The eae gene of Citrobacter freundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia.

Transmissible murine colonic hyperplasia is characterized by proliferation of anchored stem cells in the mucosa of the descending colon of laboratory mice and is caused by Citrobacter freundii biotype 4280. This bacterium produces attaching and effacing lesions in the descending colon prior to the onset of gross hyperplasia. By mutational analysis, the chromosomal eae gene of C. freundii biotype 4280 was shown to be necessary for colonic colonization. Conversely, bacteria cured of a 65-kb plasmid, which was identified in C. freundii biotype 4280, were not attenuated for colonic colonization or for the induction of colonic hyperplasia.     

Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection

The bacterial pathogen Citrobacter rodentium belongs to a family of gastrointestinal pathogens that includes enteropathogenic and enterohemorrhagic Escherichia coli and is the causative agent of transmissible colonic hyperplasia in mice. The molecular mechanisms used by these pathogens to colonize host epithelial surfaces and form attaching and effacing (A/E) lesions have undergone intense study. In contrast, little is known about the host's immune response to these infections and its importance in tissue pathology and bacterial clearance. To address these issues, wild-type mice and mice lacking T and B lymphocytes (RAG1 knockout [KO]) were infected with C. rodentium. By day 10 postinfection (p.i.), both wild-type and RAG1 KO mice developed colitis and crypt hyperplasia, and these responses became more exaggerated in wild-type mice over the next 2 weeks, as they cleared the infection. By day 24 p.i., bacterial clearance was complete, and the colitis had subsided; however, crypt heights remained increased. In contrast, inflammatory and crypt hyperplastic responses in the RAG1 KO mice were transient, subsiding after 2 weeks. By day 24 p.i., RAG1 KO mice showed no signs of bacterial clearance and infection was often fatal. Surprisingly, despite remaining heavily infected, tissues from RAG1 KO mice surviving the acute colitis showed few signs of disease. These results thus emphasize the important contribution of the host immune response during infection by A/E bacterial pathogens. While T and/or B lymphocytes are essential for host defense against C. rodentium, they also mediate much of the tissue pathology and disease symptoms that occur during infection.     

Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia, exhibits clonality: synonymy of C. rodentium and mouse-pathogenic Escherichia coli

Citrobacter rodentium (formerly Citrobacter freundii biotype 4280 and Citrobacter genomospecies 9) was described on the basis of biochemical characterization and DNA-DNA hybridization data and is the only Citrobacter species known to possess virulence factors homologous to those of the human pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli. These virulence factors are encoded on the locus of enterocyte effacement (LEE), a pathogenicity island required for the characteristic attaching and effacing (AE) pathology seen in infection with these three pathogens. C. rodentium, which apparently infects only mice, provides a useful animal model for studying the molecular basis of AE pathology. No work has been done to assess differences in pathogenicity between C. rodentium isolates from diverse sources. Here, we report the examination of 15 C. rodentium isolates using a battery of genetic and biochemical approaches. No differences were observed between the isolates by repetitive-element sequence-based PCR analysis, biochemical analysis, and possession of LEE-specific virulence factors. These data suggest that members of the species are clonal. We further characterized an atypical E. coli strain from Japan called mouse-pathogenic E. coli (MPEC) that, in our hands, caused the same disease as C. rodentium. Applying the same battery of tests, we found that MPEC possesses LEE-encoded virulence factors and is indistinguishable from the previously characterized C. rodentium isolate DBS100. These results demonstrate that MPEC is a misclassified C. rodentium isolate and that members of this species are clonal and represent the only known attaching and effacing bacterial pathogen of mice.     

Induction of anxiety-like behavior in mice during the initial stages of infection with the agent of murine colonic hyperplasia Citrobacter rodentium

Symptoms of anxiety frequently occur concomitant to the development and persistence of inflammatory bowel disease (IBD) in patients. In the present study, we utilized an animal model of IBD, infection with Citrobacter rodentium, to determine whether the infection per se can drive anxiety-like behavior. Nine-week-old CF-1 male mice were challenged orally with either saline or C. rodentium. Early in the infective process (7-8 h later), mice were tested on a hole-board open field apparatus for anxiety-like behavior measurement. Immediately following behavioral testing, plasma samples were obtained for immune cytokine analysis and colons were excised for histological analysis. In additional animals, vagal ganglia were removed and processed for c-Fos protein detection. Challenge with C. rodentium significantly increased anxiety-like behavior as evidenced by avoidance of the center area and increased risk assessment behavior. Plasma levels of the cytokines IFN-gamma, TNF-alpha and IL-12 were not different. However vagal sensory ganglia from C. rodentium-treated animals evinced significantly more c-Fos protein-positive neurons, consistent with vagal afferent transmission of C. rodentium-related signals from gut to brain. Histological examination of the colon indicated a lack of overt inflammation at the 8 h post-challenge time point, indicating that the differences in behavior were unlikely to follow from inflammation-related stress. The results of the present study demonstrate that infection with C. rodentium can induce anxiety-like symptoms that are likely mediated via vagal sensory neurons.     

CD4+-T-cell effector functions and costimulatory requirements essential for surviving mucosal infection with Citrobacter rodentium

Citrobacter rodentium causes an attaching and effacing infection of the mouse colon. Surprisingly, protective adaptive immunity against this mucosal pathogen requires a systemic T-cell-dependent antibody response. To define CD4+ T-cell effector functions promoting this systemic defense of infected epithelial surfaces, studies were undertaken in weaning-age mice lacking costimulatory molecules CD28 or CD40L or cytokines gamma interferon (IFN-gamma) or interleukin-4 (IL-4). Adoptive transfer of CD4+ T cells from wild-type, CD28(-/-), CD40L(-/-), or IFN-gamma(-/-) donors to CD4(-/-) recipients delineated functions of these CD4+ T-cell-expressed molecules on the outcome of infection. Wild-type and IL-4(-/-) mice successfully resolved infection, while 70% of IFN-gamma(-/-) mice survived. In contrast, all CD28(-/-) mice succumbed during acute infection. While fewer than half of CD40L(-/-) mice succumbed acutely, surviving mice failed to clear infection, resulting in progressive mucosal destruction, polymicrobial sepsis, and death 1 to 2 weeks later than in CD28(-/-) mice. Downstream of CD28-mediated effects, CD4+ T-cell-expressed CD40L proved essential for generating acute pathogen-specific immunoglobulin M (IgM) and early IgG, which reduced pathogen burdens. However, deficiency of CD4+ T-cell-expressed IFN-gamma did not adversely impact survival or development of protective antibody in adoptively transferred CD4(-/-) recipients, though it impacted Th1 antibody responses. These findings demonstrate that CD4+ T-cell-expressed CD40L promotes the rapid production of protective systemic antibody during acute infection, while deficiencies of IL-4 or of CD4+ T-cell-expressed IFN-gamma can be overcome. These findings have important implications for understanding the role of T-helper-cell responses during infections involving mucosal surfaces.     

Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection

The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection.     

HpaA is essential for Helicobacter pylori colonization in mice

Infection with the human gastric pathogen Helicobacter pylori can give rise to chronic gastritis, peptic ulcer, and gastric cancer. All H. pylori strains express the surface-localized protein HpaA, a promising candidate for a vaccine against H. pylori infection. To study the physiological importance of HpaA, a mutation of the hpaA gene was introduced into a mouse-adapted H. pylori strain. To justify that the interruption of the hpaA gene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavily H. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization of H. pylori in mice.     

Focal adhesion-localization of START-GAP1/DLC1 is essential for cell motility and morphology

There is a class of GTPase activating proteins for the Rho family GTPases (RhoGAPs) that contain the steroidogenic acute regulatory protein (STAR)-related lipid transfer (START) domain. In mammals three genes encode such proteins and they are designated START-GAP1–3 or deleted in liver cancer 1–3 (DLC1–3). In this study, we examined the intracellular localization and roles of START-GAP1/DLC1 in cell motility. Immunofluorescence microscopic analysis of NRK cells and HeLa cells revealed that START-GAP1 was localized in focal adhesions. Amino acid residues 265–459 of START-GAP1 were found to be necessary for focal adhesion targeting and we name the region "the focal adhesion-targeting (FAT) domain." It was previously known that ectopic expression of START-GAP1 induced cell rounding. We demonstrated that the FAT domain of START-GAP1 was partially required for this morphological change. Furthermore, expression of this domain in HeLa cells resulted in dissociation of endogenous START-GAP1 from focal adhesions as a dominant negative modulator, reducing cell migration and spreading. Taken together, START-GAP1 is targeted to focal adhesions via the FAT domain and regulates actin rearrangement through down-regulation of active RhoA and Cdc42. Its absence from focal adhesions could, therefore, cause abnormal cell motility and spreading.     

Immunohistologic demonstration of abnormal colonic crypt cell kinetics in ulcerative colitis

A monoclonal antibody, Ki-67, that reacts with cells in the proliferative phases (G1, G2, S, and M) of the cell cycle was used in an immunohistochemical labeling reaction to examine the colonic crypt epithelium in active ulcerative colitis, inactive ulcerative colitis, and normal mucosa. The proportions of labeled cells in the lower two thirds (proliferative zone) and in the upper quarter of the crypt were determined. The proportions of Ki-67-positive crypt epithelial cells in both the proliferative zone and the upper crypt were higher in biopsy specimens from patients with active ulcerative colitis than from patients with normal mucosa or with inactive ulcerative colitis. In inactive ulcerative colitis the proportion of Ki-67-positive epithelial cells in the proliferative zone of the crypt was higher than in normal mucosa. These results are similar to those obtained in studies using tritiated thymidine to determine the proportion of cells in the DNA-synthesizing thymidine to determine the proportion of cells in the DNA-synthesizing phase of the cell cycle and suggest that immuno-histochemical staining with Ki-67 may be a practical method for measuring the proliferative activity of epithelial cells in patients with ulcerative colitis and other disorders of the gastrointestinal tract.     

Involvement of T helper type 17 and regulatory T cell activity in Citrobacter rodentium invasion and inflammatory damage

Citrobacter rodentium is a murine pathogen that transiently colonizes the lumen of the large intestine. C. rodentium induces colitis, but the relative importance and temporal induction of the T helper type 17 (Th17) and regulatory T cell (T_reg) pathways in protection from the infection and inflammation have not been assessed. Our aim was to investigate the key immunological signalling events associated with successful clearance of C. rodentium. Mice were challenged with luminescent-tagged C. rodentium and killed at days 3 (early infection), 10 (peak infection) and 21 (late infection) post-infection. Bioluminescent imaging and bacterial culture determined levels of C. rodentium. Distal colon mRNA expression of interleukin (IL)-17, IL-6, IL-1β, tumour necrosis factor (TNF)-α, forkhead box P3 (FoxP3) and ghrelin were assessed using real-time polymerase chain reaction. Results were compared with age-matched non-infected mice. Low levels of C. rodentium were found at day 3, high levels at day 10, with clearance from the majority of the mice by day 21. In the distal colon, there was up-regulation of TNF-α and FoxP3 throughout the study and increases in IL-6 and IL-17 during the peak and late stages of infection. Ghrelin expression was increased at the peak and late stages of infection. This study has characterized changes to the T helper cell pathways, following the course of C. rodentium infection in mice. There were significant immunological changes, with up-regulation of the Th17 and T_reg pathways in the distal colon and an increase in ghrelin expression compared with non-infected control mice. These changes may play a role in the pathology and clearance of C. rodentium.     

Identification and characterization of Cri1, a locus controlling mortality during Citrobacter rodentium infection in mice

Infection of inbred mouse strains with Citrobacter rodentium represents an ideal model to reveal the genetic factors controlling host resistance to noninvasive enteric bacterial pathogens. We have chosen a positional cloning approach to identify putative gene(s) that control the known difference in survival between resistant C57BL/6J and susceptible C3H/HeJ and C3H/HeOuJ mice. Our work has identified one major locus within proximal chromosome 15 that is responsible for the marked susceptibility of both C3H strains, and we formally exclude Tlr4 from control of survival to this pathogen. We have named this new host resistance locus Cri1 (Citrobacter rodentium infection 1). The Cri1 genetic interval currently spans ～16 Mb and it confers survival to the infection in a recessive manner. Transfer of the Cri1 locus from the surviving B6 mice into a congenic mouse with a C3Ou genetic background confirms its overall chromosomal localization and its highly significant effect on host survival. The C3Ou.B6-Cri1 mice thus produced have also enabled us to dissociate the control of mouse survival from the control of bacterial load early in the infection as well as from control of colonic hyperplasia.     

The pathogenicity of an enteric Citrobacter rodentium Infection is enhanced by deficiencies in the antioxidants selenium and vitamin E

The pathogenesis of a Citrobacter rodentium infection was evaluated in mice fed diets with a single deficiency in either selenium or vitamin E or with a double deficiency in both selenium and vitamin E compared to mice on nutritionally adequate diets. Mice fed the selenium- and vitamin E-deficient diet for 6 weeks had increased loads of C. rodentium in the colon and spleen, which were not observed in mice fed either of the singly deficient diets or the adequate diet. Infected mice fed the doubly deficient diet had increased colon crypt hyperplasia and an influx of infiltrating cells along with gross changes to crypt architecture, including ulceration and denuding of the epithelial layer. Cytokine and chemokine mRNA levels in the colon were measured by real-time PCR. Expression of proinflammatory cytokines and chemokines was upregulated on day 12 after infection with C. rodentium in mice fed the doubly deficient diet compared to mice fed the control diet. Heme oxygenase 1, an enzyme upregulated by oxidative stress, also was more highly induced in infected mice fed the doubly deficient diet. Production of C. rodentium antigen-specific IgM and IgG antibodies was not affected by feeding the doubly deficient diet. The results indicated that selenium and vitamin E play an important role in host resistance and in the pathology induced by C. rodentium, an infection that mimics disease caused by common food-borne bacterial pathogens in humans.     

Tight junctional disruption and apoptosis in an in vitro model of Citrobacter rodentium infection

The murine model of Citrobacter rodentium infection has been used to complement in vitro studies of enteropathogenic Escherichia coli (EPEC) infections of human intestinal epithelial cells (IECs). However, the differences in epithelial cell responses between these two models are not fully understood. We used an in vitro model of C. rodentium infection to examine important, yet incompletely understood, cellular responses of murine IECs to this pathogen. C. rodentium attached to CMT-93 cells and disrupted their tight junctional expression of claudins-4 and -5. This was associated with a loss of barrier function that required live bacteria and was partially prevented by the inhibition of Rho kinase. Furthermore, C. rodentium caused an upregulation of IEC apoptosis that was associated with the cytoplasmic accumulation of apoptosis-inducing factor, but not with the activation of caspase-3. These studies demonstrate for the first time that C. rodentium affects murine IECs in ways that may be similar, but distinct, to the effects of EPEC on human IECs.     

Platelet endothelial cell adhesion molecule-1 is expressed in adenoidal crypt epithelial cells

The adenoidal epithelial crypt is a potential site of antigen transport from pharyngeal lumen to adenoidal tissue. The base of the crypt is consistently infiltrated with leucocytes, forming a reticular lymphoepithelial structure. To evaluate mechanisms that possibly mediate leucocyte infiltration, expressions of leucocyte adhesion molecules, such as platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31), vascular cell adhesion molecule-1 (VCAM-1) (CD106) and intercellular adhesion molecule-1 (ICAM-1) (CD54), were studied in the adenoidal epithelial crypt. Epithelial cells in the outer opening of the adenoidal crypt were positive for VCAM-1, whereas epithelial cells at the base of the crypt were positive for PECAM-1. Isolated ICAM-1-expressing cells were found throughout the epithelial crypt. Double immunofluorescence staining revealed that the epithelial cells positive for PECAM-1 or VCAM-1 were positive for cytokeratin. The expression of PECAM-1 in the base and VCAM-1 at the orifice of the adenoidal epithelial crypt implies that the base and the orifice of the crypt have a distinct ability to recruit leucocytes. Epithelial cells expressing PECAM-1 may have a role in the formation of the reticular lymphoepithelial structure in the epithelial crypt.     

The lpf gene cluster for long polar fimbriae is not involved in adherence of enteropathogenic Escherichia coli or virulence of Citrobacter rodentium

Using the enteropathogenic Escherichia coli (EPEC) genome sequence, we found that EPEC E2348/69 has an lpfABCDE gene cluster homologous (about 60% identical at the protein level) to the Salmonella long polar fimbria (LPF) operon. To determine whether this operon is essential for adherence, the lpfABCD(E2)(3) genes were deleted from EPEC strain E2348/69 by allelic exchange. Analysis of the resulting EPECDeltalpfABCD(E23) strain showed no change in adherence to HeLa cells or to human intestinal biopsy cells in the in vitro organ culture (IVOC) system compared to the wild type. Sera from volunteers experimentally infected with E2348/69 showed no antibody response to the major subunit protein, LpfA. These results suggested that the lpf(E23) gene cluster is not necessary for EPEC adherence and attaching/effacing (A/E) lesion formation on human biopsy samples and is not expressed during human infection. We also identified an lpf gene cluster in Citrobacter rodentium strain ICC168 (lpf(cr)). A DeltalpfA(cr) mutant of ICC168 retained wild-type adherence and A/E lesion-forming activity on HeLa cells. C3H/HeJ mice were infected with a wild-type C. rodentium strain and its lpfA(cr) isogenic mutant. Both strains were recovered at high levels in stools, and there were no significant differences between the groups both in terms of the number of CFU/organ (colon and cecum) and in terms of the amount of hyperplasia, as measured by weight. Similar results were observed in a second mouse strain, C57BL/6. These data suggest that in addition to playing no apparent role in EPEC pathogenesis, lpf(cr) is not required for C. rodentium virulence in either the C3H/HeJ or C57BL/6 mouse model.     

gamma-Glutamyltransferase is a Helicobacter pylori virulence factor but is not essential for colonization

The contribution of glutamyl transpeptidase (GGT) (gamma-glutamyltransferase [EC 2. 3. 2. 2]) to Helicobacter pylori virulence was investigated in piglets and mice using GGT-deficient isogenic strains. All animals became colonized. However, the bacterial load was significantly lower for mutant bacteria than for parent strains. These results suggest that GGT activity provides an advantage to H. pylori in colonization.