Gut microbes define liver cancer risk in mice exposed to chemical and viral transgenic hepatocarcinogens.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) frequently results from synergism between chemical and infectious liver carcinogens. Worldwide, the highest incidence of HCC is in regions endemic for the foodborne contaminant aflatoxin B1 (AFB1) and hepatitis B virus (HBV) infection. Recently, gut microbes have been implicated in multisystemic diseases including obesity and diabetes. Here, the hypothesis that specific intestinal bacteria promote liver cancer was tested in chemical and viral transgenic mouse models. METHODS: Helicobacter-free C3H/HeN mice were inoculated with AFB1 and/or Helicobacter hepaticus. The incidence, multiplicity and surface area of liver tumours were quantitated at 40 weeks. Molecular pathways involved in tumourigenesis were analysed by microarray, quantitative real-time PCR, liquid chromatography/mass spectrometry, ELISA, western blot and immunohistochemistry. In a separate experiment, C57BL/6 FL-N/35 mice harbouring a full-length hepatitis C virus (HCV) transgene were crossed with C3H/HeN mice and cancer rates compared between offspring with and without H hepaticus. RESULTS: Intestinal colonisation by H hepaticus was sufficient to promote aflatoxin- and HCV transgene-induced HCC. Neither bacterial translocation to the liver nor induction of hepatitis was necessary. From its preferred niche in the intestinal mucus layer, H hepaticus activated nuclear factor-kappaB (NF-kappaB)-regulated networks associated with innate and T helper 1 (Th1)-type adaptive immunity both in the lower bowel and liver. Biomarkers indicative of tumour progression included hepatocyte turnover, Wnt/beta-catenin activation and oxidative injury with decreased phagocytic clearance of damaged cells. CONCLUSIONS: Enteric microbiota define HCC risk in mice exposed to carcinogenic chemicals or hepatitis virus transgenes. These results have implications for human liver cancer risk assessment and prevention.

Nitric oxide and TNF-alpha trigger colonic inflammation and carcinogenesis in Helicobacter hepaticus-infected, Rag2-deficient mice.

Recombinase-activating gene-2-deficient (Rag2(-/-)) mice lacking functional lymphocytes provide a useful model of chronic inflammatory bowel disease-emulating events in human colon cancer. Infection of Rag2(-/-) mice with Helicobacter hepaticus led to accumulation of macrophages and neutrophils in the colon, a process temporally related to up-regulation of tissue inducible nitric oxide synthase (iNOS) expression at the site of infection and increased nitric oxide (NO) production, as evidenced by urinary excretion of nitrate. Progressive development of increasingly severe inflammation, hyperplasia, dysplasia, and cancer accompanied these changes. Concurrent administration of an iNOS inhibitor prevented NO production and abrogated epithelial pathology and inhibited the onset of cancer. The presence of Gr-1(+) neutrophils and elevated tumor necrosis factor-alpha (TNF-alpha) expression in colon were required for increased iNOS expression and cancer, whereas interleukin-10 (IL-10) down-regulated TNF-alpha and iNOS expression and suppressed cancer. Anti-inflammatory CD4(+) regulatory lymphocytes also down-regulated iNOS and reduced cancer formation. Collectively, these results confirm essential roles for inflammation, increased TNF-alpha expression, and elevated NO production in colon carcinogenesis.

Genomic characterization of Helicobacter hepaticus: ordered cosmid library and comparative sequence analysis.

Helicobacter hepaticus is an important pathogen in laboratory mice and induces the development of liver tumors and gastrointestinal disease in susceptible strains of mice. In this study, a miniset of 36 cosmid clones from a genomic library of H. hepaticus was ordered and grouped into four large contigs representing approximately 1 Mb of the H. hepaticus genome using PCR, DNA sequencing, Southern and dot-blot hybridization and pulsed-field gel electrophoresis. From the 200-300 terminal nucleotide sequences of 38 cosmid clones, 56 coding regions were predicted, of which 51 were found to have orthologs in the public databases and five appeared to be unique to H. hepaticus. Of these 51 genes, 36 have orthologs in Helicobacter pylori and 25 display the highest sequence similarity to H. pylori. However, chromosomal positions of these genes are not conserved between these two helicobacters. In addition, 10 H. hepaticus genes had the highest sequence similarity to orthologs in Campylobacter jejuni. The GC content in a randomly selected 21-kb H. hepaticus genomic sequence was 35.8%, which approximates the average between H. pylori (39%) and C. jejuni (30.6%). These results demonstrate that: (1) H. hepaticus is more closely related to H. pylori than C. jejuni; (2) significant genomic alterations exist between H. hepaticus and H. pylori, including gene organization, protein sequences and GC content, probably in part due to specific adaptation to distinct ecological niches.

Enteropathogenic Escherichia coli and ulcerative colitis in cotton-top tamarins (Saguinus oedipus).

The cotton-top tamarin (CTT; Saguinus oedipus) is an endangered New World primate that develops a highly prevalent idiopathic colitis resembling human ulcerative colitis. This study found that enteropathogenic Escherichia coli (EPEC) caused acute colitis in CTTs, which was associated with ulcerative colitis. EPEC clinical isolates revealed localized adherence patterns by HEp-2 assay and were devoid of Shiga-toxin production. Sequencing of the eae gene (GenBank accession no. AF319597) revealed 99.2% identity to sequences of human isolates (GenBank AF116899) and corresponded to the epsilon intimin gene subtype. Detection of intimin sequences by polymerase chain reaction on primary fecal cultures indicated widespread EPEC infection in the CTT colony. Prospective analysis revealed that animals with fecal cultures positive for intimin sequences had a higher frequency of active colitis (75.0% vs. 27.2%; P<.005, chi(2) test) and higher histological scores of colonic inflammation (0.875 vs. 0.455, respectively; P<.05, Mann-Whitney rank sum test).

Bacterial infection promotes colon tumorigenesis in Apc(Min/+) mice.

The Min mouse, which has a germ line mutation in 1 allele of the Apc tumor suppressor gene, is a model for the early steps in human colorectal cancer. Helicobacter pylori infection, a known risk factor for gastric cancer in humans, causes chronic inflammation and increased epithelial cell proliferation in the stomach. Infection with the bacterium Citrobacter rodentium is known to increase epithelial cell proliferation and to promote chemically initiated tumors in the colon of mice. Min mice infected with C. rodentium at 1 month of age were found to have a 4-fold increase in the number of colonic adenomas at 6 months of age, compared with uninfected Min mice. Most of the colonic adenomas in the infected Min mice were in the distal colon, where C. rodentium-induced hyperplasia occurs. These data demonstrate that bacterial infection promotes colon tumor formation in genetically susceptible mice.

Molecular pathogenesis of Citrobacter rodentium and transmissible murine colonic hyperplasia.

Here we review the history, clinical significance, pathology and molecular pathogenesis of Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia. C. rodentium serves as an important model pathogen for investigating the mechanisms controlling attaching and effacing pathology, epithelial hyperproliferation, and tumor promotion in the distal colon of the mouse.

Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases.

Since Helicobacter pylori was first cultivated from human gastric biopsy specimens in 1982, it has become apparent that many related species can often be found colonizing the mucosal surfaces of humans and other animals. These other Helicobacter species can be broadly grouped according to whether they colonize the gastric or enterohepatic niche. Gastric Helicobacter species are widely distributed in mammalian hosts and are often nearly universally prevalent. In many cases they cause an inflammatory response resembling that seen with H. pylori in humans. Although usually not pathogenic in their natural host, these organisms serve as models of human disease. Enterohepatic Helicobacter species are an equally diverse group of organisms that have been identified in the intestinal tract and the liver of humans, other mammals, and birds. In many cases they have been linked with inflammation or malignant transformation in immunocompetent hosts and with more severe clinical disease in immunocompromised humans and animals. The purpose of this review is to describe these other Helicobacter species, characterize their role in the pathogenesis of gastrointestinal and enterohepatic disease, and discuss their implications for our understanding of H. pylori infection in humans.

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.

Cytolethal distending toxin in avian and human isolates of Helicobacter pullorum.

Helicobacter pullorum has been isolated from the feces and livers of poultry and is associated with human gastroenteritis. Discrimination of this organism from other enterohepatic Helicobacter species and Campylobacter species has proven difficult. H. pullorum from both avian and human clinical sources has DNA sequence homology and cytotoxic activity that represent a new member of the cytolethal distending toxin (CDT) family of bacterial toxins. CDT is a potential virulence factor in H. pullorum that may serve as a distinguishing phenotype and aid in identification of this organism in veterinary and human clinical samples.

Chronic atrophic gastritis in SCID mice experimentally infected with Campylobacter fetus.

Campylobacter fetus is a cause of enteritis and invasive extraintestinal disease in humans. In order to develop an animal model of C. fetus infection, outbred ICR SCID mice were orally challenged with a clinical isolate of C. fetus. The stomachs of SCID mice were heavily colonized with C. fetus, and colonization was associated with the development of chronic atrophic gastritis. This lesion was characterized by an inflammatory infiltrate of granulocytes and macrophages that over time resulted in a loss of specialized parietal and chief cells in the corpus and the appearance of a metaplastic mucous epithelium. This lesion bears similarity to that encountered during experimental murine infection with Helicobacter pylori or Helicobacter felis. Despite colonization of the cecum and colon tissues by C. fetus in SCID mice, no lesions were noted in these tissues. A follow-up study confirmed these findings for SCID mice and also demonstrated that C. fetus could also infect the gastric mucosa of wild-type, outbred ICR mice. However, in ICR mice, the anatomic extent of colonization was more limited and the severity of inflammation and epithelial alterations was significantly less than that observed in infected SCID mice. The stomach may represent an unrecognized environmental niche for Campylobacter species.

Helicobacter hepaticus infection triggers inflammatory bowel disease in T cell receptor alphabeta mutant mice.

The T cell receptor alpha chain-deficient (TCR alpha-/-) and TCR beta chain-deficient (TCR beta-/-) mice develop chronic intestinal inflammation that resembles inflammatory bowel disease by 3 to 4 months of age. The objective of the study reported here was to determine the role of infection with the bacterial pathogen Helicobacter hepaticus in the pathogenesis of disease in TCR alphabeta mutant mice. The H. hepaticus-infected TCR alphabeta mutant mice were rederived by use of embryo transfer to produce Helicobacter-free animals. Helicobacter-free TCR alpha-/-, TCR beta-/-, and TCR alpha-/- beta-/- mice were inoculated with H. hepaticus. Experimentally infected mice and uninfected control mice were examined for intestinal lesions at 3, 6, and 9 months after inoculation. The TCR alphabeta mutant mice inoculated with H. hepaticus developed intestinal epithelial cell hyperplasia and mucosal inflammation. By 6 months after inoculation, infected animals had moderate cecal and colonic lesions. Helicobacter-free TCR alpha-/- mice, but not TCR beta-/- or TCR alpha-/- x beta-/- mice, also developed H. hepaticus-independent colitis by 9 months after inoculation. Infection with H. hepaticus is sufficient to cause chronic proliferative intestinal inflammation in TCR alphabeta mutant mice. However, H. hepaticus infection is not necessary for intestinal disease in TCR alpha-/- mice.

Cytolethal distending toxin sequence and activity in the enterohepatic pathogen Helicobacter hepaticus.

Little is known about the molecular pathogenesis of hepatitis and enterocolitis caused by enterohepatic Helicobacter species. Sonicates of the murine pathogen Helicobacter hepaticus were found to cause progressive cell distension, accumulation of filamentous actin, and G(2)/M cell cycle arrest in HeLa cell monolayers. The genes encoding this cytotoxic activity were cloned from H. hepaticus. Three open reading frames with closest homology to cdtA, cdtB, and cdtC from Campylobacter jejuni were identified. Sonicates of a laboratory strain of Escherichia coli carrying the cloned cdtABC gene cluster from H. hepaticus reproduced the cytotoxic activities seen with sonicates of H. hepaticus. Cytolethal distending toxin activity is a potential virulence determinant of H. hepaticus that may play a role in the pathogenesis of Helicobacter-associated hepatitis and enterocolitis.

The Tir-binding region of enterohaemorrhagic Escherichia coli intimin is sufficient to trigger actin condensation after bacterial-induced host cell signalling.

Enterohaemorrhagic Escherichia coli (EHEC) has emerged as an important agent of diarrhoeal disease. Attachment to host cells, an essential step during intestinal colonization by EHEC, is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (A/E) lesion, directly beneath bound bacteria. The outer membrane protein intimin is required for the formation of this structure, as is Tir, a bacterial protein that is translocated into the host cell and is thought to function as a receptor for intimin. To understand intimin function better, we fused EHEC intimin to a homologous protein, Yersinia pseudotuberculosis invasin, or to maltose-binding protein. The N-terminal 539 amino acids of intimin were sufficient to promote outer membrane localization of the C-terminus of invasin and, conversely, the N-terminal 489 amino acids of invasin were sufficient to promote the localization of the C-terminus of intimin. The C-terminal 181 residues of intimin were sufficient to bind mammalian cells that had been preinfected with an enteropathogenic E. coli strain that expresses Tir but not intimin. Binding of intimin derivatives to preinfected cells correlated with binding to recombinant Tir protein. Finally, the 181-residue minimal Tir-binding region of intimin, when purified and immobilized on latex beads, was sufficient to trigger A/E lesions on preinfected mammalian cells.

Citrobacter rodentium espB is necessary for signal transduction and for infection of laboratory mice.

Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia and contains a locus of enterocyte effacement (LEE) similar to that found in enteropathogenic Escherichia coli (EPEC). EPEC espB is necessary for intimate attachment and signal transduction between EPEC and cultured cell monolayers. Mice challenged with wild-type C. rodentium develop a mucosal immunoglobulin A response to EspB. In this study, C. rodentium espB has been cloned and its nucleotide sequence has been determined. C. rodentium espB was found to have 90% identity to EPEC espB. A nonpolar insertion mutation in C. rodentium espB was constructed and used to replace the chromosomal wild-type allele. The C. rodentium espB mutant exhibited reduced cell association and had no detectable fluorescent actin staining activity on cultured cell monolayers. The C. rodentium espB mutant also failed to colonize laboratory mice following experimental inoculation. The espB mutation could be complemented with a plasmid-encoded copy of the gene, which restored both cell association and fluorescent actin staining activity, as well as the ability to colonize laboratory mice. These studies indicate that espB is necessary for signal transduction and for colonization of laboratory mice by C. rodentium.

Phylogeny of the defined murine microbiota: altered Schaedler flora.

The "altered Schaedler flora" (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.

Development of a PCR-restriction fragment length polymorphism assay using the nucleotide sequence of the Helicobacter hepaticus urease structural genes ureAB.

Infection with Helicobacter hepaticus causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters, H. pylori and H. mustelae, H. hepaticus possesses a high level of urease activity. However, the H. hepaticus urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from H. hepaticus genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial H. hepaticus ureA gene product was found to exhibit 60% identity and 75% similarity to the predicted H. pylori UreA. The deduced amino acid sequence of a partial H. hepaticus ureB gene product exhibited 75% identity and 87% similarity to the predicted H. pylori UreB. Diversity among H. hepaticus isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the ureAB open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease HhaI. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of H. hepaticus during an 18 month in vivo colonization study, suggesting that the urease genes of H. hepaticus are stable. The urease genes among H. hepaticus strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that H. hepaticus has urease structural genes which are homologous to those of the gastric Helicobacter species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.

Proliferative enterocolitis associated with dual infection with enteropathogenic Escherichia coli and Lawsonia intracellularis in rabbits.

Both enteropathogenic Escherichia coli (EPEC) and an obligate intracellular bacterium, previously referred to as an intracellular Campylobacter-like organism and now designated Lawsonia intracellularis, have been reported as causes of enterocolitis in rabbits. An outbreak of enterocolitis in a group of rabbits, characterized by an unusually high rate of mortality, was found to be associated with dual infection with EPEC and L. intracellularis. The EPEC strain was found to have eaeA gene homology but was negative for afrA homology. The absence of the afrA gene, which encodes the structural subunit for the AF/R1 pilus, indicates that this rabbit EPEC strain is distinct from the prototypic RDEC-1 strain. This finding suggests that rabbit EPEC strains widely reported in Western Europe, which lack AF/R1 pili, are also present in rabbits in the United States. Dual infection with these two pathogens in rabbits has not been previously reported and may have contributed to the unusually high mortality observed in this outbreak.

Spontaneous inflammatory bowel disease in multiple mutant mouse lines: association with colonization by Helicobacter hepaticus.

BACKGROUND: Both genetic and microbial factors are thought to play a role in the development of inflammatory bowel disease (IBD): however, no causative microbial agent has been clearly defined for humans or animals. Normal flora or previously unrecognized intestinal pathogens may contribute to the development of disease in susceptible hosts. A newly recognized murine Helicobacter, H. hepaticus, causes hepatitis in mice and in one strain of mice is linked to liver cancer. This study investigates the association between colonization of the lower intestinal tract of multiple genetically altered lines of mice with H. hepaticus, and the occurrence of IBD. MATERIALS AND METHODS: Rectal prolapse noted clinically in multiple genetically altered mouse lines was evaluated for the presence of H. hepaticus and histologic evidence of IBD. Fifty-five mice representing 11 different genetic alterations were evaluated. RESULTS: H. hepaticus was detected in 85% of mutant mice with rectal prolapse. Histologic evidence of proliferative typhlitis, colitis or proctitis was present in 65% of the animals examined, 89% of which were positive for H. hepaticus as detected by species specific PCR. CONCLUSION: The presence of H. hepaticus in association with IBD in multiple lines of genetically altered mice suggests further studies are needed to test experimentally the role H. hepaticus plays in the development of IBD in susceptible mice. Additionally, specific mutant mouse lines infected with H. hepaticus in this study may provide additional models for elucidation of microbial and genetic factors in the pathogenesis of IBD.

Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus.

Inflammatory bowel disease (IBD) is thought to result from either an abnormal immunological response to enteric flora or a normal immunological response to a specific pathogen. No study to date has combined both factors. The present studies were carried out with an immunologically manipulated mouse model of IBD. Mice homozygous for the severe combined immunodeficiency (scid) mutation develop IBD with adoptive transfer of CD4+ T cells expressing high levels of CD45RB (CD45RB(high) CD4+ T cells). These mice do not develop IBD in germfree conditions, implicating undefined intestinal flora in the pathogenesis of lesions. In controlled duplicate studies, the influence of a single murine pathogen, Helicobacter hepaticus, in combination with the abnormal immunological response on the development of IBD was assessed. The combination of H. hepaticus infection and CD45RB(high) CD4+ T-cell reconstitution resulted in severe disease expression similar to that observed in human IBD. This study demonstrates that IBD develops in mice as a consequence of an abnormal immune response in the presence of a single murine pathogen, H. hepaticus. The interaction of host immunity and a single pathogen in this murine system provides a novel model of human IBD, an immunity-mediated condition triggered by bacterial infection.