The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing and succumbing to the infection, respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in relocalization of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumens of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon that we term antibiotic-induced bacterial commensalization (AIBC). AIBC C. rodentium was well tolerated by the host, which showed few signs of inflammation; passaged AIBC C. rodentium robustly infected naive C3H/HeN mice, suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalization in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus, which inhibited the growth of C. rodentium in vitro in a contact-dependent manner and the luminal growth of AIBC C. rodentium in vivo. Overall, our data suggest that commensal species can confer colonization resistance to closely related pathogenic species. IMPORTANCE Gut bacterial infections involve three-way interactions between virulence factors, the host immune responses, and the microbiome. While the microbiome erects colonization resistance barriers, pathogens employ virulence factors to overcome them. Treating mice infected with kanamycin-resistant Citrobacter rodentium with kanamycin caused displacement of the pathogen from the colonic mucosa to the cecal lumen. Following withdrawal of the kanamycin treatment, 65% of the mice were persistently colonized by C. rodentium, which seemed to downregulate virulence factor expression. In this model of luminal gut colonization, 35% of mice were refractory to stable C. rodentium colonization, suggesting that their microbiotas were able to confer colonization resistance. We identify a commensal bacterium of the Citrobacter genus, C. amalonaticus, which inhibits C. rodentium growth in vitro and in vivo. These results show that the line separating commensal and pathogenic lifestyles is thin and multifactorial and that commensals may play a major role in combating enteric infection.

One of the major contributors to child mortality in the world is diarrheal diseases, with an estimated 800,000 deaths per year. Many pathogens are causative agents of these illnesses, including the enteropathogenic (EPEC) or enterohemorrhagic (EHEC) forms of Escherichia coli. These bacteria are characterized by their ability to cause attaching and effacing lesions in the gut mucosa. Although much has been learned about the pathogenicity of these organisms and the immune response against them, the role of the intestinal microbiota during these infections is not well characterized. Infection of mice with E. coli requires pre-treatment with antibiotics in most mouse models, which hinders the study of the microbiota in an undisturbed environment. Using Citrobacter rodentium as a murine model for attaching and effacing bacteria, we show that C57BL/6 mice deficient in granzyme B expression are highly susceptible to severe disease caused by C. rodentium infection. Although a previous publication from our group shows that granzyme B-deficient CD4+ T cells are partially responsible for this phenotype, in this report we present data demonstrating that the microbiota, in particular members of the order Turicibacterales, have an important role in conferring resistance. Mice deficient in Turicibacter sanguinis have increased susceptibility to severe disease. However, when these mice are co-housed with resistant mice, or colonized with T. sanguinis, susceptibility to severe infection is reduced. These results clearly suggest a critical role for this commensal in the protection against entero-pathogens.

Mucosal immunity is critical to host protection from enteric pathogens and must be carefully controlled to prevent immunopathology. Regulation of immune responses can occur through a diverse range of mechanisms including bi-directional communication with the neurons. Among which include specialized sensory neurons that detect noxious stimuli due to the expression of transient receptor potential vanilloid receptor 1 (TRPV1) ion channel and have a significant role in the coordination of host-protective responses to enteric bacterial pathogens. Here we have used the mouse-adapted attaching and effacing pathogen Citrobacter rodentium to assess the specific role of the TRPV1 channel in coordinating the host response. TRPV1 knockout (TRPV1-/-) mice had a significantly higher C. rodentium burden in the distal colon and fecal pellets compared to wild-type (WT) mice. Increased bacterial burden was correlated with significantly increased colonic crypt hyperplasia and proliferating intestinal epithelial cells in TRPV1-/- mice compared to WT. Despite the increased C. rodentium burden and histopathology, the recruitment of colonic T cells producing IFNγ, IL-17, or IL-22 was similar between TRPV1-/- and WT mice. In evaluating the innate immune response, we identified that colonic neutrophil recruitment in C. rodentium infected TRPV1-/- mice was significantly reduced compared to WT mice; however, this was independent of neutrophil development and maturation within the bone marrow compartment. TRPV1-/- mice were found to have significantly decreased expression of the neutrophil-specific chemokine Cxcl6 and the adhesion molecules Icam1 in the distal colon compared to WT mice. Corroborating these findings, a significant reduction in ICAM-1 and VCAM-1, but not MAdCAM-1 protein on the surface of colonic blood endothelial cells from C. rodentium infected TRPV1-/- mice compared to WT was observed. These findings demonstrate the critical role of TRPV1 in regulating the host protective responses to enteric bacterial pathogens, and mucosal immune responses.

Endoplasmic reticulum (ER) stress is intimately linked with inflammation in response to pathogenic infections. ER stress occurs when cells experience a buildup of misfolded or unfolded protein during times of perturbation, such as infections, which facilitates the unfolded protein response (UPR). The UPR involves multiple host pathways in an attempt to reestablish homeostasis, which oftentimes leads to inflammation and cell death if unresolved. The UPR is activated to help resolve some bacterial infections, and the IRE1α pathway is especially critical in mediating inflammation. To understand the role of the IRE1α pathway of the UPR during enteric bacterial infection, we employed Citrobacter rodentium to study host-pathogen interactions in intestinal epithelial cells and the murine gastrointestinal (GI) tract. C. rodentium is an enteric mouse pathogen that is similar to the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), for which we have limited small-animal models. Here, we demonstrate that both C. rodentium and EPEC induced the UPR in intestinal epithelial cells. UPR induction during C. rodentium infection correlated with the onset of inflammation in bone marrow-derived macrophages (BMDMs). Our previous work implicated IRE1α and NOD1/2 in ER stress-induced inflammation, which we observed were also required for proinflammatory gene induction during C. rodentium infection. C. rodentium induced IRE1α-dependent inflammation in mice, and inhibiting IRE1α led to a dysregulated inflammatory response and delayed clearance of C. rodentium. This study demonstrates that ER stress aids inflammation and clearance of C. rodentium through a mechanism involving the IRE1α-NOD1/2 axis.

In vitro and in vivo studies suggest that selected Bifidobacterium bifidum strains sustain intestinal homeostasis. This study aimed to examine whether the administration of B. bifidum MIMBb75 (BB75) attenuates Citrobacter rodentium infection, a murine model for enteric infection and inflammatory bowel disease in humans. C57Bl6/J mice were randomized to receive BB75 daily starting before or after C. rodentium infection. BB75 load and infection kinetics were monitored. On day 10 post-infection (p.i.), histological parameters of the large intestine were assessed. Barrier integrity was evaluated by pathogen translocation to secondary organs and in vivo permeability test. Fecal C. rodentium load peaked at 1010 CFU/g at day 10 p.i., with clearance at day 24 p.i., regardless of probiotic treatment. BB75 administration resulted in 10⁷ cells/g of feces with no effect of timing of administration. BB75 treatment did not attenuate C. rodentium-induced crypt hyperplasia nor inflammation. C. rodentium and BB75 can co-exist in the gut with no mutual displacement. However, BB75 cannot counteract C. rodentium pathology. Our findings provide insight for the understanding of probiotics behavior and their clinical relevance in intestinal inflammation.

The mouse enteric pathogen Citrobacter rodentium, like its human counterpart, enteropathogenic Escherichia coli, causes attaching and effacing lesions in the intestinal epithelium of its host. This phenotype requires virulence factors encoded by the locus for enterocyte effacement (LEE) pathogenicity island. For timely expression of these virulence determinants at the site of infection and for efficient delivery of some virulence factors into epithelial cells, C. rodentium utilizes a positive regulatory loop involving the LEE-encoded regulatory proteins Ler, GrlA and GrlR to control LEE expression. Several transcription factors not encoded by LEE, some of which respond to specific environmental signals, also participate in this regulatory loop. Recently, we identified a non-LEE encoded, AraC-like regulatory protein, RegA, which plays a key role in the ability of C. rodentium to colonize the intestine. RegA functions by activating the transcription of a number of horizontally acquired operons encoding virulence-associated factors, such as autotransporters, fimbriae, a dispersin-like protein and its transporter. In addition, RegA represses transcription of a number of housekeeping genes. Importantly, RegA requires a gut-specific environmental signal, bicarbonate, to exert its effects on gene expression. In our proposed model, when C. rodentium senses bicarbonate ions in the gastrointestinal tract, RegA directs the bacterium to reduce the production of proteins involved in normal cellular functions, while enhancing the production of factors required for colonization and virulence.

Mucosal immunity is critical to host protection from enteric pathogens and must be carefully controlled to prevent immunopathology. Regulation of immune responses can occur through a diverse range of mechanisms including bi-directional communication with neurons. Among which include specialized sensory neurons that detect noxious stimuli due to the expression of transient receptor potential vanilloid receptor 1 (TRPV1) ion channel and have a significant role in the coordination of host-protective responses to enteric bacterial pathogens. Here we have used the mouse-adapted attaching and effacing pathogen Citrobacter rodentium to assess the specific role of TRPV1 in coordinating the host response. TRPV1 knockout (TRPV1-/-) mice had a significantly higher C. rodentium burden in the distal colon and fecal pellets compared to wild-type (WT) mice. Increased bacterial burden was correlated with significantly increased colonic crypt hyperplasia and proliferating intestinal epithelial cells in TRPV1-/- mice compared to WT. Despite the increased C. rodentium burden and histopathology, the recruitment of colonic T cells producing IFNγ, IL-17, or IL-22 was similar between TRPV1-/- and WT mice. In evaluating the innate immune response, we identified that colonic neutrophil recruitment in C. rodentium infected TRPV1-/- mice was significantly reduced compared to WT mice; however, this was independent of neutrophil development and maturation within the bone marrow compartment. TRPV1-/- mice were found to have significantly decreased expression of the neutrophil-specific chemokine Cxcl6 and the adhesion molecules Icam1 in the distal colon compared to WT mice. Corroborating these findings, a significant reduction in ICAM-1 and VCAM-1, but not MAdCAM-1 protein on the surface of colonic blood endothelial cells from C. rodentium infected TRPV1-/- mice compared to WT was observed. These findings demonstrate the critical role of TRPV1 in regulating the host protective responses to enteric bacterial pathogens, and mucosal immune responses.

We evaluated the protective effects of fermented dairy products (FDPs) in an infection model, using the mouse pathogen Citrobacter rodentium (CR). Treatment of mice with FDP formulas A, B, and C or a control product did not affect CR colonization, organ specificity, or attaching and effacing lesion formation. Fermented dairy product A (FDP-A), but neither the supernatant from FDP-A nor β-irradiated (IR) FDP-A, caused a significant reduction in colonic crypt hyperplasia and CR-associated pathology. Profiling the gut microbiota revealed that IR-FDP-A promoted higher levels of phylotypes belonging to Alcaligenaceae and a decrease in Lachnospiraceae (Ruminococcus) during CR infection. Conversely, FDP-A prevented a decrease in Ruminococcus and increased Turicibacteraceae (Turicibacter). Importantly, loss of Ruminococcus and Turicibacter has been associated with susceptibility to dextran sodium sulfate-induced colitis. Our results demonstrate that viable bacteria in FDP-A reduced CR-induced colonic crypt hyperplasia and prevented the loss of key bacterial genera that may contribute to disease pathology.

Whether type III secretion system (T3SS) effector proteins encoded by Gram-negative bacterial pathogens have intra-bacterial activities is an important and emerging area of investigation. Gram-negative bacteria interact with their mammalian hosts by using secretion systems to inject virulence proteins directly into infected host cells. Many of these injected protein effectors are enzymes that modify the structure and inhibit the function of mammalian proteins. The underlying dogma is that T3SS effectors are inactive until they are injected into host cells, where they then fold into their active conformations. We previously observed that the T3SS effectors NleB and SseK1 glycosylate Citrobacter rodentium and Salmonella enterica proteins, respectively, leading to enhanced resistance to environmental stress. Here, we sought to extend these studies to determine whether the T3SS effector protease NleC is also active within C. rodentium. To do this, we expressed the best-characterized mammalian substrate of NleC, the NF-κB p65 subunit in C. rodentium and monitored its proteolytic cleavage as a function of NleC activity. Intra-bacterial p65 cleavage was strictly dependent upon NleC. A p65 mutant lacking the known CE cleavage motif was resistant to NleC. Thus, we conclude that, in addition to NleB, NleC is also enzymatically active within C. rodentium.

We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course.

NADPH oxidase (NOX) is a membrane-bound enzyme complex that generates reactive oxygen species (ROS). Mutations in NOX subunit genes have been implicated in the pathogenesis of inflammatory bowel disease (IBD), indicating a crucial role for ROS in regulating host immune responses. In this study, we utilize genetically deficient mice to investigate whether defects in p40phox , one subunit of NOX, impair host immune response in the intestine and aggravate disease in an infection-based (Citrobacter rodentium) model of colitis. We show that p40phox deficiency does not increase susceptibility of mice to C. rodentium infection, as no differences in body weight loss, bacterial clearance, colonic pathology, cytokine production, or immune cell recruitment were observed between p40phox-/- and wild-type mice. Interestingly, higher IL-10 levels were observed in the supernatants of MLN cells and splenocytes isolated from infected p40phox -deficient mice. Further, a higher expression level of inducible nitric oxide synthase (iNOS) was also noted in mice lacking p40phox . In contrast to wild-type mice, p40phox-/- mice exhibited greater NO production after LPS or bacterial antigen re-stimulation. These results suggest that p40phox-/- mice do not develop worsened colitis. While the precise mechanisms are unclear, it may involve the observed alteration in cytokine responses and enhancement in levels of iNOS and NO.

Inflammatory bowel disease (IBD) has become a global public health challenge. Our previous study showed that barley leaf (BL) significantly reduces Citrobacter-rodentium (CR)-induced colitis, but its mechanism remains elusive. Thus, in this study, we used non-targeted metabolomics techniques to search for potentially effective metabolites. Our results demonstrated that dietary supplementation with BL significantly enriched arginine and that arginine intervention significantly ameliorated CR-induced colitis symptoms such as reduced body weight, shortened colon, wrinkled cecum, and swollen colon wall in mice; in addition, arginine intervention dramatically ameliorated CR-induced histopathological damage to the colon. The gut microbial diversity analysis showed that arginine intervention significantly decreased the relative abundance of CR and significantly increased the relative abundance of Akkermansia, Blautia, Enterorhabdus, and Lachnospiraceae, which modified the CR-induced intestinal flora disorder. Notably, arginine showed a dose-dependent effect on the improvement of colitis caused by CR.

Western-style diet (WSD), which is high in fat and low in fiber, lacks nutrients to support gut microbiota. Consequently, WSD reduces microbiota density and promotes microbiota encroachment, potentially influencing colonization resistance, immune system readiness, and thus host defense against pathogenic bacteria. Here we examined the impact of WSD on infection and colitis in response to Citrobacter rodentium. We observed that, relative to mice consuming standard rodent grain-based chow (GBC), feeding WSD starkly altered the dynamics of Citrobacter infection, reducing initial colonization and inflammation but frequently resulting in persistent infection that associated with low-grade inflammation and insulin resistance. WSD's reduction in initial Citrobacter virulence appeared to reflect that colons of GBC-fed mice contain microbiota metabolites, including short-chain fatty acids, especially acetate, that drive Citrobacter growth and virulence. Citrobacter persistence in WSD-fed mice reflected inability of resident microbiota to out-compete it from the gut lumen, likely reflecting the profound impacts of WSD on microbiota composition. These studies demonstrate potential of altering microbiota and their metabolites by diet to impact the course and consequence of infection following exposure to a gut pathogen.

Citrobacter rodentium is an attaching and effacing intestinal murine pathogen which shares similar virulence strategies with the human pathogens enteropathogenic- and enterohemorrhagic Escherichia coli to infect their host. C. rodentium is spontaneously cleared by healthy wild-type (WT) mice whereas mice lacking Muc2 or specific immune regulatory genes demonstrate an impaired ability to combat the pathogen. Here we demonstrate that apical formyl peptide receptor 2 (Fpr2) expression increases in colonic epithelial cells during C. rodentium infection. Using a conventional inoculum dose of C. rodentium, both WT and Fpr2-/- mice were infected and displayed similar signs of disease, although Fpr2-/- mice recovered more slowly than WT mice. However, Fpr2-/- mice exhibited increased susceptibility to C. rodentium colonization in response to low dose infection: 100% of the Fpr2-/- and 30% of the WT mice became colonized and Fpr2-/- mice developed more severe colitis and more C. rodentium were in contact with the colonic epithelial cells. In line with the larger amount of C. rodentium detected in the spleen in Fpr2-/- mice, more C. rodentium and enteropathogenic Escherichia coli translocated across an in vitro mucosal surface to the basolateral compartment following FPR2 inhibitor treatment. Fpr2-/- mice also lacked the striated inner mucus layer that was present in WT mice. Fpr2-/- mice had decreased mucus production and different mucin O-glycosylation in the colon compared to WT mice, which may contribute to their defect inner mucus layer. Thus, Fpr2 contributes to protection against infection and influence mucus production, secretion and organization.

The complete genomes of two virulent phages infecting Citrobacter rodentium are reported here for the first time. Both bacteriophages were isolated from local sewage treatment plant effluents. Genome analyses revealed a close relationship between both phages and allowed their classification as members of the Autographivirinae subfamily in the T7-like genus.

The incidence and prevalence of inflammatory bowel disease (IBD) have been increasing globally and progressively in recent decades. Barley leaf (BL) is a nutritional supplement that is shown to have health-promoting effects on intestinal homeostasis. Our previous study demonstrated that BL could significantly attenuate Citrobacter rodentium (CR)-induced colitis, but whether it exerts a prophylactic or therapeutic effect remains elusive. In this study, we supplemented BL before or during CR infestation to investigate which way BL acts. The results showed that BL supplementation prior to infection significantly reduced the disease activity index (DAI) score, weight loss, colon shortening, colonic wall swelling, and transmissible murine colonic hyperplasia. It significantly reduced the amount of CR in the feces and also markedly inhibited the extraintestinal transmission of CR. Meanwhile, it significantly reduced the levels and expression of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFNγ), and interleukin-1β (IL1β). In addition, pretreatment with BL improved CR-induced gut microbiota dysbiosis by reducing the content of Proteobacteria, while increasing the content of Lactobacillus. In contrast, the effect of BL supplementation during infestation on the improvement of CR-induced colitis was not as good as that of pretreatment with BL. In conclusion, BL protects against CR-caused colitis in a preventive manner.

Peptidyl arginine deiminase-4 (PAD4) is indispensable for generation of neutrophil extracellular traps (NETs), which can provide antimicrobial effects during host innate immune response; however, the role of PAD4 against gastrointestinal infection is largely unknown. Herein, we challenged PAD4-deficient (Pad4-/-) mice and wild-type (WT) littermates with Citrobacter rodentium (CR), and investigated bacteria clearance and gut pathology. Luminal colonization of CR in Pad4-/- mice peaked between 11-14 days post-infection, whereas WT mice suppressed the infection by 14 days. We demonstrated that Pad4-/- mice were unable to form NETs, whereas WT mice showed increased NETs formation in the colon during infection. Pad4-/- mice showed aggravated CR-associated inflammation as indicated by elevated systemic and colonic pro-inflammatory markers. Histological analysis revealed that transmissible colonic hyperplasia, goblet cell depletion, and apoptotic cell death were more pronounced in the colon of CR-infected Pad4-/- mice. Treating WT mice with deoxyribonuclease I, which can disrupt NETs generation, recapitulated the exacerbated CR infection and gut pathology associated with the loss of PAD4. Administration of the PAD4 inhibitor, Cl-amidine also aggravated CR infection, but to a lesser extent. Taken together, our findings highlight the importance of PAD4 in the mucosal clearance of CR and in resolving gut-associated inflammation.

Food poisoning is one of the leading causes of morbidity and mortality in the world. Citrobacter rodentium is an enteric pathogen which attaches itself to enterocytes and induces attachment and effacing (A/E) lesions. The ability of the bacterium to cause infection requires subversion of the host actin cytoskeleton. Rac-dependent actin polymerization is activated by a guanine nucleotide exchange factor known as Dedicator of cytokinesis 2 (DOCK2). However, the role of DOCK2 in infectious disease is largely unexplored. Here, we found that mice lacking DOCK2 were susceptible to C. rodentium infection. These mice harbored increased levels of C. rodentium bacteria, showed more pronounced weight loss and inflammation-associated pathology, and were prone to bacterial dissemination to the systemic organs compared with wild-type mice. We found that mice lacking DOCK2 were more susceptible to C. rodentium attachment to intestinal epithelial cells. Therefore, our results underscored an important role of DOCK2 for gastrointestinal immunity to C. rodentium infection.

The Gram-negative enteric bacterium Citrobacter rodentium is a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. For C. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novel C. rodentium colonization factor, called gut colonization fimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. A gcfA mutant shows a severe colonization defect within the first 10 days of infection. The gcf promoter is not active in C. rodentium under several in vitro growth conditions; however, it is readily expressed in a C. rodentium Δhns1 mutant lacking the closest ortholog of the Escherichia coli histone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region of gcf, further supporting its direct role as a repressor of the gcf promoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. The gcf operon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures.

Citrobacter rodentium is a natural mouse pathogen that causes attaching and effacing (A/E) lesions. It shares a common virulence strategy with the clinically significant human A/E pathogens enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) and is widely used to model this route of pathogenesis. We previously reported the complete genome sequence of C. rodentium ICC168, where we found that the genome displayed many characteristics of a newly evolved pathogen. In this study, through PFGE, sequencing of isolates showing variation, whole genome transcriptome analysis and examination of the mobile genetic elements, we found that, consistent with our previous hypothesis, the genome of C. rodentium is unstable as a result of repeat-mediated, large-scale genome recombination and because of active transposition of mobile genetic elements such as the prophages. We sequenced an additional C. rodentium strain, EX-33, to reveal that the reference strain ICC168 is representative of the species and that most of the inactivating mutations were common to both isolates and likely to have occurred early on in the evolution of this pathogen. We draw parallels with the evolution of other bacterial pathogens and conclude that C. rodentium is a recently evolved pathogen that may have emerged alongside the development of inbred mice as a model for human disease.