The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1.

Humans are frequently exposed to bacterial genotoxins of the gut microbiota, such as colibactin and cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro on HT29 intestinal cells while following the ectopic expression of the active CdtB subunit of Helicobacter hepaticus CDT. Microarray data showed a CdtB-dependent upregulation of transcripts involved in positive regulation of autophagy concomitant with the downregulation of transcripts involved in negative regulation of autophagy. CdtB promotes the activation of autophagy in intestinal and hepatic cell lines. Experiments with cells lacking autophagy related genes, ATG5 and ATG7 infected with CDT- and colibactin-producing bacteria revealed that autophagy protects cells against the genotoxin-induced apoptotic cell death. Autophagy induction could also be associated with nucleoplasmic reticulum (NR) formation following DNA damage induced by these bacterial genotoxins. In addition, both genotoxins promote the accumulation of the autophagic receptor P62/SQSTM1 aggregates, which colocalized with foci concentrating the RNA binding protein UNR/CSDE1. Some of these aggregates were deeply invaginated in NR in distended nuclei together or in the vicinity of UNR-rich foci. Interestingly, micronuclei-like structures and some vesicles containing chromatin and γH2AX foci were found surrounded with P62/SQSTM1 and/or the autophagosome marker LC3. This study suggests that autophagy and P62/SQSTM1 regulate the abundance of micronuclei-like structures and are involved in cell survival following the DNA damage induced by CDT and colibactin. Similar effects were observed in response to DNA damaging chemotherapeutic agents, offering new insights into the context of resistance of cancer cells to therapies inducing DNA damage.

A Large Polysaccharide Produced by Helicobacter hepaticus Induces an Anti-inflammatory Gene Signature in Macrophages.

Interactions between the host and its microbiota are of mutual benefit and promote health. Complex molecular pathways underlie this dialog, but the identity of microbe-derived molecules that mediate the mutualistic state remains elusive. Helicobacter hepaticus is a member of the mouse intestinal microbiota that is tolerated by the host. In the absence of an intact IL-10 signaling, H. hepaticus induces an IL-23-driven inflammatory response in the intestine. Here we investigate the interactions between H. hepaticus and host immune cells that may promote mutualism, and the microbe-derived molecule(s) involved. Our results show that H. hepaticus triggers early IL-10 induction in intestinal macrophages and produces a large soluble polysaccharide that activates a specific MSK/CREB-dependent anti-inflammatory and repair gene signature via the receptor TLR2. These data identify a host-bacterial interaction that promotes mutualistic mechanisms at the intestinal interface. Further understanding of this pathway may provide novel prevention and treatment strategies for inflammatory bowel disease.

The Cytolethal Distending Toxin Subunit CdtB of Helicobacter hepaticus Promotes Senescence and Endoreplication in Xenograft Mouse Models of Hepatic and Intestinal Cell Lines.

Cytolethal distending toxins (CDTs) are common among pathogenic bacteria of the human and animal microbiota. CDTs exert cytopathic effets, via their active CdtB subunit. No clear description of those cytopathic effects has been reported at the cellular level in the target organs in vivo. In the present study, xenograft mouse models of colon and liver cell lines were set up to study the effects of the CdtB subunit of Helicobacter hepaticus. Conditional transgenic cell lines were established, validated in vitro and then engrafted into immunodeficient mice. After successful engraftment, mice were treated with doxycyclin to induce the expression of transgenes (red fluorescent protein, CdtB, and mutated CdtB). For both engrafted cell lines, results revealed a delayed tumor growth and a reduced tumor weight in CdtB-expressing tumors compared to controls. CdtB-derived tumors showed γ-H2AX foci formation, an increase in apoptosis, senescence, p21 and Ki-67 nuclear antigen expression. No difference in proliferating cells undergoing mitosis (phospho-histone H3) was observed. CdtB intoxication was also associated with an overexpression of cytokeratins in cells at the invasive front of the tumor as well as an increase in ploidy. All these features are hallmarks of endoreplication, as well as aggressiveness in cancer. These effects were dependent on the histidine residue at position 265 of the CdtB, underlying the importance of this residue in CdtB catalytic activity. Taken together, these data indicate that the CdtB triggers senescence and cell endoreplication leading to giant polyploid cells in these xenograft mouse models.

[Preparation and identification of polyclonal antibody against methyl-accepting chemotaxis signal transduction protein of Helicobacter hepaticus].

OBJECTIVE: To prepare the polyclonal antibody against methyl-accepting chemotaxis signal transduction protein (MCP) of Helicobacter hepaticus (H.hepaticus). METHODS: The recombinant plasmid pET22b+/MCP was transformed into E.coli BL2l(DE3) to express the fusion protein His-rhMCP under the induction of IPTG. The fusion protein was purified and the antibody was obtained by immunizing rabbits. The titer of the polyclonal antibody was tested by indirect ELISA, and the specificity of the antibody was identified based on Western blotting using the prepared cell surface proteins (CSPs) of the bacteria. RESULTS: The fusion protein was successfully expressed, and the titer of the antibody reached 1:32 000. Western blotting indicated that the antibody could specifically bind to CSPs and His-rhMCP. CONCLUSION: The antibody with a high titer and specificity was prepared to facilitate further study of the pathogenicity and epidemiology of H.hepaticus in human.

Contribution of Helicobacter hepaticus cytolethal distending toxin subunits to human epithelial cell cycle arrest and apoptotic death in vitro.

BACKGROUND: Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus, the cause of chronic typhlocolitis and hepatitis leading to colonic and hepatocellular carcinomas in mice. Interaction of the tripartite polypeptide CdtA, CdtB, and CdtC subunits produced by H. hepaticus CDT (HhepCDT) causes cell cycle arrest and apoptotic death of cultured cells; however, the contribution of individual subunit to these processes has not been investigated. MATERIALS AND METHODS: The temporal relationship between cell cycle and apoptotic death of human epithelial HeLa and INT407 cells intoxicated with HhepCDT holotoxin or reconstituted recombinant HhepCDT was compared by flow cytometry. The genotoxic activity of individual and combinations of recombinant HhepCDT protein subunits or increasing concentrations of individual recombinant HhepCDT protein subunits transfected into HeLa cells was assessed at 72 hours post-treatment by flow cytometry. RESULTS: Similar time course of HhepCDT-induced G2 /M cell cycle arrest and apoptotic death was found with both cell lines which reached a maximum at 72 hours. The presence of all three HhepCDT subunits was required for maximum cell cycle arrest and apoptosis of both cell lines. Transfection of HeLa cells with HhepCdtB, but not with HhepCdtA or HhepCdtC, resulted in a dose-dependent G2 /M arrest and apoptotic death. CONCLUSION: All three subunits of HhepCDT are required for maximum epithelial cell cycle arrest and progression to apoptotic death, and HhepCdtB subunit alone is necessary and sufficient for epithelial cell genotoxicity.

Response of Helicobacter hepaticus to bovine bile.

Helicobacter hepaticus is an enterohepatic bacterium associated with inflammatory bowel disease in children and causes severe hepatobiliary disorders in mice. To elucidate the molecular response of H. hepaticus to bovine bile, a proteomic investigation was conducted. Bacteria were grown for 48 h in liquid media supplemented with different concentrations of bovine bile to determine its effects on bacterial growth and morphology. Protein expression profiles of bacteria grown at a bile concentration of 0.1% and in the absence of bile were obtained using two-dimensional gel electrophoresis. Gel spots with differences in intensities greater than 2-fold between both conditions were determined, and 55 differentially expressed proteins were identified using tandem mass spectrometry. Identified proteins participate in various biological functions including cell envelope biosynthesis, cell response to stress, iron homeostasis and transport, motility, primary and secondary metabolism, and virulence. Changes in the expression of H. hepaticus genes related to proteins involved in virulence and oxidative stress that were differentially expressed in the presence of bile were investigated using real-time reverse transcriptase PCR. The results indicated that the effects of bile on H. hepaticus included a strong response to oxidative stress and an expression of factors that can promote host colonization.

Histidine residue 94 is involved in pH sensing by histidine kinase ArsS of Helicobacter pylori.

BACKGROUND: The ArsRS two-component system is the master regulator of acid adaptation in the human gastric pathogen Helicobacter pylori. Low pH is supposed to trigger the autophosphorylation of the histidine kinase ArsS and the subsequent transfer of the phosphoryl group to its cognate response regulator ArsR which then acts as an activator or repressor of pH-responsive genes. Orthologs of the ArsRS two-component system are also present in H. pylori's close relatives H. hepaticus, Campylobacter jejuni and Wolinella succinogenes which are non-gastric colonizers. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the mechanism of acid perception by ArsS, derivatives of H. pylori 26695 expressing ArsS proteins with substitutions of the histidine residues present in its periplasmic input domain were constructed. Analysis of pH-responsive transcription of selected ArsRS target genes in these mutants revealed that H94 is relevant for pH sensing, however, our data indicate that protonatable amino acids other than histidine contribute substantially to acid perception by ArsS. By the construction and analysis of H. pylori mutants carrying arsS allels from the related epsilon-proteobacteria we demonstrate that WS1818 of W. succinogenes efficiently responds to acidic pH. CONCLUSIONS/SIGNIFICANCE: We show that H94 in the input domain of ArsS is crucial for acid perception in H. pylori 26695. In addition our data suggest that ArsS is able to adopt different conformations depending on the degree of protonation of acidic amino acids in the input domain. This might result in different activation states of the histidine kinase allowing a gradual transcriptional response to low pH conditions. Although retaining considerable similarity to ArsS the orthologous proteins of H. hepaticus and C. jejuni may have evolved to sensors of a different environmental stimulus in accordance with the non gastric habitat of these bacteria.

[Cloning and bioinformatics analysis of recombinant methyl-accepting chemotaxis signal transduction protein of Helicobacter hepaticus].

OBJECTIVE: To clone the gene encoding methyl-accepting chemotaxis signal transduction protein (MCSTP) of Helicobacter hepaticus and analyze the gene structures using bioinformatics methods. METHODS: With the specific primer of Helicobacter hepaticus MCSTP c1977, MCSTP gene was amplified by PCR from the genomic DNA of Helicobacter hepaticus and ligated to the prokaryotic expression vector pET22b(+). After sequencing, the sequence homology and structural feature of MCSTP gene were analyzed by bioinformatics method. RESULTS: A 99% similarity was identified between MCSTP gene cloned and its counterpart in standard Helicobacter hepaticus strain ATCC51449 genome DNA published by GenBank, with only a replacement of A by T at 1160 bp. A low homology was found in the MCSTP genes between Helicobacter hepaticus, Campylobacter jejuni and Helicobacter pylori by bioinformatics analysis, suggesting the specificity of MCSTP gene in Helicobacter hepaticus among the microbes. CONCLUSION: The prokaryotic expression plasmid pET22b(+)/MCSTP is constructed successfully, and the bioinformatics analysis provided evidences and clues for further study of the biological functions and pathogenic mechanism of MCSTP.

The Helicobacter hepaticus hefA gene is involved in resistance to amoxicillin.

BACKGROUND: Gastrointestinal infections with pathogenic Helicobacter species are commonly treated with combination therapies, which often include amoxicillin. Although this treatment is effective for eradication of Helicobacter pylori, the few existing reports are less clear about antibiotic susceptibility of other Helicobacter species. In this study we have determined the susceptibility of gastric and enterohepatic Helicobacter species to amoxicillin, and have investigated the mechanism of amoxicillin resistance in Helicobacter hepaticus. MATERIALS AND METHODS: The minimal inhibitory concentration (MIC) of antimicrobial compounds was determined by E-test and agar/broth dilution assays. The hefA gene of H. hepaticus was inactivated by insertion of a chloramphenicol resistance gene. Transcription was measured by quantitative real-time polymerase chain reaction. RESULTS: Three gastric Helicobacter species (H. pylori, H. mustelae, and H. acinonychis) were susceptible to amoxicillin (MIC < 0.25 mg/L). In contrast, three enterohepatic Helicobacter species (H. rappini, H. bilis, and H. hepaticus) were resistant to amoxicillin (MIC of 8, 16, and 6-64 mg/L, respectively). There was no detectable beta-lactamase activity in H. hepaticus, and inhibition of beta-lactamases did not change the MIC of amoxicillin of H. hepaticus. A H. hepaticus hefA (hh0224) mutant, encoding a TolC-component of a putative efflux system, resulted in loss of amoxicillin resistance (MIC 0.25 mg/L), and also resulted in increased sensitivity to bile acids. Finally, transcription of the hefA gene was not responsive to amoxicillin, but induced by bile acids. CONCLUSIONS: Rodents are frequently colonized by a variety of enterohepatic Helicobacter species, and this may affect their global health status and intestinal inflammatory responses. Animal facilities should have treatment strategies for Helicobacter infections, and hence resistance of enterohepatic Helicobacter species to amoxicillin should be considered when designing eradication programs.

Helicobacter hepaticus Dps protein plays an important role in protecting DNA from oxidative damage.

The ferritin-like DNA-binding protein from starved cells (Dps) family proteins are present in a number of pathogenic bacteria. Dps in the enterohepatic pathogen, Helicobacter hepaticus is characterized and a H. hepaticus dps mutant was generated by insertional mutagenesis. While the wild type H. hepaticus cells were able to survive in an atmosphere containing up to 6.0% O2, the dps mutant failed to grow in 3.0% O2, and it was also more sensitive to oxidative reagents like H2O2, cumene hydroperoxide and t-butyl hydroperoxide. Upon air exposure, the dps- cells had more damaged DNA than the wild type; they became coccoid or lysed and they contained approximately 6-fold higher amount of 8-oxoguanine (8-oxoG) DNA lesions than wild type cells. Purified H. hepaticus Dps was shown to be able to bind both iron and DNA. The iron-loaded form of Dps protein had much greater DNA binding ability than the native Dps or the iron-free Dps.

The cytolethal distending toxin B sub-unit of Helicobacter hepaticus is a Ca2+- and Mg2+-dependent neutral nuclease.

The cytolethal distending toxin B (CdtB) of the mouse pathogen Helicobacter hepaticus has cation binding and DNA catalysis residues in common with members of the mammalian deoxyribonuclease I (DNase I) family. The purpose of the present study was to characterize CdtB nuclease. To establish optimal digestion conditions and to evaluate co-factor requirements, a novel and sensitive fluorometric assay that quantitatively determines double stranded DNA digestion was developed. Although the Ca2+- and Mg2+-dependence and neutral properties of CdtB were similar to DNase I, hydrolysis of DNA by CdtB was approximately 100-fold less active than DNase I and was considerably more resistant to inhibition by ZnCl2 and G-actin.

Probiotic Lactobacillus spp. diminish Helicobacter hepaticus-induced inflammatory bowel disease in interleukin-10-deficient mice.

Clinical and experimental evidence has demonstrated the potential role of probiotics in the prevention or treatment of inflammatory bowel disease. Probiotic clones with direct immunomodulatory activity may have anti-inflammatory effects in the intestine. We investigated the roles of tumor necrosis factor alpha (TNF-alpha)-inhibitory Lactobacillus clones with a pathogen-induced murine colitis model. Murine-derived probiotic lactobacilli were selected in vitro for their ability to inhibit TNF-alpha secretion by Helicobacter hepaticus-stimulated macrophages. Interleukin-10 (IL-10)-deficient mice were treated with probiotic Lactobacillus reuteri in combination with Lactobacillus paracasei and then challenged with H. hepaticus. Ten weeks postinoculation, the severity of typhlocolitis was assessed by histologic examination of the cecocolic region. Intestinal proinflammatory cytokine responses were evaluated by real-time quantitative reverse transcriptase PCR and immunoassays, and the quantities of intestinal H. hepaticus were evaluated by real-time PCR. Intestinal colonization by TNF-alpha-inhibitory lactobacilli reduced intestinal inflammation in H. hepaticus-challenged IL-10-deficient mice despite similar quantities of H. hepaticus in cocolonized animals. Proinflammatory colonic cytokine (TNF-alpha and IL-12) levels were lowered in Lactobacillus-treated animals. In this H. hepaticus-challenged IL-10-deficient murine colitis model, lactobacilli demonstrated probiotic effects by direct modulation of mucosal inflammatory responses.

Evaluation of Helicobacter hepaticus bacterial shedding in fostered and sex-segregated C57BL/6 mice.

Neonatal fostering has been evaluated as a means of eliminating Helicobacter hepaticus infection in laboratory mouse colonies. The purpose of the present study was to evaluate cross-fostering of neonatal C57BL/6 pups from experimentally infected dams after male-absent parturition and to determine the effects of sex and housing strategy on H. hepaticus populations. Approximately 20 C57BL/6 mice (age, 1 to 4 days) were fostered daily. In all fostered mice, fecal samples collected at 21 and 42 days of age and cecal samples collected at 42 days of age tested negative for H. hepaticus by polymerase chain reaction analysis. Our results demonstrate that removal of the male prior to parturition extends the fostering period to yield Helicobacter-free mice. In a second experiment, the effects of time of infection, housing strategy, and sex on fecal H. hepaticus shedding and cecal colonization were evaluated. Neither time nor housing strategy affected bacterial shedding. In contrast, fecal and cecal bacterial loads were higher in male mice versus female mice. A novel predictive algorithm was developed to predict cecal bacterial colonization levels in light of fecal bacterial loads. Our findings likely will prove useful in Helicobacter eradication efforts and in studies designed to further elucidate the role of H. hepaticus in disease.

Expression and activity of the cytolethal distending toxin of Helicobacter hepaticus.

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.

Mechanism of proton gating of a urea channel.

The size and complexity of many pH-gated channels have frustrated the development of specific structural models. The small acid-activated six-membrane segment urea channel of Helicobacter hepaticus (HhUreI), homologous to the essential UreI of the gastric pathogen Helicobacter pylori, enables identification of all the periplasmic sites of proton gating by site-directed mutagenesis. Exposure to external acidity enhances [(14)C]urea uptake by Xenopus oocytes expressing HhUreI, with half-maximal activity (pH(0.5)) at pH 6.8. A downward shift of pH(0.5) in single site mutants identified four of six protonatable periplasmic residues (His-50 at the boundary of the second transmembrane segment TM2, Glu-56 in the first periplasmic loop, Asp-59 at the boundary of TM3, and His-170 at the boundary of TM6) that affect proton gating. Asp-59 was the only site at which a protonatable residue appeared to be essential for pH gating. Mutation of Glu-110 or Glu-114 in PL2 did not affect the pH(0.5) of gating. A chimera, where the entire periplasmic domain of HhUreI was fused to the membrane domain of Streptococcus salivarius UreI (SsUreI), retained the pH-independent properties of SsUreI. Hence, proton gating of HhUreI likely depends upon the formation of hydrogen bonds by periplasmic residues that in turn produce conformational changes of the transmembrane domain. Further studies on HhUreI may facilitate understanding of other physiologically important pH-responsive channels.