Introduction The attaching and effacing (A/E) bacteria Enteropathogenic Escherichia coli (EPEC) and Enterohemorrhagic E. coli (EHEC) are major contributors to the global disease burden caused by enteric bacterial pathogens [1]. EPEC infects the small bowel causing acute watery diarrhea, fever and nausea [1], [2] and is an important cause of infant diarrheal disease in developing countries. EPEC infections lead to the deaths of hundreds of thousands of infants annually from dehydration and other complications [1], [3]. In contrast, EHEC (O157:H7) infection is associated with sporadic outbreaks across industrialized countries, due to consumption of contaminated beef or water supplies [1], [4]. EHEC colonizes the large bowel and secretes the highly cytotoxic Shiga Toxin (Stx), which can lead to severe hemorrhagic colitis and bloody diarrhea in people of all ages [5]. Children are at an additional risk of EHEC-induced Hemolytic Uremic Syndrome, a potentially fatal complication caused by Stx-mediated acute renal failure [6]. Both EPEC and EHEC are minimally invasive, as they intimately attach to the apical plasma membrane of intestinal epithelial cells via a Type 3 Secretion System (T3SS). Infection causes localized destruction (effacement) of the epithelial microvilli to form the unique A/E lesion [7]. Significant advances have been made in delineating the mechanisms of A/E lesion formation and their requirement for disease [8]; however, the factors involved in host susceptibility to and defense against A/E pathogens remain ill defined. As EPEC and EHEC are human-specific and do not cause relevant disease in animal models [7], our understanding of innate and adaptive immunity against these pathogens has come from studying related A/E bacteria that infect other mammals. Citrobacter rodentium is a natural A/E pathogen of mice that infects epithelial cells lining the cecum, descending colon and rectum of the murine large bowel [7], [9]. C. rodentium infection leads to an acute colitis, mucosal hyperplasia, barrier disruption, and loose stools, but is resolved in 3–4 weeks in C57BL/6 mice [10]. Since C. rodentium uses similar virulence strategies to those employed by EPEC and EHEC to infect cells, including T3SS-mediated intimate attachment and A/E lesion formation, it is widely used as an in vivo model of A/E bacterial infection [10]. The C. rodentium model also allows for identification of the cells and mediators utilized by the host to control infections by A/E pathogens. While a robust adaptive immune response involving CD4+ T cells and B cells (via immunoglobulin G (IgG) secretion) is required for pathogen clearance [11], [12], studies have shown epithelial cells to be important in limiting C. rodentium colonization [13], [14]. In this regard, mounting evidence suggests epithelial-derived mucin production is an additional defense mechanism to manage enteric bacterial infections [15], [16]. Mucins are high molecular weight glycoproteins characterized by extended serine, threonine, and proline-rich domains in the protein core, which are sites of extensive O-linked glycosylation with oligosaccharides [17]. The mucin gene family contains 16 known members in humans that can be broadly divided into membrane bound or secretory forms [15]. The membrane-bound Muc1, which is produced by all intestinal epithelial cells, has been shown to play a role in host defense against Campylobacter jejuni in vivo, limiting disease and systemic spread [18]. Muc1 is also upregulated in C. rodentium infection [19], although its role in this infection is not known. However, membrane-bound MUC3 has been associated with decreased colonization of EPEC in vitro [20]. Collectively, these studies suggest that mucins may play a role in limiting the pathogenesis of A/E infections. MUC2 (mouse, Muc2) is the major colonic secretory mucin in humans and mice [21], [22]. In contrast to other epithelial mucins in the gut, MUC2 is synthesized specifically by goblet cells of the small and large intestine [22]. These cells constitutively produce MUC2 polymers, which are densely packaged into numerous apically-stored granules, and released into the intestinal lumen to form the structural basis of the mucus–gel layer [21], [23]. This layer is a biochemically complex medium, rich in carbohydrates, antimicrobial peptides and other proteins, as well as lipids and electrolytes [23], [24]. The depth of the mucus layer varies with the region of the intestinal tract, but is thickest in the colon and rectum, reaching over 800 µm in rodents [25]. Studies have revealed that Muc2-mediated mucus formation in the mammalian colon leads to 2 distinct sublayers; an inner layer that is firmly adherent to the intestinal mucosa, and an outer layer that can be washed off with minimal rinsing [26], [27]. Interestingly, commensal bacteria heavily colonize the outer of these two layers, whereas the inner layer is virtually sterile [27]. The mechanisms underlying the formation and function of these sublayers is still under investigation; however, studies in animal models have indicated that Muc2-dependent mucus production profoundly impacts intestinal physiology, as demonstrated in vivo with the generation of Muc2 deficient (Muc2−/− ) mice [28], which lack a mucus layer [27]. Depending on their genetic background, aged Muc2−/− mice may develop colorectal cancer [28] and/or spontaneous colitis [29]. Although the exact mechanisms that lead to these intestinal disorders are still elusive, deficiency in mucus production appears to alter the normal localization of commensal microbiota within the colon [27] as well as disrupt the mechanisms that govern epithelial [28], [30], [31] and immune homeostasis [29], [32]. Despite the role of Muc2 in regulating commensal and gut homeostasis, its role in host defense against epithelial-adherent pathogens such as A/E bacteria is not clear. In vitro studies have implicated MUC2 in limiting colonization of epithelial cells by EPEC [20], however the biological significance of this in vivo is undetermined. Indeed, considering that A/E pathogens colonize the mucosal surface and should therefore be constantly in contact with secreted Muc2, there is surprisingly little known about how these pathogens interact with Muc2 and the mucus layer in vivo. This is a critical question since the Muc2-dependent mucus layer is one of the first anatomical features bacteria such as A/E pathogens must encounter before reaching the intestinal epithelium [33]. Such early interactions could therefore profoundly influence the course of infection. The aim of our study was to use the C. rodentium model of A/E bacterial infection in Muc2-sufficient (wildtype) mice and Muc2-deficient (Muc2−/− ) mice to understand how A/E bacteria interact with Muc2 and the mucus layer in vivo, and for the first time to assess the role of these interactions in host defense against this important class of bacterial pathogens. Our studies reveal novel yet fundamental insights into how Muc2 is used by the host to control infection by an A/E bacterial pathogen. Results C. rodentium penetrates the mucus layer during infection While C. rodentium is known to infect the colonic mucosal surface by directly attaching to epithelial cells, its location with respect to the colonic mucus layer has not been previously assessed in situ. To study this, we infected C57BL/6 mice with a green-fluorescent protein (GFP)-expressing C. rodentium, and at 6 days post-infection (DPI) we euthanized mice and fixed large intestinal tissues in Carnoy's fixative, which preserves the mucus layer [34]. To maximize our ability to visualize the bacteria, we conducted dual immunostaining for GFP to label C. rodentium, and murine Muc2 to label the inner and outer mucus layer. In uninfected tissues, no GFP-staining was observed, confirming the specificity of the GFP antibody (Figure 1, top panels). However, during infection, we found GFP-C. rodentium widely spread in the outer mucus layer, as well as interspersed throughout the normally sterile inner mucus layer often in proximity to infected epithelial cells (Figure 1, bottom panels). These are the first studies to definitively show C. rodentium within and ultimately crossing both colonic mucus layers in situ. Since C. rodentium is able to circumvent the mucus barrier, we sought to more clearly define whether this Muc2-rich layer actually protects the host, by infecting mice genetically deficient in Muc2. 10.1371/journal.ppat.1000902.g001 Figure 1 Citrobacter rodentium penetrates the colonic mucus layer in vivo. Staining for GFP-expressing C. rodentium using an antibody that recognizes GFP (green), and murine Muc2 (red), with DAPI (blue) as a counterstain. No GFP-labeled C. rodentium can be seen in the mucus layers of uninfected mice (upper panels), but in infected mice, C rodentium is observed within the outer and inner mucus layer in regions where the underlying epithelium is infected (bottom panels). Right panels “a” and “b” are expanded images of corresponding boxed regions in left panels. o = outer mucus layer; i = inner mucus layer; Cr = C. rodentium; GC = goblet cell. Original magnification = 200×. Scale bar = 50 µm. Muc2-deficient mice exhibit heightened susceptibility to C. rodentium infection We first infected C57BL/6, Muc2+/+ mice and Muc2−/− mice with C. rodentium and monitored body weights and survival over the first 2 weeks of infection. Since we did not detect any significant phenotypic differences between C57BL/6 and Muc2+/+ mice following infection, we will subsequently refer to these mice as wildtype (WT) mice. As shown in Figure 2A, infected WT mice displayed a slight drop in weight at 2 DPI, followed by recovery and a progressive weight gain over the following week. In contrast, Muc2−/− mice steadily lost weight as their infection progressed. By 6 to 10 DPI Muc2−/− mice had lost on average over 15% of their initial body mass (Figure 2A). This was associated with several clinical signs of morbidity, including hunched posture, bloody diarrhea, and inactivity, to the point where they became moribund and had to be euthanized. Ultimately, depending on the infection, 80–100% of Muc2−/− mice required euthanization, compared to only 0–20% of WT mice (Figure 2B). 10.1371/journal.ppat.1000902.g002 Figure 2 Muc2−/− mice exhibit dramatic susceptibility to C. rodentium-induced morbidity and mortality. A. Body weights following C. rodentium infection of WT (n = 10) and Muc2−/− (n = 10) mice. Muc2−/− mice rapidly lose weight following C. rodentium infection. Results are representative of 2 independent experiments. B. Survival curve of WT mice (n = 10) and Muc2−/− mice (n = 10) following C. rodentium infection. Results are representative of 3 independent infections, each with 5–10 mice per group. C. Bioluminescent imaging showing WT and Muc2−/− mice at 4 DPI with a luciferase-expressing C. rodentium. The color bar is displayed on the left where red corresponds to the highest signal intensity and blue corresponds to the lowest signal intensity, with corresponding logarithmic units of light measurement (photons s−1 cm−2 seradian−1). Overall signal was significantly greater by 3–10 fold in the Muc2−/− mice vs. WT mice (*P = 0.039, students t-test, 3 mice per group). D. Enumeration of C. rodentium in stool at various times post-infection. Each data point represents one animal. Results are pooled from two separate infections. (2 DPI, *P = 0.013; 4 DPI, ***P 100%), epithelial integrity (0 = no change; 1 = <10 epithelial cells shedding per lesion; 2 = 11–20 epithelial cells shedding per lesion; 3 = epithelial ulceration; 4 = epithelial ulceration with severe crypt destruction); neutrophil and mononuclear cell infiltration (0 = none; 1 = mild; 2 = moderate; 3 = severe). The maximum score that could result from this scoring was 15. Statistical analysis Statistical significance was calculated by using either a two-tailed Student's t-test or the Mann-Whitney test unless otherwise indicated, with assistance from GraphPad Prism Software Version 4.00 (GraphPad Software, San Diego California USA, www.graphpad.com). A P value of ≤0.05 was considered significant. The results are expressed as the mean value with standard error of the mean (SEM). Gene accession numbers The following are the GeneIDs (Database: Entrez Gene) for each gene analyzed in this manuscript, given as gene name (official symbol GeneID #): TNF-α (Tnf GeneID: 21926); IL-23p19 (Il23a GeneID: 83430); IFN-γ (Ifng GeneID: 15978); IL-17A (Il17a GeneID: 16171), IL-17F (Il17f GeneID: 257630); IL-22 (Il22 GeneID: 50929); MCP-1 (Ccl2 GeneID: 20296); KC (Cxcl1 GeneID: 14825); iNOS (Nos2 GeneID: 18126) mCRAMP (Camp GeneID: 12796); Muc1 (Muc1 GeneID: 17829), Muc2 (Muc2 GeneID: 17831); Muc3/17 (Muc3 GeneID: 666339); Muc4 (Muc4 GeneID: 140474); Muc6 (GeneID: 353328); Muc13 (Muc13 GeneID: 17063); and Muc19 (Muc19 GeneID: 239611). Supporting Information Figure S1 Characterization of the inflammatory cell infiltrate within the colons of C. rodentium-infected WT and Muc2−/− mice. A. Immunostaining for infiltrating macrophages via F4/80 staining (top panels) and neutrophils via MPO staining (bottom panels) in descending colons of WT and Muc2−/− and mice. Original magnification = 200×. Scale Bar = 50 µm. B. Quantitative PCR analysis of pro-inflammatory chemokines and cytokines in the descending colons of WT and Muc2−/− mice at 6 DPI compared to their respective uninfected controls. Results averaged from 3 independent infections, with n = 2–4 mice per group. Error bars = SEM. C. Quantitative PCR analysis of genes that are associated with host-susceptibility to C. rodentium in the colons of WT and Muc2−/− mice at 6 DPI. Results are averaged from 4–5 mice per group, pooled from 2 independent infections. Error Bars = SEM. D. MPO staining as above in an ulcerated region of an infected Muc2−/− mouse, showing a dense population of neutrophils in direct contact with a large microcolony of C. rodentium (white asterisk, C. rodentium aggregate; arrowhead, MPO positive cell in indirect contact with the microcolony). Original magnification = 200×. Scale Bar = 50 µm. (8.58 MB TIF) Click here for additional data file. Figure S2 Analysis of Muc family gene expression and overall mucin content in colorectal tissues of uninfected or C. rodentium-infected WT and Muc2−/− mice. A. Quantitative PCR analysis of expression of genes encoding various Muc family members in the rectal tissues of WT and Muc2−/− mice under uninfected or infected (6 DPI) conditions. Results are presented as the average of 4–5 mice per group pooled from 2 independent infections. B. PAS staining of Carnoy's-fixed colorectal tissues of WT and Muc2−/− under uninfected or C. rodentium-infected (6 DPI) conditions. Very little mucin staining (magenta, arrows) can be seen in the epithelium or lumens of uninfected or infected Muc2−/− prior to or during infection. Results are representative of at least 3 independent infections with 2–3 mice per group. Original magnification = 100×. Scale bar = 100 µm. (4.68 MB TIF) Click here for additional data file.
