Figures
Abstract
Attaching and effacing (A/E) bacterial pathogens consist of human pathogens enteropathogenic Escherichia coli, enterohemorrhagic E. coli and their murine equivalent Citrobacter rodentium (CR). Emerging evidence suggests that the complex pathogen-microbiota-host interactions are critical in conferring A/E pathogen infection-induced severe symptoms and lethality in immunocompromised hosts; however, the precise underlying mechanisms remain enigmatic. Here we report that CR infection causes severe colitis and mortality in interleukin 22 knockout (Il22-/-) and Rag1 knockout (Rag1-/-) mice under germ-free (GF) conditions. In a gut microbiota-independent manner, CR colonizes in GF Il22-/- and Rag1-/- animals, triggers colonic epithelial tissue damage and systemic dissemination of CR, and results in lethal infections. Pretreatment with cefoxitin, a broad-spectrum antibiotic, exacerbates CR-induced colitis and lethality in specific-pathogen-free (SPF) Il22-/- and Rag1-/- mice. Together our results reveal that CR possesses a gut microbiota-independent virulence, which is better illustrated during infections in immunocompromised hosts associated with severe outcomes.
Author summary
We reveal that Citrobacter rodentium (CR) colonizes in the cecum and the colon and causes severe symptoms and lethality in germ-free (GF) Il22-/- and Rag1-/- mice, independent of gut microbiota. Pretreatment of antibiotics in specific-pathogen-free (SPF) immunocompromised animals exacerbates CR-induced lethality. These results suggest that CR possesses gut microbiota-independent capacity in colonization and virulence and the commensal bacteria act as the front-line defense against CR infection.
Citation: Liu Y, Xu D, Guo S, Wang S, Ding H, Siu C, et al. (2024) The gut microbiota-independent virulence of noninvasive bacterial pathogen Citrobacter rodentium. PLoS Pathog 20(12): e1012758. https://doi.org/10.1371/journal.ppat.1012758
Editor: Andreas J. Baumler, University of California Davis School of Medicine, UNITED STATES OF AMERICA
Received: July 8, 2024; Accepted: November 18, 2024; Published: December 4, 2024
Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are in the manuscript and its supporting information files.
Funding: This work was supported in part by grants from the National Institutes of Health (CA244350 and AI137719 to FW), Johns Hopkins University (Ho-Ching Yang Memorial Faculty Award to DX; Germ-Free Facility to HD), and Karen Toffler Charitable Trust (Toffler Scholar Award to DX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are among the leading etiological agents for foodborne diseases [1,2]. Murine pathogen Citrobacter rodentium (CR) shares most pathogenic mechanisms and virulence factors with EPEC and EHEC [3–8]; hence CR infection in mice has been widely used as a small animal model to study the underlying pathogenic mechanisms. Upon infection in specific-pathogen-free (SPF) mice, CR adapts to the gastrointestinal tract with expression of an array of virulence genes [9]. After reaching peak colonization, CR burden gradually declines, with complete clearance observed 2–4 weeks post infection [9]. While gut microbiota is generally regarded as a barrier against enteric pathogen colonization in SPF mice, certain Kanamycin-sensitive commensal species appear essential for CR to maintain colonic tissue association during peak of infection [10]. Of note, wild-type C57Bl/6 and many immunocompetent strains show low or no mortality, whereas immunocompromised animals, for instance interleukin-22 knockout (Il22-/-) and interleukin-22 receptor subunit alpha 1 knockout (Il22ra1-/-) mice, exhibit markedly elevated mortality following CR challenge [11,12]. Systemic spread of CR was reported in the infected SPF Il22-/- mice [11–13]; in contrast, Pham and colleagues found that systemic dissemination of Enterococcus faecalis, an intestinal opportunistic pathogen, led to lethality in Il22ra1-/- mice [11]. It has remained elusive the severe symptoms and outcomes in CR-infected immunocompromised SPF animals attribute to the systemic dissemination of CR per se, certain commensal microbes, or the synergic pathogen-microbiota interactions.
Here, utilizing germ-free (GF) Il22-/- mice lacking the critical cytokine Il22 for an acute phase response and Rag1 knockout (Rag1-/-) mice lacking mature T and B lymphocytes in adaptive immune response, both of which are susceptible to CR infection under SPF condition, we assessed the impact of gut microbiota on the CR susceptibility. Our findings reveal that in the absence of gut microbiota, CR infection sufficiently colonizes on the colonic epithelium and triggers tissue damage, thus causing systemic dissemination of CR and resulting in animal lethality. Such severe symptoms strongly indicate that CR possesses virulence independent of the host gut microbiota, which is better illustrated in the infected immunocompromised GF Il22-/- and Rag1-/- animals.
Results
CR infection causes lethality in both SPF and GF Il22-/- mice
CR infection leads to severe symptoms and lethality in an array of immunocompromised mouse strains [14]. Consistent to previously reported [11–13,15], CR infection in SPF Il22-/- mice resulted in severe body weight loss, watery diarrhea, and 100% mortality by 14 days post inoculation (dpi) (Fig 1A–1D). Moreover, systemic spread of CR associated with compromised colonic epithelial barrier integrity occurred in CR-infected mice, in contrast to no colony-forming units (CFU) of CR detected in the controls (Fig 1E). While these results highlight that CR could cause sepsis-associated lethality under SPF condition, it remains puzzling whether the lethality in SPF Il22-/- animals attributes to the spread of CR per se or the synergistic CR-gut microbiota interactions.
(A-D) Fecal live Citrobacter rodentium (CR) burden (A), body weight loss (B), clinical scores (C), and survival (D) of specific-pathogen-free (SPF) Il22-/- mice in C57Bl/6J background at indicated days post inoculation (dpi) with 2 × 109 CFU of CR or vehicle control. (E) CR burdens in the liver (top) and the spleen (bottom) derived from SPF Il22-/- mice at 10 dpi infected with vehicle control or CR. No CR burden was detected in vehicle controls. (F-I) Fecal live CR burden (F), body weight loss (G), clinical scores (H) and survival (I) of germ-free (GF) Il22-/- mice at indicated dpi with 2 × 109 CFU of CR or vehicle control. Data are representative results of at least two independent experiments. The error bars (A and F) are too small to be visible. ** p < 0.01, *** p < 0.001, **** p < 0.0001 with Student’s t tests (A-C, and G-H) and Long-rank test (D and I).
We rederived germ-free (GF) Il22-/- mice and orally infected them with CR or vehicle control, to assess the possible virulence of CR in the absence of commensal microbiota. In contrast to the gradual increase in CR burdens in SPF animals (Fig 1A), CR colonized maximally in GF Il22-/- mice as early as 2 dpi (Fig 1F). The CR-infected GF Il22-/- mice, starting from 4 dpi, exhibited severe clinical symptoms and 100% lethality by 11 dpi (Fig 1G–1I). These results demonstrate that regardless of gut microbiota, CR alone is sufficient to cause symptoms and lethality in GF Il22-/- mice, indicating the gut microbiota-independent virulence of CR.
CR colonization causes lethal colitis in GF Il22-/- mice
At necropsy, the colon lengths of CR-infected GF Il22-/- mice were substantially shortened compared to the controls (Fig 2A and 2B). Moreover, histopathological analyses on the colons revealed that CR infection led to more profound epithelial damage, goblet cell depletion, immune cell infiltration, and the hallmark colonic crypt hyperplasia [16] in the infected GF mice (Fig 2C–2E), which indicates that CR alone resulted in profound colonic inflammation in the GF Il22-/- animals. Moreover, in the infected colons, CR induced significant redistribution and degradation of Claudin-3, one of the most abundant tight junction proteins [17] (Fig 2F). Consistently, CR burdens were augmented in the liver and the spleen of CR-infected animals but remained not detectable in the controls (Fig 2G). Indeed, CR-caused damage to the integrity of colonic epithelial barrier was supported by the increased gut permeability in the infected GF Il22-/- mice, as measured by the leakage of gut-derived FITC-dextran to the serum (Fig 2H). Furthermore, utilizing ex vivo bioluminescent imaging of ICC180 strain, we demonstrated that CR attached to the cecum and the colon of infected GF Il22-/- mice at 5 dpi (Fig 2I). These results suggest that CR infection causes bona fide colonization with tight attachment to the cecal and colonic epithelia, thus triggering tight junction disruption, and leading to systemic dissemination of CR and mortality in GF Il22-/- mice.
(A) Representative images of the cecum and the colon derived from GF Il22-/- mice at 5 days post inoculation (dpi) infected with vehicle control or 2 × 109 CFU of CR. Scale bars, 1 cm. (B) Colon lengths of GF Il22-/- mice infected with vehicle control or CR. (C-D) Hematoxylin and eosin staining (C) and histopathology scores (D) of colon sections derived from GF Il22-/- mice at 5 dpi infected with vehicle control or CR. Scale bars, 100 μm. (E) Crypt lengths measured in colonic tissue sections from at least 4 GF Il22-/- mice infected with CR or vehicle control at 5 dpi. (F) Immunofluorescence micrographs of Claudin-3 and CR in the colons derived from GF Il22-/- mice at 5 dpi infected with vehicle control or CR, with nuclei counterstained by DAPI. Lum indicates the colon luminal space. Scale bars, 100 μm. (G) Live CR burdens in the liver (left) and the spleen (right) derived from GF Il22-/- mice at 5 dpi infected with vehicle control or CR. No CR burden was detected in vehicle controls. (H) FITC-dextran concentrations in the sera of GF Il22-/- mice at 5 dpi infected with vehicle control or CR, at 4 h post oral administration of FITC-dextran. (I) Representative ex vivo tissue images of GF Il22-/- mice infected with vehicle control or CR at 5 dpi, with an IVIS50 camera and displayed as pseudo colors. The color scale bar indicates bioluminescence signal intensity (photons sec-1 cm-2 sr-1). Data are representative results of at least two independent experiments. **** p < 0.0001 with Student’s t tests (B, D and E).
CR causes lethal infection in GF Rag1-/- mice
To examine whether the lethal infection of CR occurs restrictedly in Il22-/- mice, we carried out oral CR infection in GF Rag1-/- mice, another immunocompromised strain where the V(D)J recombination activation gene Rag1 was deleted [18]. Indeed, CR colonized maximally in GF Rag1-/- mice as early as 2 dpi (Fig 3A). The ex vivo bioluminescent imaging ascertained the tight attachment of CR to the cecum and the colon of infected GF Rag1-/- animals at 5 dpi (Fig 3B). Of note, such colonization sufficiently resulted in the damage to colonic epithelial barrier and CR systemic dissemination, as evidenced by the augmented serum levels of FITC-dextran leaked from the gut (Fig 3C) and the elevated CR burdens in the liver and the spleen of CR-infected GF Rag1-/- mice at 14 dpi, compared with the controls (Fig 3D). Indeed, GF Rag1-/- mice suffered severe body weight loss (Fig 3E) and succumbed to CR infection by 16–20 dpi (Fig 3F). Under GF condition, infection of CR in both Rag1-/- and Il22-/- mice is sufficient to cause severe symptoms and mortality, supporting that CR indeed harbors gut microbiota-independent virulence.
(A) Fecal live CR burden of germ-free (GF) Rag1-/- mice at indicated dpi with 2 × 109 CFU of CR or vehicle control. (B) Representative ex vivo tissue images of GF Rag1-/- mice infected with vehicle control or CR at 5 days post inoculation (dpi), with an IVIS50 camera and displayed as pseudo colors. The color scale bar indicates bioluminescence signal intensity (photons sec-1 cm-2 sr-1). (C) FITC-dextran concentrations in the sera of GF Rag1-/- mice at 14 dpi infected with vehicle control or CR, at 4 h post oral administration of FITC-dextran. (D) Live CR burdens in the liver (left) and the spleen (right) derived from GF Rag1-/- mice at 14 dpi infected with vehicle control or CR. No CR burden was detected in vehicle controls. (E-F) Body weight loss (E) and survival (F) of GF Rag1-/- mice at indicated dpi with vehicle control or CR. Data are representative results of at least two independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001, with Student’s t tests (E) and Long-rank test (F).
Gut microbiota protects CR infection-caused lethality in SPF animals
We observed that CR-caused mortality in SPF Il22-/- mice occurred noticeably slower compared with that in GF Il22-/- animals (Fig 1D and 1I), indicating that gut microbiota could protect the host against CR infection. Hence, we modified SPF Il22-/- mouse gut microbiota via pretreatment with Cefoxitin, a broad-spectrum antibiotic, and assessed its impact on the mouse susceptibility to CR challenge (Fig 4A). Fecal CR burdens were marginally augmented in Cefoxitin-administrated SPF Il22-/- mice compared to the vehicle-treated animals (Fig 4B). Notably, Cefoxitin-pretreated animals became more susceptible to CR infection, with accelerated body weight loss and mortality (Fig 4C and 4D), albeit comparable CR burdens observed in the liver and the spleen (Fig 4E). Similarly, SPF Rag1-/- mice became more vulnerable to CR infection following Cefoxitin administration, as evidenced by augmented body weight loss and mortality (Fig 4F–4G). These results indicate that in SPF Il22-/- and Rag1-/- mice, gut microbiota act as a protective barrier against CR infection.
(A) Schematic of Citrobacter rodentium (CR) infection experiment in specific-pathogen-free (SPF) Il22-/- mice. The indicated mice were pretreated with normal drinking water (Vehicle) or drinking water containing Cefoxitin (500 mg/L) for 48 h. 24 h after removal of antibiotic-containing drinking water, the mice were orally inoculated with 2 × 109 CFU of CR. (B-D) Fecal live CR burden (B), body weight loss (C) and survival (D) of vehicle control- or Cefoxitin-pretreated SPF Il22-/- mice at indicated days post inoculation (dpi) with CR. (E) CR burdens in the liver (left) and the spleen (right) derived from vehicle control- or Cefoxitin-pretreated SPF Il22-/- mice at 5 dpi infected with CR. (F-G) SPF Rag1-/- mice were pretreated with normal (Vehicle) or Cefoxitin-containing drinking water, followed with CR infection, as described in (A). Body weight loss (F) and survival (G) of vehicle control- or Cefoxitin-pretreated SPF Rag1-/- mice at indicated dpi with 2 × 109 CFU of CR. Data are representative results of at least two independent experiments. ns, not significant; * p < 0.05, ** p < 0.01, with Student’s t tests (C and F) and Long-rank test (D and G).
Discussion
CR is known to cause self-limiting infection in a wide range of immunocompetent mouse strains including C57Bl/6 mice [9]. While substantial changes in host gut microbiota composition were reported during CR infections [19,20], the impact of gut microbiota on CR colonization and virulence has been studied with controversial findings [10,21–24]. Utilizing SPF and GF C57Bl/6 mice together with CR reporter strains, Mullineaux-Sanders and colleagues proposed that while expression of virulence genes is not sufficient for CR colonization at the colonic mucosa, certain Kanamycin-sensitive species in gut microbiota seem essential for CR colonization during the peak of infection [10]. However, CR was previously shown attaching to the cecal and colonic epithelia with characterized attaching/effacing lesions in GF C57Bl/6 mice [23], indicating the gut microbiota-independent colonization of CR. Moreover, Campbell et al. reported that gut microbiota creates the dominant bottleneck in preventing CR infection [22]. The protective effect of gut microbiota on CR colonization and virulence was also supported by the evidence that antibiotic treatment predisposes the SPF C57Bl/6 mice to exacerbated CR-induced colitis [24]. Of note, the majority of these findings was performed using immunocompetent animals, which only exhibit transient and mild symptoms following CR infection. In contrast, in the immunocompromised GF Il22-/- mice, we showed that CR infection leads to 100% mortality, coinciding with severe colonic barrier damage and systemic dissemination of CR. These results demonstrate that CR per se is sufficient for the pathological infection course in GF Il22-/- mice. The tight attachment of CR to the cecum and the colon, independent of gut microbiota, most likely represents a bona fide colonization, which is further supported by the associated severe symptoms and outcomes. Moreover, CR elicits lethal infection in GF Rag1-/- mice that harbor distinct host deficiencies from GF Il22-/- animals, further strengthening the notion that CR harbors gut microbiota-independent virulence. Of note, accumulating evidence underscores that A/E pathogen infections lead to more severe symptoms and lethality in immunocompromised populations [25, 26]. Our findings illustrate a previously underappreciated gut microbiota-independent virulence of CR, which offers new insights into the complex pathogenic mechanisms during A/E pathogen infections.
Materials and Methods
Ethics statement
All animal experiments were performed according to the protocol approved by Animal Care and Use Committee at Johns Hopkins University and in direct accordance with the National Institutes of Health (NIH) guidelines. Il22-/- and Rag1-/- mice in C57Bl/6J background were maintained in a specific-pathogen-free (SPF) or germ-free (GF) mouse facility, as previously described [27].
Bacterial strains and growth conditions
Wild-type CR (DBS 100 strain) and bioluminescent CR (ICC180 strain) were described previously [28]. CR strains were grown in LB broth at 37°C overnight with shaking from single colonies on Luria–Bertani (LB) plates.
Bacterial infection in mice
CR infection in SPF and GF mice was conducted as we did previously [29, 30]. For antibiotics treatment of SPF animals, mice were given water containing 500 mg/L Cefoxitin for 48 h; after removal of antibiotic water for 24 h, mice were orally inoculated with CR.
Histology, immunohistochemistry, and immunofluorescence on colon tissue sections
GF Il22-/- and Rag1-/- mice were euthanized at 5 dpi and 14 dpi, respectively, as they succumbed to CR infection starting around 5 dpi and 14 dpi, respectively. Histology, immunohistochemistry, and immunofluorescence staining of colon tissue sections were performed as previously described [31]. Colonic crypt hyperplasia was measured as previously reported [16].
Ex vivo bioluminescent imaging
The ex vivo bioluminescent imaging of CR burden was conducted as previously described [28].
FITC-dextran assays
FITC-dextran assays for colonic permeability were performed as previously described [32].
Supporting information
S1 Data. The underlying numerical data for the figure panels.
An Excel spreadsheet containing, in separate sheets, the underlying numerical data for Figs 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 2B, 2D, 2E, 2G, 2H, 3A, 3C, 3D, 3E, 3F, 4B, 4C, 4D, 4E, 4F and 4G.
https://doi.org/10.1371/journal.ppat.1012758.s001
(XLSX)
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