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.

Non-invasive mapping of the gastrointestinal microbiota identifies children with inflammatory bowel disease.

BACKGROUND: Pediatric inflammatory bowel disease (IBD) is challenging to diagnose because of the non-specificity of symptoms; an unequivocal diagnosis can only be made using colonoscopy, which clinicians are reluctant to recommend for children. Diagnosis of pediatric IBD is therefore frequently delayed, leading to inappropriate treatment plans and poor outcomes. We investigated the use of 16S rRNA sequencing of fecal samples and new analytical methods to assess differences in the microbiota of children with IBD and other gastrointestinal disorders. METHODOLOGY/PRINCIPAL FINDINGS: We applied synthetic learning in microbial ecology (SLiME) analysis to 16S sequencing data obtained from i) published surveys of microbiota diversity in IBD and ii) fecal samples from 91 children and young adults who were treated in the gastroenterology program of Children's Hospital (Boston, USA). The developed method accurately distinguished control samples from those of patients with IBD; the area under the receiver-operating-characteristic curve (AUC) value was 0.83 (corresponding to 80.3% sensitivity and 69.7% specificity at a set threshold). The accuracy was maintained among data sets collected by different sampling and sequencing methods. The method identified taxa associated with disease states and distinguished patients with Crohn's disease from those with ulcerative colitis with reasonable accuracy. The findings were validated using samples from an additional group of 68 patients; the validation test identified patients with IBD with an AUC value of 0.84 (e.g. 92% sensitivity, 58.5% specificity). CONCLUSIONS/SIGNIFICANCE: Microbiome-based diagnostics can distinguish pediatric patients with IBD from patients with similar symptoms. Although this test can not replace endoscopy and histological examination as diagnostic tools, classification based on microbial diversity is an effective complementary technique for IBD detection in pediatric patients.

Enteric infection with Citrobacter rodentium induces coagulative liver necrosis and hepatic inflammation prior to peak infection and colonic disease.

Acute and chronic forms of inflammation are known to affect liver responses and susceptibility to disease and injury. Furthermore, intestinal microbiota has been shown critical in mediating inflammatory host responses in various animal models. Using C. rodentium, a known enteric bacterial pathogen, we examined liver responses to gastrointestinal infection at various stages of disease pathogenesis. For the first time, to our knowledge, we show distinct liver pathology associated with enteric infection with C. rodentium in C57BL/6 mice, characterized by increased inflammation and hepatitis index scores as well as prominent periportal hepatocellular coagulative necrosis indicative of thrombotic ischemic injury in a subset of animals during the early course of C. rodentium pathogenesis. Histologic changes in the liver correlated with serum elevation of liver transaminases, systemic and liver resident cytokines, as well as signal transduction changes prior to peak bacterial colonization and colonic disease. C. rodentium infection in C57BL/6 mice provides a potentially useful model to study acute liver injury and inflammatory stress under conditions of gastrointestinal infection analogous to enteropathogenic E. coli infection in humans.

Allele- and tir-independent functions of intimin in diverse animal infection models.

Upon binding to intestinal epithelial cells, enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium trigger formation of actin pedestals beneath bound bacteria. Pedestal formation has been associated with enhanced colonization, and requires intimin, an adhesin that binds to the bacterial effector translocated intimin receptor (Tir), which is translocated to the host cell membrane and promotes bacterial adherence and pedestal formation. Intimin has been suggested to also promote cell adhesion by binding one or more host receptors, and allelic differences in intimin have been associated with differences in tissue and host specificity. We assessed the function of EHEC, EPEC, or C. rodentium intimin, or a set of intimin derivatives with varying Tir-binding abilities in animal models of infection. We found that EPEC and EHEC intimin were functionally indistinguishable during infection of gnotobiotic piglets by EHEC, and that EPEC, EHEC, and C. rodentium intimin were functionally indistinguishable during infection of C57BL/6 mice by C. rodentium. A derivative of EHEC intimin that bound Tir but did not promote robust pedestal formation on cultured cells was unable to promote C. rodentium colonization of conventional mice, indicating that the ability to trigger actin assembly, not simply to bind Tir, is required for intimin-mediated intestinal colonization. Interestingly, streptomycin pre-treatment of mice eliminated the requirement for Tir but not intimin during colonization, and intimin derivatives that were defective in Tir-binding still promoted colonization of these mice. These results indicate that EPEC, EHEC, and C. rodentium intimin are functionally interchangeable during infection of gnotobiotic piglets or conventional C57BL/6 mice, and that whereas the ability to trigger Tir-mediated pedestal formation is essential for colonization of conventional mice, intimin provides a Tir-independent activity during colonization of streptomycin pre-treated mice.

Alkyl hydroperoxide reductase is required for Helicobacter cinaedi intestinal colonization and survival under oxidative stress in BALB/c and BALB/c interleukin-10-/- mice.

Helicobacter cinaedi, a common human intestinal bacterium, has been implicated in various enteric and systemic diseases in normal and immunocompromised patients. Protection against oxidative stress is a crucial component of bacterium-host interactions. Alkyl hydroperoxide reductase C (AhpC) is an enzyme responsible for detoxification of peroxides and is important in protection from peroxide-induced stress. H. cinaedi possesses a single ahpC, which was investigated with respect to its role in bacterial survival during oxidative stress. The H. cinaedi ahpC mutant had diminished resistance to organic hydroperoxide toxicity but increased hydrogen peroxide resistance compared with the wild-type (WT) strain. The mutant also exhibited an oxygen-sensitive phenotype and was more susceptible to killing by macrophages than the WT strain. In vivo experiments in BALB/c and BALB/c interleukin-10 (IL-10)(-/-) mice revealed that the cecal colonizing ability of the ahpC mutant was significantly reduced. The mutant also had diminished ability to induce bacterium-specific immune responses in vivo, as shown by immunoglobulin (IgG2a and IgG1) serum levels. Collectively, these data suggest that H. cinaedi ahpC not only contributes to protecting the organism against oxidative stress but also alters its pathogenic properties in vivo.

The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.

Multivariate modeling identifies neutrophil- and Th17-related factors as differential serum biomarkers of chronic murine colitis.

BACKGROUND: Diagnosis of chronic intestinal inflammation, which characterizes inflammatory bowel disease (IBD), along with prediction of disease state is hindered by the availability of predictive serum biomarker. Serum biomarkers predictive of disease state will improve trials for therapeutic intervention, and disease monitoring, particularly in genetically susceptible individuals. Chronic inflammation during IBD is considered distinct from infectious intestinal inflammation thereby requiring biomarkers to provide differential diagnosis. To address whether differential serum biomarkers could be identified in murine models of colitis, immunological profiles from both chronic spontaneous and acute infectious colitis were compared and predictive serum biomarkers identified via multivariate modeling. METHODOLOGY/PRINCIPAL FINDINGS: Discriminatory multivariate modeling of 23 cytokines plus chlorotyrosine and nitrotyrosine (protein adducts from reactive nitrogen species and hypochlorite) in serum and tissue from two murine models of colitis was performed to identify disease-associated biomarkers. Acute C. rodentium-induced colitis in C57BL/6J mice and chronic spontaneous Helicobacter-dependent colitis in TLR4(-/-) x IL-10(-/-) mice were utilized for evaluation. Colon profiles of both colitis models were nearly identical with chemokines, neutrophil- and Th17-related factors highly associated with intestinal disease. In acute colitis, discriminatory disease-associated serum factors were not those identified in the colon. In contrast, the discriminatory predictive serum factors for chronic colitis were neutrophil- and Th17-related factors (KC, IL-12/23p40, IL-17, G-CSF, and chlorotyrosine) that were also elevated in colon tissue. Chronic colitis serum biomarkers were specific to chronic colitis as they were not discriminatory for acute colitis. CONCLUSIONS/SIGNIFICANCE: Immunological profiling revealed strikingly similar colon profiles, yet distinctly different serum profiles for acute and chronic colitis. Neutrophil- and Th17-related factors were identified as predictive serum biomarkers of chronic colitis, but not acute colitis, despite their presence in colitic tissue of both diseases thereby demonstrating the utility of mathematical modeling for identifying disease-associated serum biomarkers.

Mutagenic potency of Helicobacter pylori in the gastric mucosa of mice is determined by sex and duration of infection.

Helicobacter pylori is a human carcinogen, but the mechanisms evoked in carcinogenesis during this chronic inflammatory disease remain incompletely characterized. We determined whether chronic H. pylori infection induced mutations in the gastric mucosa of male and female gpt delta C57BL/6 mice infected for 6 or 12 mo. Point mutations were increased in females infected for 12 mo. The mutation frequency in this group was 1.6-fold higher than in uninfected mice of both sexes (P < 0.05). A:T-to-G:C transitions and G:C-to-T:A transversions were 3.8 and 2.0 times, respectively, more frequent in this group than in controls. Both mutations are consistent with DNA damage induced by oxidative stress. No increase in the frequency of deletions was observed. Females had more severe gastric lesions than males at 6 mo postinfection (MPI; P < 0.05), but this difference was absent at 12 MPI. In all mice, infection significantly increased expression of IFNgamma, IL-17, TNFalpha, and iNOS at 6 and 12 mo, as well as H. pylori-specific IgG1 levels at 12 MPI (P < 0.05) and IgG2c levels at 6 and 12 MPI (P < 0.01 and P < 0.001). At 12 MPI, IgG2c levels in infected females were higher than at 6 MPI (P < 0.05) and also than those in infected males at 12 MPI (P < 0.05). Intensity of responses was mediated by sex and duration of infection. Lower H. pylori colonization indicated a more robust host response in females than in males. Earlier onset of severe gastric lesions and proinflammatory, Th1-biased responses in female C57BL/6 mice may have promoted mutagenesis by exposing the stomach to prolonged oxidative stress.

Nitric oxide, oxygen, and superoxide formation and consumption in macrophages and colonic epithelial cells.

Knowledge of the rates at which macrophages and epithelial cells synthesize NO is critical for predicting the concentrations of NO and other reactive nitrogen species in colonic crypts during inflammation, and elucidating the linkage between inflammatory bowel disease, NO, and cancer. Macrophage-like RAW264.7 cells, primary bone marrow-derived macrophages (BMDM), and HCT116 colonic epithelial cells were subjected to simulated inflammatory conditions, and rates of formation and consumption were determined for NO, O(2), and O(2)(-). Production rates of NO were determined in either of two ways: continuous monitoring of NO concentrations in a closed chamber with corrections for autoxidation, or NO(2)(-) accumulation measurements in an open system with corrections for diffusional losses of NO. The results obtained using the two methods were in excellent agreement. Rates of NO synthesis (2.3 +/- 0.6 pmol s(-1) 10(6) cells(-1)), NO consumption (1.3 +/- 0.3 s(-1)), and O(2) consumption (59 +/- 17 pmol s(-1) 10(6) cells(-1) when NO is negligible) for activated BMDM were indistinguishable from those of activated RAW264.7 cells. NO production rates calculated from NO(2)(-) accumulation data for HCT116 cells infected with Helicobacter cinaedi (3.9 +/- 0.1 pmol s(-1) 10(6) cells(-1)) were somewhat greater than those of RAW264.7 macrophages infected under similar conditions (2.6 +/- 0.1 pmol s(-1) 10(6) cells(-1)). Thus, RAW264.7 cells have NO kinetics nearly identical to those of primary macrophages, and stimulated epithelial cells are capable of synthesizing NO at rates comparable to those of macrophages. Using these cellular kinetic parameters, simulations of NO diffusion and reaction in a colonic crypt during inflammation predict maximum NO concentrations of about 0.2 microM at the base of a crypt.

Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization by Citrobacter rodentium.

The gastrointestinal tract is colonized by an enormous array of microbes that are known to have many beneficial effects on the host. Previous studies have indicated that stressor exposure can disrupt the stability of the intestinal microbiota, but the extent of these changes, as well as the effects on enteric infection, has not been well characterized. In order to examine the ability of stressors to induce changes in the gut microbiota, we exposed mice to a prolonged restraint stressor and then characterized microbial populations in the intestines using both traditional culture techniques and bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Exposure to the stressor led to an overgrowth of facultatively anaerobic microbiota while at the same time significantly reducing microbial richness and diversity in the ceca of stressed mice. Some of these effects could be explained by a stressor-induced reduction in the relative abundance of bacteria in the family Porphyromonadaceae. To determine whether these alterations would lead to increased pathogen colonization, stressed mice, as well as nonstressed controls, were challenged orally with the enteric murine pathogen Citrobacter rodentium. Exposure to the restraint stressor led to a significant increase in C. rodentium colonization over that in nonstressed control mice. The increased colonization was associated with increased tumor necrosis factor alpha (TNF-alpha) gene expression in colonic tissue. Together, these data demonstrate that a prolonged stressor can significantly change the composition of the intestinal microbiota and suggest that this disruption of the microbiota increases susceptibility to an enteric pathogen.

Myc is required for activation of the ATM-dependent checkpoints in response to DNA damage.

BACKGROUND: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown. PRINCIPAL FINDINGS: In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status. CONCLUSION: These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.

Detergent-resistant microdomains mediate activation of host cell signaling in response to attaching-effacing bacteria.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes outbreaks of bloody diarrhea and the hemolytic-uremic syndrome. EHEC intimately adheres to epithelial cells, effaces microvilli and induces attaching-effacing (AE) lesions. Detergent-resistant microdomains (lipid rafts) serve as membrane platforms for the recruitment of signaling complexes to mediate host responses to infection. The aim of this study was to define the role of lipid rafts in activating signal transduction pathways in response to AE bacterial pathogens. Epithelial cell monolayers were infected with EHEC (MOI 100:1, 3 h, 37 degrees C) and lipid rafts isolated by buoyant density ultracentrifugation. Phosphoinositide 3-kinase (PI3K) localization to lipid rafts was confirmed using PI3K and anti-caveolin-1 antibodies. Mice with cholesterol storage disease Niemann-Pick, type C were used as in vivo models to confirm the role of lipid rafts in mediating signaling response to AE organisms. In contrast to uninfected cells, PI3K was recruited to lipid rafts in response to EHEC infection. Metabolically active bacteria and cells with intact cholesterol-rich microdomains were necessary for the recruitment of second messengers to lipid rafts. Recruitment of PI3K to lipid rafts was independent of the intimin (eaeA) gene, type III secretion system, and production of Shiga-like toxins. Colonization of NPC(-/-) colonic mucosa by Citrobacter rodentium and AE lesion formation were both delayed, compared with wild-type mice infected with the murine-specific AE bacterial pathogen. C. rodentium-infected NPC(-/-) mice had reduced colonic epithelial hyperplasia (64+/-8.251 vs 112+/-2.958 microm; P<0.05) and decreased secretion of IFN-gamma (17.6+/-17.6 vs 71+/-26.3 pg/ml, P<0.001). Lipid rafts mediate host cell signal transduction responses to AE bacterial infections both in vitro and in vivo. These findings advance the current understanding of microbial-eukaryotic cell interactions in response to enteric pathogens that hijack signaling responses mediated through lipid rafts.

Toll-like receptor 4-mediated regulation of spontaneous Helicobacter-dependent colitis in IL-10-deficient mice.

BACKGROUND & AIMS: The commensal microbiota is believed to have an important role in regulating immune responsiveness and preventing intestinal inflammation. Intestinal microbes produce signals that regulate inflammation via Toll-like receptor (TLR) signaling, but the mechanisms of this process are poorly understood. We investigated the role of the anti-inflammatory cytokine interleukin (IL)-10 in this signaling pathway using a mouse model of colitis. METHODS: Clinical, histopathologic, and functional parameters of intestinal inflammation were evaluated in TLR4(-/-), IL-10(-/-), and TLR4(-/-) x IL-10(-/-) mice that were free of specific pathogens and in TLR4(-/-) x IL-10(-/-) mice following eradication and reintroduction of Helicobacter hepaticus. Regulatory T-cell (Treg) function was evaluated by crossing each of the lines with transgenic mice that express green fluorescent protein under control of the endogenous regulatory elements of Foxp3. Apoptotic cells in the colonic lamina propria were detected by a TUNEL assay. RESULTS: TLR4-mediated signals have 2 interrelated roles in promoting inflammation in TLR4(-/-) x IL-10(-/-) mice. In the absence of TLR4-mediated signals, secretion of proinflammatory and immunoregulatory cytokines is dysregulated. Tregs (Foxp3(+)) that secrete interferon-gamma and IL-17 accumulate in the colonic lamina propria of TLR4(-/-) x IL-10(-/-) mice and do not prevent inflammation. Aberrant control of epithelial cell turnover results in the persistence of antigen-presenting cells that contain apoptotic epithelial fragments in the colonic lamina propria of Helicobacter-infected TLR4(-/-) mice. CONCLUSIONS: In mice that lack both IL-10- and TLR4-mediated signals, aberrant regulatory T-cell function and dysregulated control of epithelial homeostasis combine to exacerbate intestinal inflammation.

Decreased expression of colonic Slc26a3 and carbonic anhydrase iv as a cause of fatal infectious diarrhea in mice.

Citrobacter rodentium causes epithelial hyperplasia and colitis and is used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections. Little or no mortality develops in most inbred strains of mice, but C3H and FVB/N mice exhibit fatal outcomes of infection. Here we test the hypothesis that decreased intestinal transport activity during C. rodentium infection results in fatality in C3H/HeOu and FVB/N mice. Susceptible strains were compared to resistant C57BL/6 mice and to inbred strains SWR and SJL of Swiss origin, which have not been previously characterized for outcomes of C. rodentium infection. Mortality in susceptible strains C3H/HeOu and FVB/N was associated with significant fluid loss in feces, a remarkable downregulation of Slc26a3 and carbonic anhydrase IV (CAIV) message and protein expression, retention of chloride in stool, and hypochloremia, suggesting defects in intestinal chloride absorption. SWR, SJL, and C57BL/6 mice were resistant and survived the infection. Fluid therapy fully prevented mortality in C3H/HeOu and FVB/N mice without affecting clinical disease. Common pathogenic mechanisms, such as decreased levels of expression of Slc26a3 and CAIV, affect intestinal ion transport in C. rodentium-infected FVB and C3H mice, resulting in profound electrolyte loss, dehydration, and mortality. Intestinal chloride absorption pathways are likely a potential target for the treatment of infectious diarrhea.

Determination of bacterial antibiotic resistance based on osmotic shock response.

We investigate the buoyant mass of bacterial cells in real time with the suspended microchannel resonator (SMR) as the population recovers from an osmotic shock. The density of the culture medium is chosen such that the bacteria initially have a positive buoyant mass which becomes negative as they recover from the hyperosmotic stress. This behavior can be used to differentiate between an antibiotic-resistant and an antibiotic-susceptible strain of the pathogenic bacteria Citrobacter rodentium, and we propose a general approach for exploiting the high precision of the SMR for rapid detection of antibiotic resistance.

Modulation of acute diarrheal illness by persistent bacterial infection.

Acute diarrheal illness is a global health problem that may be exacerbated by concurrent infection. Using Citrobacter rodentium, a murine model of attaching and effacing diarrheagenic Escherichia coli, we demonstrate that persistent Helicobacter hepaticus infection modulates host responses to diarrheal disease, resulting in delayed recovery from weight loss and from tissue damage. Chronic colitis in concurrently infected mice is characterized by macrophage and Foxp3(+) regulatory T-cell accumulation. Prolonged disease is also associated with increased interleukin-17 expression, which may be due to suppression of gamma interferon during the acute phase of diarrheal infection. This new model of polymicrobial infection provides insight into the mechanism by which subclinical infection can exacerbate morbidity due to an unrelated self-limiting infection.

Diarrhea as a cause of mortality in a mouse model of infectious colitis.

BACKGROUND: Comparative characterization of genome-wide transcriptional changes during infection can help elucidate the mechanisms underlying host susceptibility. In this study, transcriptional profiling of the mouse colon was carried out in two cognate lines of mice that differ in their response to Citrobacter rodentium infection; susceptible inbred FVB/N and resistant outbred Swiss Webster mice. Gene expression in the distal colon was determined prior to infection, and at four and nine days post-inoculation using a whole mouse genome Affymetrix array. RESULTS: Computational analysis identified 462 probe sets more than 2-fold differentially expressed between uninoculated resistant and susceptible mice. In response to C. rodentium infection, 5,123 probe sets were differentially expressed in one or both lines of mice. Microarray data were validated by quantitative real-time RT-PCR for 35 selected genes and were found to have a 94% concordance rate. Transcripts represented by 1,547 probe sets were differentially expressed between susceptible and resistant mice regardless of infection status, a host effect. Genes associated with transport were over-represented to a greater extent than even immune response-related genes. Electrolyte analysis revealed reduction in serum levels of chloride and sodium in susceptible animals. CONCLUSION: The results support the hypothesis that mortality in C. rodentium-infected susceptible mice is associated with impaired intestinal ion transport and development of fatal fluid loss and dehydration. These studies contribute to our understanding of the pathogenesis of C. rodentium and suggest novel strategies for the prevention and treatment of diarrhea associated with intestinal bacterial infections.

Role of the Helicobacter hepaticus flagellar sigma factor FliA in gene regulation and murine colonization.

The enterohepatic Helicobacter species Helicobacter hepaticus colonizes the murine intestinal and hepatobiliary tract and is associated with chronic intestinal inflammation, gall stone formation, hepatitis, and hepatocellular carcinoma. Thus far, the role of H. hepaticus motility and flagella in intestinal colonization is unknown. In other, closely related bacteria, late flagellar genes are mainly regulated by the sigma factor FliA (sigma(28)). We investigated the function of the H. hepaticus FliA in gene regulation, flagellar biosynthesis, motility, and murine colonization. Competitive microarray analysis of the wild type versus an isogenic fliA mutant revealed that 11 genes were significantly more highly expressed in wild-type bacteria and 2 genes were significantly more highly expressed in the fliA mutant. Most of these were flagellar genes, but four novel FliA-regulated genes of unknown function were identified. H. hepaticus possesses two identical copies of the gene encoding the FliA-dependent major flagellin subunit FlaA (open reading frames HH1364 and HH1653). We characterized the phenotypes of mutants in which fliA or one or both copies of the flaA gene were knocked out. flaA_1 flaA_2 double mutants and fliA mutants did not synthesize detectable amounts of FlaA and possessed severely truncated flagella. Also, both mutants were nonmotile and unable to colonize mice. Mutants with either flaA gene knocked out produced flagella morphologically similar to those of wild-type bacteria and expressed FlaA and FlaB. flaA_1 mutants which had flagella but displayed reduced motility did not colonize mice, indicating that motility is required for intestinal colonization by H. hepaticus and that the presence of flagella alone is not sufficient.

Helicobacter hepaticus HHGI1 is a pathogenicity island associated with typhlocolitis in B6.129-IL10 tm1Cgn mice.

Helicobacter hepaticus strain 3B1 (H. hepaticus) contains a genomic island of approximately 71 kb, HHGI1, with some of the common features shared among known bacterial pathogenicity islands. In this study, we characterized the pathogenic potential of HHGI1 by infecting B6.129-IL10 tm1Cgn (IL10-/-) mice with an isogenic mutant (namely HhPAId1) lacking 19 predicted genes within HHGI1. In contrast to H. hepaticus (P<0.001), HhPAId1 did not cause typhlocolitis and hyperplasia in IL10-/- mice. Colonization levels of HhPAId1 were significantly higher in the cecum (P<0.007) and similar in the colon (P=0.27) when compared to H. hepaticus by 13 or 16 weeks post inoculation (WPI). The magnitude of the Th1-associated IgG2c response against HhPAId1 was less than that against H. hepaticus (P<0.004). There was no significant difference in Th2-associated IgG1 responses against these two strains. Cecal and colonic mRNA levels of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-17a in the HhPAId1-infected mice were significantly lower than those in the H. hepaticus-infected mice (P<0.05) at 13 WPI. These results demonstrate that genes in the HHGI1 contribute to the pathogenicity of H. hepaticus, at least in part via up-regulation of proinflammatory mediators IFN-gamma, TNF-alpha and IL-17a.

DNA damage induced by chronic inflammation contributes to colon carcinogenesis in mice.

Chronic inflammation increases cancer risk. While it is clear that cell signaling elicited by inflammatory cytokines promotes tumor development, the impact of DNA damage production resulting from inflammation-associated reactive oxygen and nitrogen species (RONS) on tumor development has not been directly tested. RONS induce DNA damage that can be recognized by alkyladenine DNA glycosylase (Aag) to initiate base excision repair. Using a mouse model of episodic inflammatory bowel disease by repeated administration of dextran sulfate sodium in the drinking water, we show that Aag-mediated DNA repair prevents colonic epithelial damage and reduces the severity of dextran sulfate sodium-induced colon tumorigenesis. Importantly, DNA base lesions expected to be induced by RONS and recognized by Aag accumulated to higher levels in Aag-deficient animals following stimulation of colonic inflammation. Finally, as a test of the generality of this effect we show that Aag-deficient animals display more severe gastric lesions that are precursors of gastric cancer after chronic infection with Helicobacter pylori. These data demonstrate that the repair of DNA lesions formed by RONS during chronic inflammation is important for protection against colon carcinogenesis.