A novel murine infection model for Shiga toxin–producing Escherichia coli

Enterohemorrhagic E. coli (EHEC) is an important subset of Shiga toxin–producing (Stx-producing) E. coli (STEC), pathogens that have been implicated in outbreaks of food-borne illness and can cause intestinal and systemic disease, including severe renal damage. Upon attachment to intestinal epithelium, EHEC generates “attaching and effacing” (AE) lesions characterized by intimate attachment and actin rearrangement upon host cell binding. Stx produced in the gut transverses the intestinal epithelium, causing vascular damage that leads to systemic disease. Models of EHEC infection in conventional mice do not manifest key features of disease, such as AE lesions, intestinal damage, and systemic illness. In order to develop an infection model that better reflects the pathogenesis of this subset of STEC, we constructed an Stx-producing strain of Citrobacter rodentium, a murine AE pathogen that otherwise lacks Stx. Mice infected with Stx-producing C. rodentium developed AE lesions on the intestinal epithelium and Stx-dependent intestinal inflammatory damage. Further, the mice experienced lethal infection characterized by histopathological and functional kidney damage. The development of a murine model that encompasses AE lesion formation and Stx-mediated tissue damage will provide a new platform upon which to identify EHEC alterations of host epithelium that contribute to systemic disease.

Shiga toxin (Stx) is a potent phage-encoded cytotoxin, and Stx-producing Escherichia coli (STEC) have been implicated in serious outbreaks of food-borne disease. For example, an E. coli serotype O104 strain lysogenized with an Stx phage was responsible for a recent large outbreak of diarrheal illness with serious and in some instances lethal systemic complications (1). Enterohemorrhagic E. coli (EHEC) represent a subset of STEC, and EHEC, particularly serotype O157:H7, have been responsible for both sporadic cases and epidemic outbreaks of diarrheal illness in the United States as well as worldwide (2, 3). Human EHEC infection can cause severe local disease, such as hemorrhagic colitis, characterized by damage to the intestinal epithelium, acute abdominal pain, and bloody diarrhea (4–7). EHEC infection may also result in life-threatening systemic disease, most notably hemolytic uremic syndrome (HUS), characterized by the triad of hemolytic anemia, thrombocytopenia, and renal failure (6, 8). In fact, HUS is the leading cause of renal failure in children (9, 10).

A characteristic pathogenic feature of EHEC — one that it shares with enteropathogenic E. coli (EPEC), a cause of infantile diarrhea in the developing world, and Citrobacter rodentium, a natural murine pathogen — is the ability to generate attaching and effacing (AE) lesions (2, 11). These histopathological lesions are characterized by intimate adherence of the bacterium to the host cell, effacement of brush border microvilli, and the formation of filamentous actin–rich pedestals beneath bound bacteria (ref. 12; for review, see ref. 2). The formation of AE lesions requires translocation of effector proteins into the mammalian cell via a type III secretion system (13, 14). A critical type III effector protein is Tir, which localizes in the host plasma membrane and acts as a receptor for the bacterial outer membrane protein intimin, promoting intimate adherence and filamentous actin assembly beneath bound bacteria (15–19). Both Tir and intimin are essential for colonization and virulence in several animal models of EHEC and Citrobacter infection (20–25).

Of the three AE pathogens, only EHEC produces Stx, which is largely responsible for the extensive tissue damage and systemic disease generated by EHEC (6, 26, 27). Given that Stx is encoded by lambdoid prophages, its production is often enhanced upon phage induction (28–30). Stx traverses the intestinal epithelium and targets the endothelium, leading to vascular damage, particularly in the kidney and CNS, tissues that express high levels of the Stx receptor and the glycolipid globotriaosylceramide, or Gb₃ (31–33). Once inside eukaryotic cells, Stx inhibits protein synthesis by inactivating ribosomes. The action of Stx on endothelial cells leads to the induction of proinflammatory cytokines, thrombus formation in the vasculature, and renal damage (34–38). Stx1 and Stx2 are the two major serotypes of Stx, and EHEC that express exclusively Stx2 are associated with a greater risk of HUS (39–41). There are several variants of Stx2, of which Stx2c and Stx2dact are particularly important in human disease (42–44). In particular, Stx2dact is proteolytically activated in the intestinal mucus and is associated with STEC that cause toxigenic illness in humans despite the fact that they lack the ability to generate AE lesions (45, 46).

Injection of Stx into mice results in the induction of proinflammatory cytokines, vascular damage, hemolysis, and renal damage (47–50), but in contrast to piglet and rabbit infection models (23, 51–53), murine oral EHEC infection models do not recapitulate AE lesion formation, extensive mucosal colonization, or intestinal damage (54–56). For example, in conventional mice, Stx-mediated mortality occurs only after administration of large numbers of EHEC, numbers that diminish in the days following inoculation (55, 56). Disruption of the normal flora by antibiotic pretreatment, or its complete elimination by rearing of the animals in a germ-free environment, sensitizes mice to lethal infection after relatively low-dose oral inoculation (57–59). However, only STEC that produce the mucus-activatable allele Stx2dact consistently cause lethal infection of streptomycin-pretreated mice (58, 59). Furthermore, EHEC infection of altered-flora mice does not result in well-documented AE lesions, nor have bacterial genes that promote AE lesion formation been demonstrated to be required for intestinal colonization or disease (57–61). Indeed, a laboratory E. coli K12 strain that overproduces Stx2 but lacks Tir, intimin, and the type III secretion apparatus is capable of lethal infection of streptomycin-pretreated mice (61).

The AE pathogen Citrobacter rodentium has been used extensively as a murine model for EHEC and EPEC colonization (20, 62–65) but does not express Stx. To establish an infection model that reflects both the characteristic mode of intestinal colonization and Stx-mediated disease, we constructed a C. rodentium strain lysogenized by an Stx2dact-producing phage. Administration of C. rodentium (λstx_2dact) to mice by oral gavage recapitulated Tir-dependent AE lesion formation, as well as important aspects of systemic Stx-mediated disease, such as intestinal and renal damage. Thus, C. rodentium (λstx_2dact) provides a model for EHEC colonization and toxin-mediated disease in conventional mice.

Cultured C. rodentium (λstx_2dact), a C. rodentium strain lysogenized with a λ Stx phage, produces mucus-activatable Stx2dact at levels comparable to STEC. In order to develop a murine model of Stx-mediated disease using C. rodentium, we generated C. rodentium strains expressing Stx1 or any of several alleles of Stx2, as well as Stx1/Stx2 hybrids (data not shown; see Methods). Two phages, Φ1720a-02 and the previously identified Φ1720a-01, were derived from EC1720a, a STEC strain originally isolated from ground beef (66). In preliminary screening of these C. rodentium lysogens, only C. rodentium (Φ1720a-02) produced significant amounts of toxin in vitro or caused systemic disease in mice (data not shown; see Figure 1). In order to facilitate the generation of lysogens of Φ1720a-02, the phage was marked with a chloramphenicol resistance cassette (cat, Cm^R) (see Methods). C. rodentium DBS100 (11) (referred to herein as C. rodentium) was lysogenized with Φ1720a-02 Rz::cat to create C. rodentium DBS770 (Φ1720a-02 Rz::cat), which, for simplicity, we herein refer to as C. rodentium (λstx_2dact). In addition, a C. rodentium lysogen of an Stx-deficient control phage, λstx_2dact::kan^R, was also generated (see Methods).

Figure 1

C. rodentium (λstx_2dact) produces high levels of Shiga toxin upon prophage induction at levels comparable to EHEC isolates. Stx2 in supernatants (S) or pellets (P) of untreated or mitomycin C–treated cultures of the indicated strains was measured by capture ELISA, and results are expressed relative to bacterial number (see Methods). ND, not detected. Cr, C. rodentium. Data shown are the averages ± SEM of quadruplicate samples. Data show results of 1 experiment representative of 3 independent experiments.

To test whether C. rodentium (λstx_2dact) was able to produce Stx2 upon in vitro growth, the level of Stx2 was quantified by ELISA. Spontaneous prophage induction is predicted to release toxin into culture supernatants, so supernatants and bacterial pellets were analyzed separately. EDL933, an EHEC O157:H7 strain that harbors both stx₁ and stx₂ (67), produced high levels of Stx2 in both the supernatant and pellet (Figure 1). In contrast, TUV93-0, the stx₁- and stx₂-deficient derivative of EDL933 (68), was completely unable to produce Stx2. EC1720a, which harbors Φ1720a-01 and Φ1720a-02, produced easily detectable levels of Stx2 in both supernatant and pellet, albeit at levels several orders of magnitude lower than those found for EDL933, suggesting that baseline expression and/or prophage induction is lower in this strain. Importantly, C. rodentium (λstx_2dact) produced high levels of Stx2, the vast majority of which was present in the culture supernatant. As predicted, C. rodentium (λstx_2dact::kan^R) did not produce detectable toxin.

Induction of the Stx-producing prophage often promotes high-level production of Stx by EHEC strains (28–30), so each of the above bacterial strains was treated with mitomycin C to induce lysogenic phage. Compared with uninduced samples, cultures of EDL933, EC1720a, and C. rodentium (λstx_2dact) treated with mitomycin C produced several orders of magnitude more Stx2, particularly in the supernatant, consistent with lytic induction of the stx-encoding prophage (Figure 1). Strain TV93-0 and C. rodentium (λstx_2dact::kan^R) did not produce any Stx upon mitomycin C treatment, as predicted. Notably, the levels of Stx produced by C. rodentium (λstx_2dact) were comparable to or higher than the levels generated by the EHEC strains EC1720a and EDL933, suggesting that this strain might produce Stx-mediated disease upon animal infection.

The sequences of stxA (Stx2d activatable A subunit) and stxB (Stx2d activatable B subunit) of Φ1720a-02 were determined, and although the inferred amino acid sequences aligned well with non-mucus-activatable Stx2d (69), the A subunit contained the C-terminal glutamic acid necessary for activation of Stx2dact (refs. 46, 58, and Supplemental Figure 1, A and B; supplemental material available online with this article; doi: 10.1172/JCI62746DS1). Thus, we tested the capacity of the toxin produced by C. rodentium (λstx_2dact) to be activated in the presence of intestinal mucus. Strains B2F1, which encodes the mucus-activatable toxin Stx2dact, and EH250, which encodes the non-activatable Stx2d, were used as positive and negative controls, respectively (46, 69, 70). Culture supernatants from each strain were incubated with murine intestinal mucus or with buffer alone, and the Vero cell cytotoxicity of serially diluted samples was determined. Toxin activity in culture supernatants from E. coli strain B2F1 increased approximately 15-fold upon incubation with murine mucus (Table 1). In contrast, the toxin activity of EH250, which does not contain mucus-activatable toxin, increased only minimally in the presence of the mucus (Table 1). Toxin activity in the culture supernatant of C. rodentium (λstx_2dact) increased approximately 18-fold, comparable to the activity of E. coli B2F1, upon incubation with mucus, indicating that the Stx produced by λstx_2dact is indeed mucus activatable (Table 1).

Table 1

Stx2d from C. rodentium (λstx_2dact) is mucus activatable

C. rodentium (λstx_2dact) causes lethal infection in mice. To determine whether C. rodentium (λstx_2dact) would provide an Stx-mediated disease model, we infected 5- to 8-week old C57BL/6 mice with approximately 1 × 10⁹ CFU of this strain via oral gavage. As controls, the Stx-deficient derivative C. rodentium (λstx_2dact::kan^R) and the parental nonlysogen C. rodentium DBS100 were inoculated in parallel. Fecal shedding of C. rodentium DBS100 and C. rodentium (λstx_2dact::kan^R) reached approximately 3 × 10⁹ to 5 × 10⁹ per gram of feces at day 6 after infection and remained relatively constant to the end of monitoring, 10 days after infection (Figure 2A). C. rodentium (λstx_2dact) colonized the feces with similar kinetics, reaching a peak of approximately 10¹⁰ CFU per gram (Figure 2A).

Figure 2

C. rodentium (λstx_2dact) causes lethal infection in mice. (A) Colonization of 5-week-old C57BL/6 mice by C. rodentium (λstx_2dact), C. rodentium (λstx_2dact::kan^R), and C. rodentium (DBS100) was determined by viable stool counts. Shown are the average CFU (±SEM) of 5 mice. (B) Fecal water content of mock-infected 5-week-old mice or mice infected with C. rodentium (λstx_2dact), C. rodentium (λstx_2dact::kan^R), or C. rodentium (DBS100). Shown are the averages (±SEM) of groups of 5 mice. Range of average fecal water content of uninfected mice is represented by the vertical bar at 0 days after infection. *P < 0.05 compared with all other groups as determined by 2-way ANOVA followed by Bonferroni post tests. (C) Body weight during infection of 8-week-old mice, expressed as percent change from day 0. Shown are the averages (±SEM) of 5 mice per group. **P < 0.01, ***P < 0.001 compared with all other groups as determined by 2-way ANOVA followed by Bonferroni post tests. (D) Percent survival of groups of five 8-week-old mice that were mock infected or infected with C. rodentium (λstx_2dact), C. rodentium (λstx_2dact::kan^R), or C. rodentium (DBS100). For all panels, results are representative of at least 5 independent experiments.

To test whether mice infected with C. rodentium (λstx_2dact) experience Stx-mediated intestinal disease, we monitored fecal water content. As expected, feces from uninfected mice contained 45%–60% water throughout the course of infection (Figure 2B). Feces from mice infected with C. rodentium DBS100 or C. rodentium (λstx_2dact::kan^R) had similar water content through day 4 after infection but starting at post-infection day 6, a time point at which maximal bacterial burden was reached, contained elevated levels of water, as previously observed for non-Stx-producing C. rodentium (71). In contrast, water content of feces from mice infected with C. rodentium (λstx_2dact) was significantly elevated (>70%) at 4 days after infection (Figure 2B), indicating that the production of Stx was associated with an early increase in fecal water content. By 8 days after infection, fecal water content declined to normal values, which could be due to the putative increasing dehydration and concomitant water reabsorption that may occur in these mice during later stages of infection (see Figure 2B).

Weight loss is a notable feature in mice after Stx injection or infection in mice with an intact commensal flora with high doses of STEC (47, 48, 54). We found that mock-infected mice, as well as mice infected with C. rodentium DBS100 or C. rodentium (λstx_2dact::kan^R), maintained body weight throughout infection, indicating that infection with C. rodentium strains that do not produce Stx were not associated with weight loss (Figure 2C). In contrast, mice infected with C. rodentium (λstx_2dact) lost >10% of their starting weight by 6 days after infection and >20% by 8 days after infection.

Finally, the severe weight loss in mice infected with C. rodentium (λstx_2dact) corresponded temporally with mortality or severe disease warranting euthanasia (Figure 2D). All mock-infected mice or mice infected with C. rodentium DBS100 or C. rodentium (λstx_2dact::kan^R) survived. In contrast, mortality was observed in mice infected with C. rodentium (λstx_2dact) starting at day 4 after infection, and (in the experiment represented in Figure 2D) no mice survived past day 9. Consistent with the previous correlation between weight loss and serious systemic disease (47, 48, 54), nearly all (~95%) of mice that died or were euthanized had lost >20% body weight at the preceding weight assessment (data not shown).

Stx-mediated intestinal damage during murine infection with C. rodentium (λstx_2dact). Although EHEC infection of humans is associated with hemorrhagic colitis, EHEC and/or STEC infection of streptomycin-pretreated or gnotobiotic mice does not typically result in obvious AE lesion formation or striking intestinal damage (refs. 57, 59, and A. Melton-Celsa, unpublished observations). Efforts to document Stx-dependent hemorrhagic colitis using a guaiac fecal occult blood test were complicated by the fact that this nonquantitative assay also detected occult blood in the stool of mice infected with the non-Stx-producing C. rodentium (λstx_2dact::kan^R) (Supplemental Figure 2).

To assess the presence of AE lesions in mice infected with C. rodentium (λstx_2dact), we examined colonic tissue by transmission electron microscopy (TEM) at 6 days after infection. Indeed, intimate bacterial attachment, microvillar effacement, and robust actin pedestals were produced on the intestinal epithelium of mice infected with C. rodentium (λstx_2dact), in contrast to the normal intestinal architecture observed in mock-infected mice (Figure 3A). As predicted from previous work (11), Stx was not required for the development of AE lesion formation, because mice infected with C. rodentium (λstx_2dact::kan^R) developed distinct AE lesions beneath bound bacteria (Figure 3A).

Figure 3

Stx-mediated intestinal damage during murine infection with C. rodentium (λstx_2dact). (A) TEM of the proximal large intestine of C57BL/6 mice 6 days after mock infection or infection by the indicated strain. White arrowheads indicate the presence of actin-rich pedestals. Scale bars: 0.5 μm. (B) H&E-stained large intestinal section of mock-infected mice or mice infected with the designated strain 7 to 10 days after infection; original magnification, ×100 (top row), ×200 (bottom row, left and right panels), ×400 (bottom row, middle panel). Scale bars: 50 μm. Black box indicates area of complete crypt loss and abscess formation. Asterisks indicate goblet cells that are prominent in normal intestinal tissue and diminished in mice infected with C. rodentium (λstx_2dact). Arrowhead indicates infiltration of inflammatory cells into the mucscularis mucosa and muscularis propria layers.

To investigate intestinal disease caused by infection with C. rodentium (λstx_2dact), the large intestines of mice infected with C. rodentium (λstx_2dact) for 7–10 days were examined histologically. In accordance with previous studies, mice infected with C. rodentium DBS100 or C. rodentium (λstx_2dact::kan^R) showed limited intestinal pathology, with only minimal hyperplasia and inflammatory infiltrates compared with mock-infected mice (data not shown and Figure 3B). (C. rodentium infection typically results in colonic hyperplasia only after approximately 14 days of infection [refs. 63, 72] and was not observed here). In contrast, gross evaluation of intestines from mice infected with C. rodentium (λstx_2dact) showed severe destruction of the intestinal mucosa, colitis, and acute ischemic injury, with neutrophil infiltration into the muscularis layer and lamina propria and intestinal abscesses (Figure 3B). Also present were areas of necrosis and degenerative changes in the epithelial cells, with crypt withering and an overall loss of goblet cells. These data show that C. rodentium (λstx_2dact) infection is associated with AE lesion formation and severe Stx-mediated intestinal damage.

Stx-mediated systemic induction of proinflammatory cytokines during murine infection with C. rodentium (λstx_2dact). Although Stx is capable of activating complement (73–75), serum levels of complement proteins C3 and Bb in mice infected with C. rodentium (λstx_2dact) were not altered, and sC5b-9 was elevated only transiently at 7 days after infection (data not shown; see Methods). Instead, in measuring 23 cytokines in the sera of mice infected with C. rodentium (λstx_2dact) at 7–10 days after infection (see Methods), we found that several proinflammatory cytokines and chemokines commonly associated with disease in humans and animal models of HUS (47, 48, 50, 76–78) were elevated relative to those in mock-infected mice and mice infected with C. rodentium (λstx_2dact::kan^R). Serum levels of the proinflammatory cytokine TNF-α were elevated, as were the neutrophil chemoattractants IL-17 and KC (Figure 4). Additionally, macrophage/monocyte chemoattractants RANTES, MCP-1, and MIP-1β were elevated 2- to 5-fold in serum from C. rodentium (λstx_2dact)–infected mice relative to mock-infected mice or mice infected with C. rodentium (λstx_2dact::kan^R) (Figure 4). Thus, induction of the cytokines/chemokines was attributable to the production of Stx2dact rather than to simple colonization by C. rodentium.

Figure 4

Stx-mediated systemic induction of proinflammatory cytokines during murine infection with C. rodentium (λstx_2dact). The concentrations of TNF-α, KC, MIP-1β, IL-17, RANTES, and MCP-1 in serum at 7–10 days after infection in mock-infected mice or mice infected with the indicated strain were determined by Luminex (see Methods). Data are pooled from 2 independent experiments, each using 3- to 8-week-old female mice and 5–10 mice per group. **P < 0.01, ***P < 0.001, 1-way ANOVA and Tukey’s multiple comparison tests.

The kidney is a specific target of Stx-mediated damage during murine infection with C. rodentium (λstx_2dact). Although thrombocytopenia and hemolytic anemia are two prominent manifestations of HUS, the platelet and red blood cell blood counts of mice infected with C. rodentium (λstx_2dact) were not significantly elevated relative to those of mock-infected mice or mice infected with C. rodentium (λstx_2dact::kan^R) (data not shown; see Methods). Red blood cells were morphologically normal, and evidence of thrombotic microangiopathy, such as platelet aggregates, excessive clotting, or elevated levels of fibrin D-dimers, was not observed (data not shown).

We also histologically evaluated several extraintestinal tissues for damage upon intestinal infection by C. rodentium (λstx_2dact). The lungs and heart of mice infected with C. rodentium (λstx_2dact) revealed no pathology (Supplemental Figure 3, A and B). Nasal turbinates express Gb₃ receptors (79) but appeared normal after C. rodentium (λstx_2dact) infection (Supplemental Figure 3C). Although the spleens of mice infected with C. rodentium (λstx_2dact) showed reactive germinal center formation and germinal center hyperplasia with large follicles, mice infected with C. rodentium (λstx_2dact::kan^R) also showed considerable germinal center activity (Supplemental Figure 3D). Finally, examination of the livers of mice infected with C. rodentium (λstx_2dact) or C. rodentium (λstx_2dact::kan^R) revealed foci of neutrophilic and lymphocytic inflammation as well as scattered hepatocellular death (Supplemental Figure 3E). This injury in mice infected with C. rodentium (λstx_2dact) was somewhat more severe than in mice infected with C. rodentium (λstx_2dact::kan^R), consistent with the elevation of serum proinflammatory cytokines. Consistent with the lack of thrombocytopenia, we found no platelet aggregates in any of the tissues (data not shown).

Kidney damage is a hallmark of HUS, and Stx causes renal pathology in mice after direct injection or upon absorption from intestinal STEC (47, 48, 54, 59). Renal cytokines and chemokines are elevated in murine models of Stx intoxication, so we measured cytokine and chemokine levels in the kidneys of mice infected with C. rodentium (λstx_2dact) at 7–10 days after infection. Although the levels of the cytokines IL-6 and G-CSF varied considerably from mouse to mouse, they were significantly increased in the kidneys of mice infected with C. rodentium (λstx_2dact) compared with mock-infected controls, as were the chemokines RANTES and KC (Figure 5A). In all cases, the increase in cytokine/chemokine levels was dependent on the production of Stx2dact, because infection by C. rodentium (λstx_2dact::kan^R) was not associated with any significant elevation in levels (Figure 5A). Furthermore, the increase in renal IL-6 and RANTES was at least in part a reflection of increased transcription of the corresponding genes, because RT-PCR analysis of kidney mRNA revealed elevated levels of Il6 and Rantes mRNA relative to mock-infected mice or mice infected with by C. rodentium (λstx_2dact::kan^R) (data not shown).

Figure 5

Stx-mediated renal damage during murine infection with C. rodentium (λstx_2dact). (A) Renal cytokines in mock-infected mice or mice infected with the indicated strain. Data are pooled from 3 independent experiments. *P < 0.05, **P < 0.01. (B) Micrographs (original magnification, ×600) of H&E-stained kidney sections from mock-infected mice or the indicated infected mice infected at 7–10 days after infection. Arrowheads indicate attenuation and sloughing of epithelial cells within the proximal tubules. Scale bars: 50 μm. (C) BUN of 8-week-old mice, each represented as a single data point, infected with indicated strain or mock infected at day of necropsy (7–10 days after infection). Shown is the pool of 4 independent experiments using 5 mice per group. **P < 0.01. (D) Proteinuria in mock-infected mice or the indicated infected mice. Data shown are average protein indices ± SEM of 5 mice per group. The range of protein measured was 0–500 mg/dl, with 0 indicating undetectable protein, 0.5 indicating trace, 1.0 indicating ~30 mg/dl, 2.0 indicating ~100 mg/dl, and 3.0 indicating ~500 mg/dl. Shown is a representative of 5 independent experiments. *P < 0.05, **P < 0.01 compared with C. rodentium (λstx_2dact::kan^R). Hematuria in mock-infected mice or the indicated infected mice infected. Shown are the averages (±SEM) of groups of 10 mice. ***P < 0.001 compared with the other groups. Urine Kim-1 concentration in mock-infected mice or the indicated infected mice. Shown are the averages (±SEM) of groups of 3–6 mice. **P < 0.01 compared with the other groups.

To evaluate renal pathology associated with the C. rodentium (λstx_2dact) infection model, kidney sections were taken between 7 and 10 days after infection from mice infected with C. rodentium (λstx_2dact) and were stained with H&E and PAS. Although renal cytokines were elevated, infection with C. rodentium (λstx_2dact) was not associated with a striking renal inflammatory response (Figure 5B and data not shown), nor was CCR1, a chemokine receptor associated with renal inflammation (80), elevated in the kidney (data not shown and see Methods). Instead, consistent with the observation that Stx intoxication of mice results in tubular damage (47–49), kidneys of mice infected with C. rodentium (λstx_2dact) showed proximal tubule injury that was not evident in the kidneys of mock-infected mice or mice infected with C. rodentium (λstx_2dact::kan^R) (Figure 5B). Their proximal tubular epithelium was flattened, with prominent pyknotic nuclear material, loss of luminal brush border, and sloughing of dead cells in the tubular lumen (Figure 5B, arrowheads). Mitotic activity, indicating regenerative repopulation of the proximal tubules, was also observed in some mice (data not shown).

To assess renal function in mice infected with C. rodentium (λstx_2dact), we measured blood urea nitrogen (BUN) during the phase of terminal illness (7–10 days after infection) (see Methods). For each mouse euthanized due to severe disease prior to 10 days after infection, 1 mouse each from the mock-infected and C. rodentium (λstx_2dact::kan^R)–infected groups was euthanized, and they were analyzed in parallel. The mean BUN of mice infected with C. rodentium (λstx_2dact) was significantly (P < 0.01) elevated relative to mock-infected mice, and was also elevated relative to mice infected with C. rodentium (λstx_2dact::kan^R), although the latter comparison did not reach statistical significance (Figure 5C).

Renal tubular damage, with both leakage of protein and sloughing of cells into the lumen, may result in proteinuria, hematuria, and elevated levels of the renal tubule–specific biomarker kidney injury molecule-1 (Kim-1) (refs. 81–83 and see Methods). Whereas urine of mock-infected mice or mice infected with C. rodentium (λstx_2dact::kan^R) contained no or only trace levels of protein (protein index of 0–0.5 indicating <30 mg/dl), urine of mice infected with C. rodentium (λstx_2dact) contained significantly elevated protein indices as early as 2 days after infection (Figure 5D). Between 4 and 7 days after infection, average urine protein indices of the colorimetric assay were as high 1 to 1.5, corresponding to approximately 30–100 mg/dl. Hematuria, also assessed using a colorimetric assay, was moderately elevated on days 7 and 8 after infection. Finally, the marker for tubular damage Kim-1 was also elevated late in infection compared with mock-infected mice or mice infected with C. rodentium (λstx_2dact::kan^R). These results indicate that the tubular damage sustained during infection by C. rodentium (λstx_2dact) reflected by histological analysis in fact results in functional renal compromise.

Tir is required for colonization and disease in the C. rodentium (λstx_2dact) infection model. The ability to generate of AE lesions is a well-established virulence attribute of EHEC, but STEC strains that produce Stx2dact are capable of causing disease in humans (and mice) in the absence of AE lesion formation (45, 58, 59). To test whether murine infection with C. rodentium (λstx_2dact) provided a disease model that was dependent on AE lesion formation, we determined whether a C. rodentium (λstx_2dact) defective for Tir production was also defective for colonization and disease. Whereas C. rodentium (λstx_2dact) and C. rodentium (λstx_2dact::kan^R) were capable of generating actin pedestals indistinguishable from those formed by wild-type C. rodentium DBS100 on monolayers of mouse embryonic fibroblasts (MEFs), C. rodentium (λstx_2dact)Δtir was impaired in cell binding and unable to form pedestals (data not shown). Pedestal formation was restored to this strain by pTir (pEM129), a Tir-expressing plasmid (data not shown; see Supplemental Table 2 and Methods). To confirm that Tir was also required for AE lesion formation during infection, we compared the intestinal epithelia of mice infected with C. rodentium (λstx_2dact)Δtir and the parental strain C. rodentium (λstx_2dact) by TEM at 6 days after infection. Whereas infection by C. rodentium (λstx_2dact) resulted in effacement of the intestinal brush-border microvilli and the formation of robust pedestals (Figure 3A), infection by C. rodentium (λstx_2dact)Δtir left the epithelial cytoskeletal architecture indistinguishable from that of mock-infected controls. When tir was introduced in trans on pTir, the intestines of mice infected with C. rodentium (λstx_2dact)Δtir/pTir showed many bound bacteria elevated on actin pedestals (Figure 3B) with concomitant destruction of microvilli, indicating that, as expected, Tir is required for AE lesion formation.

The lack of mucosal attachment by C. rodentium (λstx_2dact)Δtir revealed by TEM reflected a significant colonization defect when assessed quantitatively, because mice infected with C. rodentium (λstx_2dact)Δtir had vastly fewer fecal bacteria than mice infected with C. rodentium (λstx_2dact) (Figure 6A). At 3 days after infection, fecal counts of mice infected with C. rodentium (λstx_2dact)Δtir were approximately 3 orders of magnitude lower than those of mice infected with C. rodentium (λstx_2dact), and this difference grew to approximately 7 orders of magnitude 4 days later. In contrast, C. rodentium (λstx_2dact)Δtir/pTir was capable of efficiently colonizing mice, with peak bacterial burdens reaching almost 10¹⁰ per gram of stool (Figure 6A). Notably, the kinetics of colonization by C. rodentium (λstx_2dact)Δtir/pTir were somewhat delayed relative to C. rodentium (λstx_2dact), consistent with previous reports that plasmid complementation of a C. rodentiumtir mutant is incomplete (20, 84).

Figure 6

Tir is required for colonization and disease upon C. rodentium (λstx_2dact) infection of mice. (A) Colonization of 8-week-old C57BL/6 mice that were mock infected mice or infected with the indicated strain was determined by plating stool for viable counts. Shown are the averages CFU (±SEM) of 5 mice. Data are representative of 5 independent experiments. Statistical significance was determined using 2-way ANOVA and Bonferroni post tests and indicates that C. rodentium (λstx_2dact)Δtir colonized mice significantly (*P < 0.05) less efficiently than C. rodentium (λstx_2dact) and C. rodentium (λstx_2dact)Δtir/pTir). (B) Body weight during infection of 8-week-old mice is expressed as percent change from day 0. Shown are the averages (±SEM) of 5 mice per group. Data are representative of 5 independent experiments. (C) Percent survival of mock-infected 8-week-old mice or mice infected with C. rodentium (λstx_2dact), C. rodentium (λstx_2dact)Δtir, or C. rodentium (λstx_2dact)Δtir/pTir. Groups of 5 mice were analyzed. Data are representative of 5 independent experiments. (D) H&E-stained large intestinal sections of mock-infected mice or mice infected with the indicated strain. Scale bars: 50 μm. Asterisks indicate goblet cells that are prominent in normal intestinal tissue. Arrowhead indicates infiltration of inflammatory cells into the mucscularis mucosa and muscularis propria layers. (E) H&E-stained micrograph (original magnification, ×600) of kidney sections from mock-infected mice or mice infected with the indicated strain. Arrowhead indicates attenuation and sloughing of epithelial cells within the proximal tubules. Scale bars: 50 μm.

The defect of C. rodentium (λstx_2dact)Δtir in intestinal colonization was reflected in all aspects of Stx-mediated disease. Histological analyses of the intestines of mice infected with C. rodentium (λstx_2dact)Δtir were indistinguishable from those of mock-infected mice, in contrast to those of mice infected with C. rodentium (λstx_2dact)Δtir/pTir, which showed areas of necrosis, inflammatory infiltrates, and the loss of goblet cells (Figure 6D). Renal damage was associated only with infection by C. rodentium (λstx_2dact)Δtir/pTir, not with the Tir-deficient mutant (Figure 6E). These histological assessments of disease correlated with weight loss, because mice infected with C. rodentium (λstx_2dact)Δtir maintained weight throughout infection (Figure 6B). In contrast, mice infected with C. rodentium (λstx_2dact)Δtir/pTir lost on average 18% body weight by 15 days after infection. The kinetics of weight loss by mice infected with the Tir-complemented strain, similar to the kinetics of colonization, were delayed several days relative to mice infected with C. rodentium (λstx_2dact), but once average weight began to decrease at approximately 10 days post-infection, it declined steadily (Figure 6B). Finally, whereas infection by C. rodentium (λstx_2dact)Δtir caused no mortality, infection by C. rodentium (λstx_2dact)Δtir/pTir, like C. rodentium (λstx_2dact), was uniformly fatal, with the predicted temporal delay (Figure 6C). Thus, Tir is an essential virulence factor during lethal infection by C. rodentium (λstx_2dact).

STEC have been implicated in serious outbreaks of diarrheal disease and systemic damage, including a European outbreak due to a serotype O104 STEC strain (1). An important subclass of STEC is EHEC, which triggers the formation of AE lesions on the intestinal mucosa. Some rabbit and piglet models manifest both AE lesion formation and Stx-mediated disease (21, 23, 52, 53, 85), but no such model has yet been developed in conventional mice. We show here that C. rodentium lysogenized with λStx2dact, which produces a mucus-activatable Stx previously associated with severe disease in mice (59–61), provides a model that features prototypic AE lesions during intestinal colonization and Stx-mediated weight loss and death, recapitulating renal dysfunction observed in human HUS and murine toxin injection models (4, 54).

Systemic Stx-mediated disease in humans is associated with the induction of inflammatory cytokines (77, 86), and patients with a preponderance of proinflammatory cytokines often have more severe disease (87, 88). Indeed, one model of Stx-mediated disease in mice requires the co-administration of LPS and Stx (50). Several means by which inflammatory cytokines may promote systemic disease have been proposed, including induction of the Gb₃ glycolipid Stx receptor on host cells (89–91), alteration of fibrinolysis and anticoagulation, or recruitment of inflammatory cells into damaged tissues (77). In response to Stx, cultured monocytes secrete TNF-α and IL-6 (90, 92, 93), and we found that these cytokines were elevated in the serum or kidneys of mice infected with C. rodentium (λstx_2dact). This response was dependent on Stx production, because neither TNF-α nor IL-6 was elevated in mice infected with C. rodentium (λstx_2dact::kan^R). MCP-1 and KC were also elevated in C. rodentium (λstx_2dact)–infected mice in an Stx-dependent manner. Notably, MCP-1 and IL-8, a functional human analog of KC, are found at elevated levels in the urine of children with HUS (94).

Renal damage is central to human HUS (9), and the induction of kidney cytokines in the C. rodentium (λstx_2dact) model was accompanied by renal compromise. Glomerulonephritis is a prominent feature of human HUS, a manifestation that correlates with the relative abundance of Gb₃ receptors on glomerular endothelium (95). In contrast, Gb₃ is enriched in the renal tubules in mice (49), and the tubular damage we observed in infected mice likely reflects this localization. Consistent with this hypothesis, tubular damage is also typically observed in EHEC-infected germ-free or streptomycin-treated mice (49, 57–59). Elevated serum creatinine, an indicator of renal dysfunction, has been observed in some other murine models for Stx-mediated disease (47, 48, 59), but we found that serum creatinine was not significantly elevated in mice infected with C. rodentium (λstx_2dact) (E. Mallick, unpublished observations). A possible explanation is that elevated serum creatinine is a late marker of renal injury (82) and affected by many non-renal factors (81, 96). In fact, the renal histopathological findings were reflected in other measures of functional compromise, because mice infected with C. rodentium (λstx_2dact), but not C. rodentium (λstx_2dact::kan^R), demonstrated elevated BUN, proteinuria, hematuria, and urinary Kim-1, a marker of tubular damage, consistent with other murine models of HUS (47, 48, 54, 57, 59).

Several manifestations of murine Stx-mediated disease and human HUS are absent in the C. rodentium (λstx_2dact) murine model. For example, neurological damage is associated with HUS, and cerebral hemorrhage and edema have been reported in EHEC-infected, streptomycin-pretreated mice (97, 98). Although mice infected with C. rodentium (λstx_2dact) occasionally displayed tremors and seizures, we did not observe consistent pathological changes in the nasal turbinates, a region of the CNS rich in the Stx receptor (79). In addition, contrary to what has been observed in human disease (6, 8) and Stx administration models (47–49, 88, 99–101), mice infected with C. rodentium (λstx_2dact) did not display renal platelet aggregation, red cell congestion, or fibrin deposition and did not show evidence of thrombocytopenia or anemia. Given the hypothesis that erythrocytes are ruptured upon passage through damaged renal glomeruli (102), it is possible that the lack of glomerulonephritis in our model contributes to the lack of observable anemia. We also found no hematological evidence of thrombotic disease, such as prolonged clotting time or fibrin D-dimer formation. Following systemic administration of Stx, mice that are deficient in ADAMTS13, a protease that cleaves the hemostatic von Willebrand factor, demonstrate wide strain-specific variation in the degree of microvascular thrombosis, thrombocytopenia, and hemolytic anemia (88, 101). It is possible that infection by C. rodentium (λstx_2dact) might trigger more overt microangiopathy and thus more closely resemble human HUS, in murine strains different from the C57BL/6 strain utilized here.

Given that a central goal of this study was to test whether C. rodentium (λstx_2dact) could recapitulate EHEC toxin–mediated disease, an important finding was the requirement for Tir in promoting Stx-mediated disease in this model. Colonization by a Tir-deficient derivative of C. rodentium (λstx_2dact) was many orders of magnitude lower than colonization by the parental strain. A Tir-encoding plasmid complemented the mutant for the ability to colonize mice and cause disease. The kinetics of colonization, weight loss, and death were somewhat slower than with C. rodentium (λstx_2dact), suggesting that, as observed in previous studies (20, 84), plasmid-based complementation of a Tir-deficient C. rodentium mutant may result in only partial restoration of the wild-type phenotype.

Interestingly, in spite of the reports that the Stx receptor Gb₃ is expressed on cells in the distal colon of the mouse (32, 103), the streptomycin pretreatment and gnotobiotic mouse models of EHEC infection do not typically result in obvious intestinal damage (refs. 57, 59, and A. Melton-Celsa, unpublished observations). Mild inflammatory cell infiltration, goblet cell depletion, and intestinal epithelial necrosis have been reported in a few studies (97, 104–106). In contrast, we found here dramatic intestinal damage in mice infected with C. rodentium (λstx_2dact); indeed, these pathological changes were similar to those commonly observed in human infection by EHEC, which in some cases can mimic inflammatory bowel disease or ischemic colitis (107–110). It is tempting to speculate that AE lesion formation, a prominent feature of this murine infection model, is critical to facilitating Stx-mediated intestinal epithelial damage. The development of a murine model that encompasses both AE lesion formation and severe Stx-mediated tissue damage provides an opportunity to more fully explore the relationship between the pathogen-induced alteration in host cytoskeletal structure and subsequent local and systemic disease.

View Supplemental data

We thank the UMass Diabetes and Endocrinology Research Center facility for fixing, sectioning, and staining all tissue sections and particularly Lara Strittmatter and Gregory Hendricks of the UMass Electron Microscopy Core Facility for the invaluable electron microscopy work. The UMass Mouse Phenotyping Core Facility performed Luminex cytokine and blood chemistry studies. This work was supported by NIH R21 AI92009 and R01 AI46454 to J.M. Leong; R37 AI20148 to A. O’Brien; R01 049842, R01 AI049842, and 026731 PHS P01CA026731 to D.B. Schauer; and NIH/New England Regional Center for Excellence, Biodefense and Emerging Infectious Diseases Cooperative Pilot Projects Program R01 DK59101 to J.R. Butterton and D.B. Schauer.

Address correspondence to: John M. Leong, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA. Phone: 617.636.0488; Fax: 617.636.0337; E-mail: john.leong@tufts.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(11):4012–4024. doi:10.1172/JCI62746.

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