therefore, we performed a host cell invasion assay to further analyze the virulence of the serovar Enteritidis and Typhimurium strains. We recently developed a high-throughput format for quantitative analysis of Salmonella invasion into HeLa cells (26). In this assay, cells are infected with serovar Enteritidis and Typhimurium strains harboring a plasmid expressing GFP under the control of a SPI-2 promoter (pM975) (see Materials and Methods), and invading bacteria are detected by automated microscopy by virtue of their GFP expression. Using this assay, we found that both wild-type and TTSS-2⁻ mutant strains invaded with equivalent efficiencies. In contrast, both TTSS-1⁻ mutants and both TTSS-1⁻ TTSS-2⁻ double mutants did not invade (Fig. 4). The invasion defect of the TTSS-1⁻ mutants could be complemented (Fig. 4). In conclusion, TTSS-1-mediated virulence, both in vitro and in vivo, was equivalent between serovar Enteritidis strain P125109 and serovar Typhimurium strain SL1344.

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FIG. 4.

HeLa cell invasion assay confirming that TTSS-1-mediated host invasion is equivalent in serovar Enteritidis P125109 and serovar Typhimurium SL1344. HeLa cells were grown in 96-well plates and infected for 20 min with the indicated strains (at the indicated multiplicity of infection [MOI]) of serovar Enteritidis (open symbols) or serovar Typhimurium (filled symbols) harboring the reporter plasmid pM975. Invasion was detected by virtue of GFP expression from pM975, using automated imaging, image processing, and data processing (see Materials and Methods). This yielded the percentage of infected cells at each MOI tested. Three pieces of data were obtained per strain at each MOI (data shown are averages ± standard deviations). Negative control wells harbored HeLa cells and medium supplemented with gentamicin (400 μg/ml) prior to the addition of Salmonella (MOI, 125). wt, wild type; re-compl., recomplemented strain.

TTSS-1⁻ mutants of serovars Enteritidis and Typhimurium show similar DC dependency for enteropathogenesis via the alternative pathway.The TTSS-1⁻ mutant of serovar Enteritidis P125109 triggered mucosal inflammation with surprisingly fast kinetics (see above). The mechanism explaining this phenomenon remained to be analyzed. Earlier work by us had established that TTSS-1⁻ mutants of serovar Typhimurium SL1344 trigger colitis via the “alternative” pathway and that this pathway strictly requires transepithelial transport facilitated by mucosal DCs and MyD88-dependent signaling by mucosal lymphocytes or leukocytes (17, 18). However, it remained to be shown whether this also holds true for TTSS-1-defective serovar Enteritidis strains. This was addressed using appropriate mouse lines. To test the requirement for mucosal DCs, we employed a transgenic mouse line (DTR^tg) expressing the primate diphtheria toxin receptor under the control of the CD11c promoter (21). In the cecal mucosae of these mice, i.p. injection of diphtheria toxin leads to specific depletion of DCs while leaving most other cell populations undisturbed (17).

Two groups of streptomycin-pretreated mice were infected with the P125109 TTSS-1⁻ mutant (5 × 10⁷ CFU by gavage). The first group of mice carried the DTR transgene, and DCs were depleted by DTX injection (see Materials and Methods). The second group consisted of littermates lacking the transgene. In the absence of DCs, the P125109 TTSS-1⁻ mutant could colonize the intestinal lumen (Fig. 5A), but it failed to trigger mucosal inflammation (Fig. 5B). This was in line with earlier data on equivalent serovar Typhimurium strains (17). Furthermore, spread to the MLNs was significantly impaired in the absence of DCs (Fig. 5A). These data verified that the TTSS-1⁻ mutant of serovar Enteritidis P125109 indeed employed the same “alternative” pathway for eliciting enteropathogenesis as that described earlier for serovar Typhimurium and suggested that it was simply more efficient at doing so.

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FIG. 5.

DC depletion abrogates enteropathogenesis of the TTSS-1⁻ mutant of serovar Enteritidis P125109. Streptomycin-pretreated DTR^tg mice (open circles; n = 8 mice) and wild-type littermates (filled circles; n = 7 mice) were pretreated with streptomycin. DTX was administered to both groups, 1 day before and 1 day after infection with the TTSS-1⁻ mutant of serovar Enteritidis P125109. Mice were sacrificed at day 3 p.i., and cecal pathology (B) and colonization levels in the cecal lumen, the MLNs, the spleen, and the liver (A) were analyzed. *, statistically significant difference (P < 0.05).

DC localization of serovar Enteritidis P125109 TTSS-1⁻.The serovar Typhimurium TTSS-1⁻ mutant localizes in CX3CR1⁺ DCs at day 1 p.i. and switches to CX3CR1⁻ host cells, presumably macrophages, by day 3 p.i. (17). We analyzed whether this also holds true for serovar Enteritidis P125109 TTSS-1⁻ by using CX3CR1^gfp/+ mice (20). These mice express GFP in the cecum lamina propria DCs (17). Streptomycin-pretreated CX3CR1^gfp/+ mice were infected with TTSS-1⁻ mutants of serovar Enteritidis P125109 or serovar Typhimurium SL1344 harboring pDsRed (see Materials and Methods). This plasmid yields bright red fluorescence after bacteria have entered the mucosal tissue (17). By fluorescence microscopy, we found that both strains were localized exclusively in DCs at day 1 p.i. Again, serovar Enteritidis P125109 was present in significantly larger numbers (data not shown). This was in line with the data presented above (Fig. 3B). At day 2 p.i., 90% of the serovar Enteritidis P125109 TTSS-1⁻ bacteria were already located in CX3CR1⁻ host cells. In the case of serovar Typhimurium SL1344, this process took 1 day longer (Fig. 6) (17). The faster relocalization into CX3CR1⁻ host cells provided additional evidence for why serovar Enteritidis P125109 triggers the alternative pathway so efficiently.

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FIG. 6.

DC localization of serovar Enteritidis P125109 TTSS-1⁻. Streptomycin-pretreated CX3CR1^gfp/+ mice (n = 5) were pretreated with streptomycin and infected with the indicated Salmonella strains harboring pDsRed. At day 1 or 2 p.i., localization in CX3CR1⁺ DCs or CX3CR1⁻ cells of the lamina propria (Lp) was analyzed by fluorescence microscopy (see Materials and Methods) (six cecum tissue sections per mouse).

The TTSS-1⁻ mutant of serovar Enteritidis P125109 requires MyD88 to trigger colitis.A second hallmark of the “alternative” pathway is the requirement for MyD88. MyD88 is a central adapter protein that is shared by the interleukin-1 receptor and many Toll-like receptors of the innate immune system (2). Previous work on TTSS-1⁻ mutants of serovar Typhimurium established that MyD88 signaling is required for induction of enterocolitis via the alternative pathway, while it is dispensable for the “classical” pathway (18). Here we analyzed whether the P125109 TTSS-1⁻ mutant also requires MyD88 signaling to trigger mucosal inflammation.

Streptomycin-pretreated MyD88^−/− mice (C57BL/6 background) and their wild-type (MyD88^+/+) littermates were infected for 3 days with wild-type P125109 or the isogenic mutants lacking TTSS-1 and/or TTSS-2 function (5 × 10⁷ CFU by gavage). Cecal pathology and colonization of the cecal lumen, the MLNs, and the spleen were analyzed. In line with our previous findings for serovar Typhimurium, we observed that the wild-type serovar Enteritidis strain P125109 and the TTSS-2⁻ mutant triggered colitis in MyD88^−/− and wild-type mice alike (P > 0.05) (Fig. 7A). In contrast, the TTSS-1⁻ mutant of P125109 triggered colitis in wild-type but not in MyD88^−/− mice. Thus, the TTSS-1⁻ mutant of P125109 triggers colitis in a MyD88-dependent fashion. Therefore, we concluded that it employs the “alternative” pathway, as outlined in Fig. 1. Nevertheless, it did so with faster kinetics than equivalent serovar Typhimurium SL1344 mutants (Fig. 2) (17, 18). We speculate that this strain may express features which are missing from serovar Typhimurium SL1344 (e.g., an additional TTSS-2 effector protein may be present in strain P125109). These hypothetical features might enhance DC-mediated transport or protect serovar Enteritidis P125109 (but not serovar Typhimurium SL1344) from innate immune defenses in the gut mucosa and thereby enhance the kinetics of mucosa colonization and trigger mucosal inflammation via this pathway.

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FIG. 7.

Enteropathogenesis of the TTSS-1⁻ mutant of serovar Enteritidis P125109 is abrogated in MyD88^−/− mice. Streptomycin-pretreated MyD88^−/− mice (empty circles; n = 4 to 8 per group) and wild-type (wt) littermates (filled circles; n = 5 to 7 per group) were pretreated with streptomycin and infected with the indicated serovar Enteritidis P125109 strains. Mice were sacrificed at day 3 p.i., and we analyzed cecal pathology (A) and the bacterial loads in the cecal lumen (B), the spleen (C), and the MLNs (D). The origin of the y axis represents the detection limit. *, statistically significant difference (P < 0.05).