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Helicobacter hepaticus expresses a member of the cytolethal distending toxin (CDT) family of bacterial cytotoxins. To investigate the role of CDT in the pathogenesis of H. hepaticus, transposon mutagenesis was used to generate a series of isogenic mutants in and around the cdtABC gene cluster. An H. hepaticus transposon mutant with a disrupted cdtABC coding region no longer produced CDT activity. Conversely, a transposon insertion outside of the cluster did not affect the CDT activity. An examination of these mutants demonstrated that CDT represents the previously described granulating cytotoxin in H. hepaticus. Challenge of C57BL/6 interleukin 10−/− mice with isogenic H. hepaticus mutants revealed that CDT expression is not required for colonization of the murine gut. However, a CDT-negative H. hepaticus mutant had a significantly diminished capacity to induce lesions in this murine model of inflammatory bowel disease.
Enterohepatic Helicobacter species (EHS) are emerging pathogens of the intestinal tracts and/or livers of humans, other mammals, and birds (11, 24, 29). They are responsible for a number of acute disease syndromes, including gastroenteritis and bacteremia or sepsis. In addition, a subset of the EHS also cause persistent infections associated with chronic inflammation and neoplasia, which is analogous to the association between Helicobacter pylori infection and chronic gastritis and gastric cancer (3).
Helicobacter hepaticus is an EHS that naturally infects the distal gastrointestinal tracts of mice. This organism was originally discovered in male A/JCr mice that were serving as controls in a long-term carcinogenesis assay (13, 32). These mice developed liver tumors at a higher rate than expected and in addition had chronic active hepatitis.
Once H. hepaticus was recognized, it was found to be prevalent in production and research mouse colonies (28). In some strains of mice, H. hepaticus infection causes subclinical hepatic disease and enteritis (generally typhlitis or colitis). Susceptible strains develop chronic active hepatitis with or without tumors but usually only mild enteritis. However, in mice with altered immune function, including interleukin 10−/− (IL-10−/−), T-cell receptor alpha/beta−/−, SCID or RAG2−/− mice reconstituted with CD45RBhigh T cells, and NF-κB (p50−/− p65+/−) mice, the typhlitis and colitis associated with an H. hepaticus infection can be severe and progressive, leading to rectal prolapse, adenocarcinoma, weight loss, and death (4, 5, 7, 10, 21).
Candidate virulence determinants in EHS have begun to be identified. H. hepaticus and a subset of the other EHS produce cytolethal distending toxin (CDT) (6, 35, 36). CDT is a potential virulence factor elaborated by a heterogeneous group of pathogenic bacteria including Campylobacter jejuni and other Campylobacter species, certain Escherichia coli strains, Shigella dysenteriae, Haemophilus ducreyi, and Actinobacillus actinomycetemcomitans (reviewed in references 8, 18, 25, and 37). CDT induces progressive cell enlargement and eventual death in cultured mammalian cells. Cytotoxicity is accompanied by G2/M cell cycle arrest. In all bacterial species in which CDT activity has been demonstrated, three linked genes (cdtA, cdtB, and cdtC) encode cytotoxic activities. Genetic studies indicate that all three genes are necessary to transfer CDT activity to a laboratory strain of E. coli. CdtB has position-specific homology to type I mammalian DNases and exhibits nuclease activity in vitro (9, 16). Mammalian cells treated with CDT have evidence of the activation of DNA repair mechanisms (15, 20). Transfection of plasmids expressing CdtB or microinjection of purified CdtB into mammalian cells reproduces the cytopathic effect (CPE) of CDT. There is evidence that CdtA, CdtB, and CdtC form a tripartite AB2 toxin, with CdtB as the active A subunit and CdtA and CdtC forming a heterodimeric B subunit (17).
Although CDT occurs in a diverse group of pathogenic bacteria, no role for CDT in pathogenesis has been convincingly demonstrated in vivo. Isogenic CDT mutations have been generated in Haemophilus ducreyi (30, 34) and C. jejuni (26), but minimal attenuation of virulence has been seen. One possible reason for this is that the model systems employed for infection in these organisms do not accurately reflect the natural infectious process. Here, we examined the role of CDT in the pathogenesis of H. hepaticus, a bona fide murine pathogen, employing experimental infection of IL-10−/− mice.
E. coli strain TOP10 was grown at 37°C in Luria-Bertani broth or on Luria-Bertani agar supplemented with 100 μg of ampicillin per ml or 20 μg of chloramphenicol per ml where indicated below. The wild-type H. hepaticus strain 3B1 was obtained from the American Type Culture Collection (ATCC 51448). Wild-type H. hepaticus and isogenic mutant strains were grown at 37°C for 3 to 4 days in a microaerobic environment, which was maintained in vented GasPak jars without a catalyst after evacuation to −20 mm Hg and equilibration with a gas mixture consisting of 80% N2, 10% CO2, and 10% H2. H. hepaticus was grown on tryptic soy agar supplemented with 5% sheep blood and with 20 μg of chloramphenicol per ml (all from Sigma, St. Louis, Mo.) for chloramphenicol-resistant strains.
A transposon with a selectable marker that can be employed in H. hepaticus was constructed starting with the Tn5-based transposon construction vector pMOD-2 <MCS> (Epicentre Technologies, Madison, Wis.). The Campylobacter coli Cmr cassette was obtained by digesting plasmid pBHpC8 (14) with HincII. The resulting 800-bp blunt-ended fragment was ligated into the HincII site in the multiple cloning site of pMOD-2 <MCS>. The resultant plasmid was named pMOD-2Cm, and the resultant transposon is referred to as EZ::TnCm.
In vitro transposition of plasmid DNA was performed with purified hyperactive Tn5 transposase (Epicentre) according to the manufacturer's recommendations. pVBY9 is a plasmid that carries the entire cdtABC gene cluster from H. hepaticus (36). pVBY9 was mutagenized in vitro with EZ::TnCm and then transformed into E. coli strain TOP-10, with selection for chloramphenicol resistance. About 30 independent transposon mutants were selected, and the transposition site was localized by restriction mapping. For selected mutants, the precise site of transposition was determined by DNA sequence analysis.
Four confluent, 100-mm-diameter plates of H. hepaticus were harvested with a cell scraper. Bacteria were resuspended in 1 ml of sterile, ice-cold wash buffer (15% [wt/vol] glycerol, 7% [wt/vol] sucrose), pelleted for 3 min at 15,800 × g, and washed twice before being resuspended in 160 μl of ice-cold wash buffer. Recombinant plasmids were isolated from E. coli with a QIAGEN (Santa Clarita, Calif.) plasmid kit. Two micrograms of plasmid DNA was used to transform 40 μl of H. hepaticus by high-voltage electroporation with a 0.2-cm-gap-size cuvette and a 2.5-kV peak output for a field strength of 12.5 kV/cm in an E. coli pulser (Bio-Rad, Hercules, Calif.). The transformed bacteria were resuspended in 100 μl of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 10 mM glucose) and then plated without selection. Eight hours later, the bacteria were harvested, resuspended, and plated with antibiotic selection.
H. hepaticus was harvested from agar plates with a cell scraper and was resuspended in Hanks' balanced salt solution (Invitrogen Life Technologies, Carlsbad, Calif.). Total bacterial proteins were solubilized with a nonionic detergent (bacterial protein extraction reagent [B-PER]; Pierce Chemical Co., Rockford, Ill.) according to the recommendations of the manufacturer. Bacteria were pelleted by centrifugation at 2,040 × g for 10 min, resuspended in 300 μl of B-PER, and vortexed. Insoluble material was removed by centrifugation at 13,800 × g for 5 min, and soluble proteins contained in the supernatant were collected and stored at −80°C. Alternately, H. hepaticus sonicates were prepared as previously described (36). H. hepaticus growth from three 100-mm-diameter plates was harvested into 1 ml of phosphate-buffered saline and disrupted by six 30-s pulses on ice. Debris was removed by centrifugation, and the cleared sonicates were filtered through a 0.2-μm-pore-size filter before being stored at −80°C.
CDT assays using B-PER extracts or sonicates were performed as described previously (36). Cultured mammalian cells were seeded on glass coverslips or in 25-cm2 tissue culture flasks treated with B-PER extracts, sonicates from wild-type H. hepaticus, or the isogenic CDT mutants. CDT activity was determined by direct microscopic examination of stained monolayers or cell cycle analysis by flow cytometry (36).
All animal protocols were reviewed and approved by the Michigan State University All University Committee on Animal Use and Care. Female C57BL/6 IL-10−/− mice were obtained from the Jackson Laboratories (Bar Harbor, Maine) and housed with autoclaved food, bedding, and water, with cage changes performed in a laminar flow hood at the University Research Containment Facility at Michigan State University. Animals were housed in groups of five per microisolator cage.
H. hepaticus was harvested after 48 h of growth on agar plates and resuspended in a small volume of tryptic soy broth. The optical density at 600 nm of the inoculum was measured, and 10-fold serial dilutions of the inoculum were plated to quantify the CFU used for infection. Mice were inoculated with a suspension of bacteria with an optical density of 1.0 at 600 nm (~108 CFU) in a volume of 0.2 to 0.3 ml. Bacteria were introduced directly into the stomach with a 24-gauge ball-tipped gavage needle. Mice were challenged with a total of three doses of bacteria on three alternating days. Control mice were inoculated with sterile tryptic soy broth. The mouse infection study was performed twice, each time with five mice in each experimental group.
Fresh fecal pellets were collected from each mouse. Culture for H. hepaticus was accomplished by homogenizing feces in 0.5 ml of phosphate-buffered saline and plating 50 μl on tryptic soy agar supplemented with 5% sheep blood, 20 μg of cefoperazone per ml, 10 μg of vancomycin per ml, and 2 μg of amphotericin B per ml. DNA was isolated from fecal pellets as described previously (33). PCR amplification was performed using the H. hepaticus-specific primers 5′ GCA TTT GAA ACT GTT ACT CTG 3′ (B38) and 5′ CTG TTT TCA AGC TCC CC 3′ (B39), which produce a 417-bp amplicon (28). PCR was performed with 5 μl of the template at approximately 250 ng/μl. Each 25-μl PCR mixture contained 20 pmol of each primer, a 200 μM concentration of each deoxynucleoside triphosphate, and 1.5 U of Taq DNA polymerase in a solution of 10 mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl2 (final concentrations; Ready To Go PCR beads; Amersham Pharmacia Biotech, Piscataway, N.J.). Cycling conditions included 30 cycles of 30 s at 94°C, 45 s at 54°C, and 45 s at 72°C. PCR products were visualized by agarose gel electrophoresis.
Six weeks after infection, mice were euthanized by CO2 asphyxiation. Collection of the ileocecocolic junction from each mouse was performed as follows. After the opening of the peritoneal cavity, the cecum was externalized and removed by transection of the terminal ileum and the proximal colon. A 1-cm piece of the terminal ileum and 2 cm of the proximal colon were left attached to the cecum, and the tissue was placed in a sterile petri dish. The cecum was transected approximately halfway down its length, at the sharpest point of its curve. The intestinal contents were gently expressed with the back of a scalpel blade. Neutral buffered formalin (10%) was infused gently into the transected cecal opening until fixative was seen emerging from the colonic and/or ileal openings. The tissue was placed intact into a histologic tissue cassette. The tissue was processed, and paraffin was embedded. The tissue blocks were sectioned until the lumen of the ileocecocolic junction was reached. Sections were placed on glass slides and stained routinely with hematoxylin and eosin.
Histologic sections were scored by a veterinary pathologist who was blind to the sample source and who used the following scoring system for inflammation: 0, normal; 1, small multifocal lamina proprial and/or transepithelial leukocyte accumulations; 2, coalescing mucosal inflammation with or without early submucosal extension; 3, coalescing mucosal inflammation with a prominent multifocal submucosal extension with or without follicle formation; and 4, severe diffuse inflammation of the mucosa, submucosa, and deeper layers.
Categorical inflammation scores were compared by the nonparametric Wilcoxon rank sum test using the JMP statistical package (SAS, Cary, N.C.). Statistical significance was set at a P value of <0.05.
Isogenic CDT mutants were constructed to study the role of CDT in the pathogenesis of H. hepaticus infection. A transposon was developed based on a commercial Tn5-based transposition system. The resultant transposon carries a chloramphenicol resistance marker from C. coli (14). This transposon (EZ::TnCm) was used to mutagenize a plasmid, pVBY9 (36), that carries the entire cdtABC gene cluster from H. hepaticus and is sufficient to transfer CDT activity to a laboratory strain of E. coli. For selected mutants, the transposon insertion site was determined by restriction mapping and DNA sequence analysis. The mutant plasmids were also tested for the ability to transfer CDT activity to E. coli (Fig. (Fig.11).
Two mutant pVBY9 plasmids were selected to make isogenic H. hepaticus mutants by allelic exchange. The plasmid pVBY9::Tn20 has EZ::TnCm inserted at the beginning of the cdtA open reading frame (Fig. (Fig.1).1). The plasmid pVBY9::Tn16 has EZ::TnCm inserted downstream of the cdtC open reading frame. Detergent extracts or sonicates of E. coli harboring pVBY9::Tn16 retain CDT activity, but extracts or sonicates of E. coli carrying pVBY9::Tn20 no longer produce CDT CPE when they are added to HeLa cell monolayers (Fig. (Fig.11).
The plasmids pVBY9::Tn16 (CDT+) and pVBY9::Tn20 (CDT−) were used to transform H. hepaticus strain 3B1 by high-voltage electroporation. Following electroporation and a period of outgrowth on nonselective media, bacteria were transferred to chloramphenicol-containing media. No chloramphenicol-resistant transformants were seen following a mock transformation control without DNA. Transformation with either pVBY9::Tn16 or pVBY9::Tn20 resulted in between 10 and 20 chloramphenicol-resistant colonies/μg of plasmid DNA. Representative chloramphenicol-resistant colonies were analyzed by Southern hybridization, confirming that the wild-type CDT coding region of the H. hepaticus chromosome was replaced by the transposon-mutagenized CDT coding regions carried on the plasmids (data not shown). These isogenic H. hepaticus mutants were named 3B1::Tn16 and 3B1::Tn20.
The isogenic mutants 3B1::Tn16 and 3B1::Tn20 were tested for the expression of CDT. Detergent extracts of 3B1::Tn16 produced CDT CPE on HeLa cell monolayers identical to those of wild-type H. hepaticus 3B1, but extracts of 3B1::Tn20 did not possess detectable CDT activity (Fig. (Fig.2).2). The 3B1::Tn20 mutant had a normal morphology and growth characteristics identical to those of wild-type 3B1 and 3B1::Tn16 in vitro (data not shown).
It has previously been reported that H. hepaticus does not possess vacuolating cytotoxin (Vac) activity or sequence homology to vacA of H. pylori. A distinct cytotoxic activity referred to as granulating cytotoxin (GCT) had been described to occur in H. hepaticus (31). This activity was observed after 48 to 72 h when culture supernatants and cell extracts of H. hepaticus were added to a cloned liver cell line derived from a newborn male C3H/An mouse (ATCC CCL-9.1; clone NCTC 1469). The facts that both GCT activity and CDT activity were present in culture supernatants and that both required 48 to 72 h before a CPE was visually apparent led us to test the hypothesis that GCT activity is mediated by CDT.
CCL-9.1 cells were treated with sonicates of wild-type H. hepaticus 3B1 as well as the two transposon mutants 3B1::Tn16 (CDT+) and 3B1::Tn20 (CDT−). Sonicates of either wild-type 3B1 or 3B1::Tn16 caused CPE on CCL-9.1 cells (Fig. (Fig.3).3). Conversely, sonicates from the CDT-negative mutant failed to induce CPE on CCL-9.1 cells. Additionally, CCL-9.1 cells that exhibited CPE following treatment with sonicates from 3B1 or 3B1::Tn16 were arrested at G2/M. Furthermore, CCL-9.1 cells treated with sonicates from an E. coli clone carrying pVBY9 exhibited CPE.
H. hepaticus has been shown to be sufficient to induce inflammatory bowel disease (IBD) in several strains of mice with altered immune function. Specific-pathogen-free IL-10−/− mice develop severe typhlocolitis when they are infected with H. hepaticus. In order to determine if CDT plays a role in the development of murine IBD, IL-10−/− mice were experimentally infected with the isogenic H. hepaticus CDT mutants.
Specific-pathogen-free C57BL/6 IL-10−/− mice, free of known Helicobacter spp., were orally challenged with either wild-type H. hepaticus 3B1, 3B1::Tn16, or 3B1::Tn20. A control group received sterile culture broth. Colonization was monitored by culture and PCR. H. hepaticus isolated by culture from feces was tested with PCR primers spanning regions of the CDT locus to ensure that there was no cross-contamination of the H. hepaticus strains used in the experiment.
Six weeks after challenge, all animals that received H. hepaticus remained colonized with the bacterium. In all cases, only the specific challenge strain was recovered from infected animals. At this time, all animals were euthanized and subjected to necropsy. By 6 weeks postinfection, none of the control animals developed significant typhlocolitis (Fig. (Fig.4A).4A). One control animal had a small focus of chronic inflammatory cells at the ileocecal junction, a lesion that can occur spontaneously in uninoculated animals. All IL-10−/− mice infected with wild-type H. hepaticus 3B1 developed lesions resembling IBD that were consistent with those described by other researchers. These animals developed marked mucosal hyperplasia accompanied by inflammation of the lamina propria with various degrees of submucosal extension (Fig. (Fig.4B).4B). IL-10−/− mice infected with the transposon-marked CDT-producing isogenic mutant 3B1::Tn16 also developed typhlocolitis with a severity equivalent to that of mice infected with wild-type H. hepaticus 3B1 (Fig. (Fig.4C4C and and5).5). IL-10−/− mice that were infected with the CDT-negative mutant 3B1::Tn20, however, exhibited significantly less severe typhlocolitis than mice infected with CDT-producing H. hepaticus strains (Fig. (Fig.4D4D and and5).5). Although all mice were infected with the mutant H. hepaticus (as judged by fecal culture and PCR), one mouse was histologically normal, and the majority of animals exhibited only mild, patchy mucosal inflammation (Fig. (Fig.5).5). Although two mice infected with 3B1::Tn20 did develop diffuse mucosal inflammation, none of the mice infected with the CDT-negative mutant developed the submucosal inflammation encountered in a subset of the mice infected with CDT-positive strains. Mesenteric perivasculitis was encountered in infected animals with the most severe inflammation but never in uninfected controls (Fig. (Fig.4E4E and F).
Subsequent to the recognition of H. pylori and other gastric Helicobacter species, the EHS were identified in the distinct environment of the intestinal tracts and/or livers of humans, other mammals, and birds (11, 24, 29). EHS are emerging as important veterinary and human pathogens that are responsible for a number of acute disease syndromes, including gastroenteritis and bacteremia or sepsis. In addition, a subset of EHS also cause persistent infections associated with chronic inflammation and neoplasia, which is analogous to the association between H. pylori infection and subsequent chronic gastritis and gastric cancer (3). There is growing evidence that EHS can be associated with chronic liver diseases in humans, including chronic hepatitis, liver carcinoma, chronic cholecystitis, and cholangiocarcinoma (1, 2, 12, 22, 23).
In order to study the molecular pathogenesis of EHS infections, genetic tools for the manipulation of these organisms are needed. We describe here a method for the generation of targeted isogenic mutations in the EHS H. hepaticus. The transposon mutagenesis system described here is also useful for the generation of random mutations throughout the genome in addition to the generation of targeted mutations in potential virulence factors.
The production of toxins is a critical feature of the pathogenesis of a number of enteric pathogens (reviewed in reference 27). Toxins have been theorized to play a role in a number of pathogenic processes, including tissue invasion and the stimulation of fluid secretion to enhance nutrient delivery and/or to facilitate the spread to new hosts and escape from immune surveillance. Since we identified CDT in H. hepaticus, we sought to determine if this toxin plays a role in the pathogenesis of a disease associated with murine infection by this pathogen.
H. hepaticus was previously reported to possess GCT activity (31). Given the ability of H. hepaticus to colonize the murine biliary tree and cause chronic hepatitis and hepatic cancer, the organisms were tested for the ability to cause CPE on CCL-9.1 cells, which are cloned liver cells derived from a newborn C3H/An mouse.
We demonstrate here that the cytopathic activity initially described as GCT is mediated by CDT. Three lines of evidence support this conclusion. First, a transposon mutant of H. hepaticus targeting the cdtABC gene cluster lacks both CDT and GCT activity. Second, an E. coli strain that carries the cloned cdtABC gene cluster from H. hepaticus produces GCT activity as assayed on CCL-9.1 cells. Third, cell cycle analysis of CCL-9.1 cells that exhibit GCT CPE reveals that the cells arrested at G2/M.
Although CDT activity and gene sequences appear to be widely distributed among a diverse group of bacterial pathogens, there has been little experimental evidence that suggests that CDT plays a critical role in the production of disease in mammalian hosts infected with CDT-producing organisms. The elimination of CDT activity in Haemophilus ducreyi did not alter the virulence of this organism in either the temperature-dependent rabbit model for experimental chancroid (19) or human volunteers (34). An isogenic C. jejuni CDT mutant was found to be as efficient as the wild type at colonizing the gastrointestinal tracts of mice (26). It was suggested that the C. jejuni CDT mutant was less invasive since it was found in spleen, liver, and blood samples of only 4 of 15 infected mice 2 h after challenge, whereas wild-type C. jejuni was found in spleen, liver, and blood samples of 8 of 15 infected mice.
The data presented here indicate that CDT plays a role in the development of IBD in immune-altered mice infected with H. hepaticus. Although an isogenic H. hepaticus CDT mutant retained the ability to colonize C57BL/6 IL-10−/− mice, animals infected with the mutant developed significantly less severe disease than littermates infected with a wild-type H. hepaticus strain. Additionally, there were notable qualitative differences in the natures of the typhlocolitis seen in mice infected with 3B1::Tn20. Although each individual mouse was infected with the mutant H. hepaticus strain, the gastrointestinal tract of one mouse was histologically normal, and another exhibited only mild, patchy mucosal inflammation. Although two mice infected with 3B1::Tn20 did develop diffuse mucosal inflammation, none of the mice infected with the CDT-negative mutant developed significant submucosal inflammation similar to that observed in a subset of the mice infected with CDT-positive strains.
In summary, we have provided a description of a transposon-mediated shuttle mutagenesis system for the construction of isogenic mutants of the murine pathogen H. hepaticus. Using this genetic system we have demonstrated that the previously described GCT activity of H. hepaticus is mediated by CDT. In addition, we have demonstrated a role for CDT in mediating the IBD seen in IL-10−/− mice infected with H. hepaticus. The mechanism by which CDT leads to intestinal inflammation is not clear, but it may be that CDT targets a particular cell type involved in innate and/or adaptive immunity to escape immune surveillance. In mice with altered immune systems, such as IL-10−/− mice, this leads to a marked dysregulation of the immune response that results in the clinical entity of IBD.
This work was supported by Public Health Service grants CA67529 and AI50952 to J.G.F. and DK52413 to D.B.S. and a Michigan State University Intramural Research Grant Program New Investigator Award to V.B.Y.
Editor: D. L. Burns