Helicobacter hepaticus infection in BALB/c mice abolishes subunit-vaccine-induced protection against M. tuberculosis.

BCG, the only licensed vaccine against tuberculosis (TB), provides geographically variable protection, an effect ascribed to exposure to environmental mycobacteria (EM). Here we show that altering the intestinal microbiota of mice by early-life infection with the commensal bacterium Helicobacter hepaticus (Hh) increases their susceptibility to challenge with Mycobacterium tuberculosis (Mtb). Furthermore Hh-infected mice immunised parenterally with the recombinant subunit vaccine, human adenovirus type 5 expressing the immunodominant antigen 85A of Mtb (Ad85A), display a reduced lung immune response and protection against Mtb challenge is also reduced. Expression of interleukin 10 (IL10) messenger RNA is increased in the colon of Hh infected mice. Treatment of Hh-infected Ad85A-immunised mice with anti-IL10 receptor antibody, following challenge with Mtb, restores the protective effect of the vaccine. These data show for the first time that alteration of the intestinal microbiota by addition of a single commensal organism can profoundly influence protection induced by a TB subunit vaccine via an IL10-dependent mechanism, a result with implications for the deployment of such vaccines in the field.

Helicobacter hepaticus cholesterol-α-glucosyltransferase is essential for establishing colonization in male A/JCr mice.

BACKGROUND: Helicobacter pylori cholesterol-α-glucosyltransferase (cgt) is essential for survival of H. pylori in mice. Enterohepatic H. hepaticus, the cause of colonic and hepatocellular carcinoma in susceptible mouse strains, contains an ortholog of the H. pylori cgt. However, the role of cgt in the pathogenesis of H. hepaticus has not been investigated. MATERIALS AND METHODS: Two cgt-deficient isogenic mutants of wild-type H. hepaticus (WT) 3B1 were generated and used to inoculate male A/JCr mice. Cecal and hepatic colonization levels of the mutants and WT 3B1 as well as select inflammation-associated cytokines were measured by qPCR at 4 months postinoculation. RESULTS: Both mutants were undetectable in the cecum of any inoculated mice (10 per mutant) but were detected in two livers (one for each mutant); by contrast, 9 and 7 of 10 mice inoculated with WT 3B1 were qPCR positive in the ceca and livers, respectively. The mice inoculated with the mutants developed significantly less severe hepatic inflammation (p < .05) and also produced significantly lower hepatic mRNA levels of proinflammatory cytokines Ifn-γ (p < .01) and Tnf-α (p ≤ .02) as well as anti-inflammatory factors Il10 and Foxp3 compared with the WT 3B1-inoculated mice. Additionally, the WT 3B1-inoculated mice developed significantly higher Th1-associated IgG2a (p < .0001) and Th2-associated IgG1 responses (p < .0001) to H. hepaticus infection than mice dosed with isogenic cgt mutants. CONCLUSION: Our data indicate that the cholesterol-α-glucosyltransferase is required for establishing colonization of the intestine and liver and therefore plays a critical role in the pathogenesis of H. hepaticus.

Inhibitory effect of Lactobacillus acidophilus on Helicobacter hepaticus in vitro.

Helicobacter hepaticus and Helicobacter pylori both belong to Helicobacter species. Strains of Lactobacillus acidophilus, including L4 and L6, have shown significant inhibitory effects on H. pylori. Based on this phenomenon, we aim to investigate the inhibitory effect of L. acidophilus on H. hepaticus. Both standard and isolated H. hepaticus strains were grown under microaerophilic conditions at 37 °C in the presence of L. acidophilus supernatant, or lactic acid. The diameters of the inhibition zones were measured on the solid culture media. In liquid culture, the cell concentrations were measured and the urease activity was determined by phenol red staining. Sixteen strains of L. acidophilus isolated from human feces (named as L1-L16) showed anti-H. hepaticus effects. Two of them (L4 and L6) exhibited the most apparent effects on H. hepaticus inhibition. The L. acidophilus supernatant of L4 and L6 significantly increased the diameters of the inhibition zones compared with that of the lactic acid control (P < 0.05). The inhibitory role of L. acidophilus supernatant was independent of the pH value of solution (P > 0.05). Moreover, in liquid culture, L. acidophilus supernatant significantly reduced the cell growth rate and the urease activity of H. hepaticus cells in a time-dependent pattern (P < 0.05 compared with lactic acid control). No obvious difference was observed between the standard and isolated strain of H. hepaticus (P > 0.05). Our results indicate that L. acidophilus can decrease the viability and urease activity of H. hepaticus in vitro and this inhibition is independent of pH levels. This provides evidence for developing novel approaches for the prevention and treatment of H. hepaticus infection.

Helicobacter hepaticus NikR controls urease and hydrogenase activities via the NikABDE and HH0418 putative nickel import proteins.

Helicobacter hepaticus open reading frame HH0352 was identified as a nickel-responsive regulator NikR. The gene was disrupted by insertion of an erythromycin resistance cassette. The H. hepaticus nikR mutant had five- to sixfold higher urease activity and at least twofold greater hydrogenase activity than the wild-type strain. However, the urease apo-protein levels were similar in both the wild-type and the mutant, suggesting the increase in urease activity in the mutant was due to enhanced Ni-maturation of the urease. Compared with the wild-type strain, the nikR strain had increased cytoplasmic nickel levels. Transcription of nikABDE (putative inner membrane Ni transport system) and hh0418 (putative outer membrane Ni transporter) was nickel- and NikR-repressed. Electrophoretic mobility shift assays (EMSAs) revealed that purified HhNikR could bind to the nikABDE promoter (P(nikA)), but not to the urease or the hydrogenase promoter; NikR-P(nikA) binding was enhanced in the presence of nickel. Also, qRT-PCR and EMSAs indicated that neither nikR nor the exbB-exbD-tonB were under the control of the NikR regulator, in contrast with their Helicobacter pylori homologues. Taken together, our results suggest that HhNikR modulates urease and hydrogenase activities by repressing the nickel transport/nickel internalization systems in H. hepaticus, without direct regulation of the Ni-enzyme genes (the latter is the case for H. pylori). Finally, the nikR strain had a two- to threefold lower growth yield than the parent, suggesting that the regulatory protein might play additional roles in the mouse liver pathogen.

Characterization of a Helicobacter hepaticus putA mutant strain in host colonization and oxidative stress.

Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, L-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.

Nickel enzyme maturation in Helicobacter hepaticus: roles of accessory proteins in hydrogenase and urease activities.

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, possesses a hydrogenase and a urease, both of which are nickel-containing enzymes. Analysis of the genome sequence of H. hepaticus revealed a full set of accessory genes which are required for the nickel maturation of each enzyme in other micro-organisms. Erythromycin-resistant mutants were constructed in four of these genes, hypA, hypB, ureE and ureG. Controls for polar effect were provided for hypA or hypB mutants by disrupting each gene located immediately downstream, i.e. hp0809 or hypC, respectively. Urease and hydrogenase activities were determined for each strain with or without supplemented nickel in the medium. As expected, the ureE and the ureG mutants had negligible urease activity, but they retained normal levels of hydrogenase activity. Urease levels could not be increased by the addition of nickel to the medium. The H. hepaticus hypA and hypB strains were deficient in both urease and hydrogenase activities, suggesting that both gene products act in a similar fashion as their counterparts in H. pylori. However, in contrast with the analogous mutants of H. pylori, the addition of nickel into the growth medium failed to restore either urease or hydrogenase enzyme levels in the H. hepaticus hypA or hypB mutants, indicating a probably unique role for these genes in the mouse liver pathogen.

Eradication of Helicobacter bilis and H. hepaticus from infected mice by using a medicated diet.

Infection of laboratory mice with Helicobacter spp. is a serious problem for many laboratory animal facilities worldwide. Rederivation and antibiotic treatment are two of the most common methods used to eliminate the bacterial infection from rodent colonies. Forty-seven newly imported mice were suspected to be positive for Helicobacter infection based on PCR analysis of pooled fecal samples from sentinel animals. We treated the mice with a medicated feed containing four antibiotic compounds (amoxicillin, clarithromycin, metronidazole, omeprazole). After eight weeks of continuous administration the animals were negative for H. bilis and H. hepaticus. Frequent retesting of the animals for up to one year proved that the mouse colony remained negative for Helicobacter spp.

Inhibition of Helicobacter hepaticus-induced colitis by IL-10 requires the p50/p105 subunit of NF-kappa B.

Defects within the innate immune system sensitize NF-kappaB-deficient (p50(-/-); p65(+/-)) mice to Helicobacter hepaticus (Hh)-induced colitis. Because IL-10 plays a central role in the inhibition of Hh-induced colitis, we hypothesized that the ability of IL-10 to inhibit the innate inflammatory response to Hh may be compromised in NF-kappaB-deficient mice. To test this hypothesis, we evaluated the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit Hh-induced colitis in RAG-2(-/-) (RAG) and p50(-/-); p65(+/-); RAG-2(-/-) (3X/RAG) mice. As expected, IL-10-Ig efficiently inhibited the development of colitis in RAG mice. In contrast, the ability of IL-10-Ig to inhibit colitis was compromised in 3X/RAG mice. The defect in response to IL-10-Ig appeared to be primarily the result of the absence of the p50/p105 subunit, because the ability of IL-10-Ig to inhibit colitis was also compromised in p50(-/-); RAG-2(-/-) (p50/RAG) mice. Radiation chimeras demonstrated that the presence of p50/p105 within hemopoietic cells of the innate immune system was necessary for efficient inhibition of colitis by IL-10-Ig. Consistent with a defect in the suppressive effects of IL-10 in the absence of p50/p105, we found that the ability of IL-10 to control LPS-induced expression of IL-12 p40 was significantly compromised in macrophages lacking p50/p105. These results suggest that the absence of the p50/p105 subunit of NF-kappaB within hemopoietic cells of the innate immune system interferes with the ability of IL-10 to suppress inflammatory gene expression and Hh-induced colitis.

Modulation of host immune responses by the cytolethal distending toxin of Helicobacter hepaticus.

Persistent murine infection with Helicobacter hepaticus leads to chronic gastrointestinal inflammation and neoplasia in susceptible strains. To determine the role of the virulence factor cytolethal distending toxin (CDT) in the pathogenesis of this organism, interleukin-10-deficient (IL-10-/-) mice were experimentally infected with wild-type H. hepaticus and a CDT-deficient isogenic mutant. Both wild-type H. hepaticus and the CDT-deficient mutant successfully colonized IL-10-/- mice, and they reached similar tissue levels by 6 weeks after infection. Only animals infected with wild-type type H. hepaticus developed significant typhlocolitis. However, by 4 months after infection, the CDT-deficient mutant was no longer detectable in IL-10-/- mice, whereas wild-type H. hepaticus persisted for the 8-month duration of the experiment. Animals infected with wild-type H. hepaticus exhibited severe typhlocolitis at 8 months after infection, while animals originally challenged with the CDT-deficient mutant had minimal cecal inflammation at this time point. In follow-up experiments, animals that cleared infection with the CDT-deficient mutant were protected from rechallenge with either mutant or wild-type H. hepaticus. Animals infected with wild-type H. hepaticus developed serum immunoglobulin G1 (IgG1) and IgG2c responses against H. hepaticus, while animals challenged with the CDT-deficient mutant developed significantly lower IgG2c responses and failed to mount IgG1 responses against H. hepaticus. These results suggest that CDT plays a key immunomodulatory role that allows persistence of H. hepaticus and that in IL-10-/- mice this alteration of the host immune response results in the development of colitis.

Colonization dynamics of altered Schaedler flora is influenced by gender, aging, and Helicobacter hepaticus infection in the intestines of Swiss Webster mice.

The distribution and colonization levels of the altered Schaedler flora (ASF) in their natural hosts are poorly understood. Intestinal colonization levels of the eight ASF strains in outbred Swiss Webster mice with or without Helicobacter hepaticus infection were characterized by real-time quantitative PCR. All ASF strains were detected in the cecum and colon, but some strains displayed significant variation in colonization levels with host age, gender, and H. hepaticus infection status.

Differential regulation of urease activity in Helicobacter hepaticus and Helicobacter pylori.

Helicobacter hepaticus is a pathogen of rodents, which causes diverse enteric and hepatic inflammatory diseases and malignancies. The urease enzyme is an important colonization factor of gastric Helicobacter species like Helicobacter pylori, but little is known about the role and regulation of urease in enterohepatic Helicobacter species. Here it is reported that urease activity of H. hepaticus does not contribute to acid resistance, and that it is nickel-responsive at the post-translational level. H. hepaticus strain ATCC 51449 did not grow or survive at pH 3.0, and supplementation with urea or NiCl2 did not abrogate this acid sensitivity. Furthermore, urease enzyme activity of H. hepaticus was acid-independent, which contrasts with the acid-induced urease system of H. pylori. Nickel supplementation of Brucella medium resulted in a tenfold increase in urease activity in both H. hepaticus and H. pylori, but the maximum level of urease activity in H. hepaticus was still three- to fivefold lower when compared to H. pylori in the same conditions. The increase in urease activity of H. hepaticus was not associated with elevation of urease mRNA or protein levels. Inhibition of protein synthesis by chloramphenicol did not affect nickel-responsive induction of urease activity in H. hepaticus, and confirmed that nickel induction occurs at the post-translational level, probably by activation of preformed apo-enzyme. In conclusion, both the role of the urease enzyme and the regulation of urease activity differ between the enterohepatic pathogen H. hepaticus and the gastric pathogen H. pylori.

Colonization of the cecal mucosa by Helicobacter hepaticus impacts the diversity of the indigenous microbiota.

Establishment of mucosal and/or luminal colonization is the first step in the pathogenesis of many gastrointestinal bacterial pathogens. The pathogen must be able to establish itself in the face of competition from the complex microbial community that is already in place. We used culture-independent methods to monitor the colonization of the cecal mucosa of Helicobacter-free mice following experimental infection with the pathogen Helicobacter hepaticus. Two days after infection, H. hepaticus comprised a minor component of the mucosa-associated microbiota, but within 14 days, it became the dominant member of the community. Colonization of the mucosa by H. hepaticus was associated with a decrease in the overall diversity of the microbial community, in large part due to changes in evenness resulting from the relative dominance of H. hepaticus as a member of the community. Our results demonstrate that invasion of the complex gastrointestinal microbial community by a pathogenic microorganism causes reproducible and significant disturbances in the community structure. The use of non-culture-based methods to monitor these changes should lead to a greater understanding of the ecological principles that govern pathogen invasion and may lead to novel methods for the prevention and control of gastrointestinal pathogens.

Helicobacter hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and in causing liver lesions in A/J mice.

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, expresses a nickel-containing hydrogen-oxidizing hydrogenase enzyme. Growth of a hyaB gene-targeted mutant was unaffected by the presence of hydrogen, unlike the wild-type strain, which showed an enhanced growth rate when supplied with H(2). Hydrogenase activities in H. hepaticus were constitutive and not dependent on the inclusion of H(2) during growth. Addition of nickel during growth significantly stimulated both urease (for wild-type and hyaB) and hydrogenase (for wild-type) activities. In a 5-h period, the extent of (14)C-labeled amino acid uptake by the wild type was markedly enhanced in the presence of hydrogen and was >5-fold greater than that of the hyaB mutant strain. In the presence of H(2), the short-term whole-cell amino acid uptake V(max) of the parent strain was about 2.2-fold greater than for the mutant, but the half-saturation affinity for amino acid transport was the same for the parent and mutant strain. The liver- and cecum-colonizing abilities of the strains was estimated by real-time PCR quantitation of the H. hepaticus-specific cytolethal distending toxin gene and showed similar animal colonization for the hyaB mutant and the wild type. However, at 21 weeks postinoculation, the livers from mice inoculated with wild type exhibited moderate lobular lymphoplasmacytic hepatitis with hepatocytic coagulative necrosis, but the hydrogenase mutants exhibited no histological evidence of lobular inflammation or necrosis.