ABSTRACT
Chronic Helicobacter pylori infection is the leading cause of intestinal-type adenocarcinoma, as prolonged Helicobacter colonization triggers chronic active gastritis, which may evolve into adenocarcinoma of the intestinal type. In this environment, cytokines play a significant role in determining the evolution of the infection. In combination with other factors (genetic, environmental and nutritional), the pro-inflammatory response may trigger pro-oncogenic mechanisms that lead to the silencing of tumour-suppressor genes, such as trefoil factor 1 (TFF1). The latter is known to play a protective role by maintaining the gastric mucosa integrity and retaining H. pylori in the mucus layer, preventing the progression of infection and, consequently, the development of gastric cancer (GC). Since TFF1 expression is reduced during chronic Helicobacter infection with a loss of gastric mucosa protection, we investigated the molecular pathways involved in this reduction. Specifically, we evaluated the effect of some pro-inflammatory cytokines on TFF1 regulation in GC and primary gastric cells by RT-qPCR and luciferase reporter assay analyses and the repressor role of the transcription factor C/EBPß, overexpressed in gastric-intestinal cancer. Our results show that, among several cytokines, IFNγ stimulates C/EBPß expression, which acts as a negative regulator of TFF1 by binding its promoter at three different sites.
Subject(s)
Adenocarcinoma , Helicobacter Infections , Helicobacter pylori , Stomach Neoplasms , Humans , Helicobacter Infections/genetics , Helicobacter Infections/metabolism , Helicobacter Infections/pathology , Helicobacter pylori/metabolism , Trefoil Factor-1/genetics , Trefoil Factor-1/metabolism , Trefoil Factor-1/pharmacology , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Adenocarcinoma/genetics , Cytokines/metabolismABSTRACT
BACKGROUND: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. METHODS: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2x10(5) cagPAI(-) H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. RESULTS: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. CONCLUSION: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.
Subject(s)
Bacterial Vaccines/immunology , Helicobacter Infections/prevention & control , Helicobacter pylori/immunology , Salmonella typhi/immunology , T-Lymphocyte Subsets/immunology , Adult , Antigens, Bacterial/immunology , Breath Tests , CD4-Positive T-Lymphocytes/immunology , Double-Blind Method , Gastritis/immunology , Gastritis/microbiology , Gastritis/pathology , Helicobacter Infections/complications , Helicobacter Infections/immunology , Helicobacter Infections/pathology , Helicobacter pylori/enzymology , Helicobacter pylori/isolation & purification , Humans , Immunity, Cellular , Male , Middle Aged , Prospective Studies , Salmonella Vaccines/immunology , Urease/immunology , Vaccination/adverse effects , Vaccines, Synthetic/immunologyABSTRACT
BACKGROUND: Despite intensive research, the etiology of acute anterior uveitis (AAU) remains poorly defined. Infection with gram-negative bacteria such as Yersinia, Salmonella, Shigella, and Chlamydia have already been suggested as a possible trigger event for AAU. Helicobacter pylori is also a gram-negative bacterium, shares the lipopolysaccharides, but did not attract the attention of many ophthalmologists until recently. Having in mind the relatively high incidence of H. pylori infection in the population, we propose that H. pylori may also be a trigger factor for AAU. PATIENTS AND METHODS: The presence of anti-H. pylori antibodies in matching serum and aqueous humor samples of 15 idiopathic AAU patients was determined using a commercial Western blot assay. Control serum and aqueous humor were obtained from five patients undergoing cataract surgery. RESULTS: Six out of 15 AAU patients (40%) were serum-positive for H. pylori, and half of these (n = 3) also had anti-H. pylori antibodies in the aqueous humor. All five aqueous humor and sera controls tested negative for H. pylori infection. CONCLUSION: These are the first results demonstrating anti-H. pylori antibodies in the aqueous humor of AAU patients. Further studies are needed to demonstrate whether this antibody is indeed locally produced. Our data may provide first evidence for a causative link between H. pylori infection and AAU.
Subject(s)
Helicobacter Infections/diagnosis , Helicobacter pylori/pathogenicity , Uveitis, Anterior/microbiology , Adult , Aged , Antibodies, Bacterial/analysis , Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Aqueous Humor/immunology , Aqueous Humor/microbiology , Bacterial Proteins/immunology , Female , Humans , Male , Middle Aged , Uveitis, Anterior/immunologyABSTRACT
Orally administered recombinant Salmonella vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, it is crucial to gather knowledge about the possible impact of preexisiting immunity to carrier antigens on the immunogenicity of recombinant vaccines. Thirteen volunteers were preimmunized with Salmonella typhi Ty21a in order to evaluate the effects of prior immunization with the carrier strain. Then, they received three doses of 1-2 x 10(10) viable organisms of either the vaccine strain S. typhi Ty21a (pDB1) expressing subunits A and B of recombinant Helicobacter pylori urease (n = 9), or placebo strain S. typhi Ty21a (n = 4). Four volunteers were preimmunized and boosted with the vaccine strain S. typhi Ty21a (pDB1). No serious adverse effects were observed in any of the volunteers. Whereas none of the volunteers primed and boosted with the vaccine strain responded to the recombinant antigen, five of the nine volunteers preimmunized with the carrier strain showed cellular immune responses to H. pylori urease (56%). This supports the results of a previous study in non-preimmunized volunteers where 56% (five of nine) of the volunteers showed a cellular immune response to urease after immunisation with S. typhi Ty21a (pDB1).
Subject(s)
Bacterial Vaccines/immunology , Helicobacter pylori/immunology , Salmonella Vaccines/immunology , Urease/immunology , Adult , Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Bacterial Vaccines/adverse effects , Bacterial Vaccines/genetics , CD4-Positive T-Lymphocytes/immunology , Female , Helicobacter Infections/prevention & control , Helicobacter pylori/enzymology , Helicobacter pylori/genetics , Humans , Immunity, Cellular , Immunoglobulin G/blood , Interferon-gamma/analysis , Lymphocyte Activation , Male , Plasmids , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Salmonella Vaccines/administration & dosage , Salmonella Vaccines/adverse effects , Salmonella Vaccines/genetics , Salmonella typhi/immunology , Urease/genetics , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/immunologyABSTRACT
An IgA1 protease is produced by the human pathogens Neisseria gonorrhoeae and N. meningitidis but not by related non-pathogenic, commensal, Neisseria species. In this study, the chromosomal iga locus was characterized in the N. gonorrhoeae strain MS11 and compared to corresponding loci in N. meningitidis and commensal Neisseria species. In N. gonorrhoeae, the genes trpB and ksgA were found immediately downstream of iga. In addition to comL and comA, a homolog of the Escherichia coli YFII gene was identified upstream of iga. Each gene in the iga region (YFII and comL, comA and iga, and trpB and ksgA) is transcribed in the opposite direction to its neighbors. The comL/ comA and iga/ trpB pairs each have a transcriptional terminator in the correct position for joint use. These terminators contain the common gonococcal DNA uptake sequence (DUS). A highly conserved direct repeat of 25 bp located immediately adjacent to the iga gene in N. gonorrhoeae was also found in N. meningitidis. In Southern hybridization experiments, no homology to iga was detectable in the chromosomal DNAs of the commensal species N. mucosa, N. lactamica, N. flavescens, N. cinerea, N. subflava, N. flava, N. sicca or N. elongata. When N. gonorrhoeae comL and trpB were used as probes, signals were detected on the same restriction fragment in six of the eight species. This indicated that commensal Neisseria species share a possible integration site for the iga gene between comA and trpB. The region between comA and trpB was therefore amplified by PCR. The fragment obtained from N. lactamica showed a high degree of homology to gonococcal comA and trpB, respectively, but iga was replaced by a sequence of 13 bp that shows no homology to any known gonococcal sequence. Our data suggest that iga was acquired by a common ancestor of N. gonorrhoeae and N. meningitidis rather than being distributed by horizontal gene transfer. N. lactamica, which is more closely related to N. gonorrhoeae than other commensals, may have lost iga by deletion.
Subject(s)
Neisseria/enzymology , Neisseria/genetics , Serine Endopeptidases/genetics , Bacterial Proteins/genetics , Base Sequence , Blotting, Southern , DNA/metabolism , DNA Primers/chemistry , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Genetic Linkage , Models, Genetic , Molecular Sequence Data , Open Reading Frames , Phylogeny , Physical Chromosome Mapping , Plasmids/metabolism , Polymerase Chain Reaction , Recombination, Genetic , Sequence Homology, Nucleic Acid , Species SpecificityABSTRACT
A multiparameter selection of Helicobacter pylori antigens for vaccine development identified 15 candidates, 6 of which are known protective antigens. Two novel antigens with low homology to other organisms (HP0231 and HP0410) were overexpressed and purified with high yields. Both confer protective immunity in the mouse Helicobacter infection model.
Subject(s)
Antigens, Bacterial/immunology , Helicobacter pylori/immunology , Animals , Antigens, Bacterial/analysis , Bacterial Vaccines/immunology , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred BALB C , T-Lymphocytes/immunologyABSTRACT
Helicobacter pylori urease was expressed in the common live typhoid vaccine Ty21a yielding Ty21a(pDB1). Nine volunteers received Ty21a(pDB1) and three control volunteers received Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed responses in one or two out of three assays for cellular responses to the carrier (proliferation, IFN-gamma-secretion, IFN-gamma-ELISPOT). Three of the volunteers that had received Ty21a(pDB1) showed a weak but significant T-cell response to Helicobacter urease, while no volunteer had detectable humoral responses to urease. Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.
Subject(s)
Bacterial Vaccines/pharmacology , Helicobacter pylori/enzymology , Helicobacter pylori/immunology , Salmonella typhi/genetics , Urease/genetics , Urease/immunology , Adolescent , Adult , Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/genetics , B-Lymphocytes/immunology , Bacterial Vaccines/adverse effects , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Double-Blind Method , Female , Gene Expression , Helicobacter pylori/genetics , Humans , Interferon-gamma/biosynthesis , Lymphocyte Activation , Male , Safety , T-Lymphocytes/immunology , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/pharmacologyABSTRACT
The obligate intracellular pathogen Chlamydophila pneumoniae (Chlamydia pneumoniae) initiates infections in humans via the mucosal epithelia of the respiratory tract. Here, we report that epithelial cells infected with C. pneumoniae are resistant to apoptosis induced by treatment with drugs or by death receptor ligation. The induction of protection from apoptosis depended on the infection conditions since only cells containing large inclusions were protected. The underlying mechanism of infection-induced apoptosis resistance probably involves mitochondria, the major integrators of apoptotic signaling. In the infected cells, mitochondria did not respond to apoptotic stimuli by the release of apoptogenic factors required for the activation of caspases. Consequently, active caspase-3 was absent in infected cells. Our data suggest a direct modulation of apoptotic pathways in epithelial cells by C. pneumoniae.
Subject(s)
Apoptosis , Chlamydophila pneumoniae/growth & development , Epithelial Cells/microbiology , Apoptosis/drug effects , Caspase 3 , Caspases/metabolism , Cytochrome c Group/metabolism , Enzyme Activation , Humans , Mitochondria/drug effects , Staurosporine/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor-like ('hummingbird') phenotype. In this study, using a cagA mutant complemented with wild-type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti-phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA-green fluorescent protein (GFP) fusion proteins and two-dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C-terminus. Site-directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).
Subject(s)
Antigens, Bacterial , Bacterial Proteins/metabolism , Gastric Mucosa/microbiology , Helicobacter pylori/classification , Helicobacter pylori/pathogenicity , Tyrosine/metabolism , Adenocarcinoma/microbiology , Bacterial Proteins/genetics , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Gastric Mucosa/metabolism , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Humans , Immunoblotting , Microscopy, Confocal , Phenotype , Phosphorylation , Stomach Neoplasms/microbiology , Tumor Cells, Cultured , VirulenceABSTRACT
Previously we described a recombinant attenuated Salmonella typhimurium aroA strain (SL3261[pYZ97]) with constitutive expression of plasmid encoded Helicobacter pylori urease subunits A and B (UreAB). Single dose oral vaccination effectively induced prophylactic immunity against bacterial challenge in BALB/c mice. Here we successfully extended this approach to several mouse strains with allelic differences in NRAMP-1 and H-2 genes. The respective host determinants are known to influence the immune response against S. typhimurium. A comparative analysis of the vaccine efficacy in C57BL/6 and BALB/c mice showed that the live vaccine confers long lasting immunity in both strains (>18 weeks). In C57BL/6 mice, protection was still observed 54 weeks while not all vaccinated BALB/c were immune when challenged after this time. BALB/c mice also needed higher doses of SL3261[pYZ97] for full protection. We also demonstrate a therapeutic potential of SL3261[pYZ97] in H. pylori infected BALB/c and C57BL/6 mice. Urease- and carrier-specific serum antibody responses as well as the level of colonization by the Salmonella were analyzed in both mouse strains after immunization with low (4 x 10(7)CFU) or high (1 x 10(9)CFU) vaccine doses. The results are discussed in the context of inoculum size and the mode of antigen supply required for effective vaccination with recombinant Salmonella.
Subject(s)
Bacterial Vaccines/immunology , Helicobacter pylori/immunology , Salmonella typhimurium/genetics , Urease/immunology , Vaccines, Synthetic/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Female , Helicobacter pylori/enzymology , Mice , Mice, Inbred Strains , Recombinant Proteins/immunology , Species Specificity , VaccinationABSTRACT
The common human pathogen Helicobacter pylori is the major cause of gastric and duodenal ulcers. Based on the complete genome sequences of two independent isolates more than 1800 protein species have been resolved by two-dimensional gel electrophoresis and more than 200 of them have been identified (http://www.mpiib-berlin.mpg.de/2D-PAGE). Using these data, a large range of research areas including strain fingerprinting, protein composition and subcellular localization, gene regulation, and pathogen-host interactions have been investigated. The results that have been obtained led to a more detailed understanding of the Helicobacter biology and pathology and open further interesting fields for future work.
Subject(s)
Helicobacter pylori/genetics , Proteome/genetics , Proteome/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Databases, Protein , Electrophoresis, Gel, Two-Dimensional , Genome, Bacterial , Helicobacter Infections/etiology , Helicobacter pylori/pathogenicity , Humans , Peptide MappingABSTRACT
Helicobacter pylori is one of the most common bacterial pathogens that causes a variety of gastric diseases. During infection, the immuno-dominant H. pylori CagA protein is translocated and tyrosine-phosphorylated in gastric epithelial cells. We compared tyrosine phosphorylation patterns of five CagA variants by two-dimensional electrophoresis (2-DE) and immunoblotting studies. Tyrosine-phosphorylated CagA was detected as two distinct protein species in strains P12, P227, G27 and 26695 suggesting that two tyrosine residues of CagA can be phosphorylated both separately and simultaneously. Prediction programs revealed the presence of three putative tyrosine phosphorylation motifs in the sequences of CagA. Mutations in these motifs were identified suggesting that only two putative phosphorylation-relevant tyrosines are present in each CagA variant. CagA of strain J99 was found to be unique because essential codons were mutated in each of the three motifs and, consequently, revealed no tyrosine phosphorylation signals at all. These findings support the view that CagA from different H. pylori strains can be tyrosine-phosphorylated at one or two out of three predicted positions. Additionally, truncated CagA protein species of about 100-105 kDa (p100CagA) have been detected after infection with some of the H. pylori strains. The isoelectric point determined by both 2-DE and sequence analysis suggested that p100CagA represents the amino (N)-terminal part of the protein. Translocation, tyrosine phosphorylation and size modification of CagA might be involved in host signal transduction and development of gastric disease.
Subject(s)
Antigens, Bacterial , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Helicobacter pylori/metabolism , Amino Acid Motifs , Amino Acid Sequence , Bacterial Proteins/genetics , Base Sequence , Cell Line , Cloning, Molecular , DNA, Bacterial/genetics , Electrophoresis, Gel, Two-Dimensional , Epithelial Cells/metabolism , Genes, Bacterial , Helicobacter pylori/genetics , Helicobacter pylori/pathogenicity , Humans , Immunoblotting , Molecular Weight , Phosphorylation , Proteome , Spectrometry, Mass, Electrospray Ionization , Tyrosine/metabolismABSTRACT
Helicobacter pylori is a very common bacterial pathogen that causes gastric disease by inducing the infiltration of immune cells as an initial event. Virulent H. pylori strains express a type IV secretion system composed of several virulence (Vir) proteins encoded by the cag pathogenicity island (cag PAI). During infection of phagocytic cells (U937, Josk-M and J774A.1) we have detected a de novo tyrosine-phosphorylated protein (p35p-Tyr) with sizes of 30 kDa, 38 kDa or 40 kDa, depending on the H. pylori strain. p35p-Tyr occurrence required functional virB4, virB7, virB10, virB11, virD4 and cagA (cytotoxin-associated gene A) genes encoded by the cag PAI suggesting that p35p-Tyr is a bacterial protein of variable size. We have biochemically purified p35p-Tyr from infected U937 cells. Tryptic peptides of p35p-Tyr determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) identified the carboxy (C)-terminal part of the H. pylori CagA protein. Subsequent analysis by two-dimensional electrophoresis (2-DE) and immunoblotting using anti-CagA antibodies revealed the presence of three stable CagA protein species in phagocytes: (i) 130-140 kDa full-length CagA (p135CagA), (ii) a 100-105 kDa fragment (p100CagA) and (iii) a 30-40 kDa fragment (p35CagA). Unlike p135CagA, p35CagA and p100CagA were also detected in much lower amounts in H. pylori without host cell contact. Therefore, breakage or processing leads to the production of p35CagA and p100CagA, a process that is enhanced after translocation into host cells. MALDI-MS data and the isoelectric point determined by both 2-DE and sequence analysis suggested that p35CagA represents the C-terminal part of CagA and p100CagA corresponds to the remaining amino (N)-terminal fragment. The possible function of CagA in host signal transduction and development of gastric disease is discussed.
Subject(s)
Antigens, Bacterial , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Helicobacter pylori/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Cell Line , Electrophoresis, Gel, Two-Dimensional , Genes, Bacterial , Helicobacter Infections/etiology , Helicobacter pylori/genetics , Helicobacter pylori/pathogenicity , Humans , Immunoblotting , Molecular Sequence Data , Molecular Weight , Peptide Fragments/genetics , Peptide Mapping , Phagocytes/metabolism , Phagocytes/microbiology , Phosphorylation , Protein Processing, Post-Translational , Proteome , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tyrosine/metabolism , VirulenceABSTRACT
Chlamydiae are obligate intracellular bacteria residing exclusively in host cell vesicles termed inclusions. We have investigated the effects of deferoxamine mesylate (DAM)-induced iron deficiency on the growth of Chlamydia pneumoniae and Chlamydia trachomatis serovar L2. In epithelial cells subjected to iron starvation and infected with either C. pneumoniae or C. trachomatis L2, small inclusions were formed, and the infectivity of chlamydial progeny was impaired. Moreover, for C. trachomatis L2, we observed a delay in homotypic fusion of inclusions. The inhibitory effects of DAM were reversed by adding exogenous iron-saturated transferrin, which restored the production of infectious chlamydiae. Electron microscopy examination of iron-deprived specimens revealed that the small inclusions contained reduced numbers of C. pneumoniae that were mostly reticulate bodies. We have previously reported specific accumulation of transferrin receptors (TfRs) around C. pneumoniae inclusions within cells grown under normal conditions. Using confocal and electron microscopy, we show here a remarkable increase in the amount of TfRs surrounding the inclusions in iron-starved cultures. It has been shown that iron is an essential factor in the growth and survival of C. trachomatis. Here, we postulate that, for C. pneumoniae also, iron is an indispensable element and that Chlamydia may use iron transport pathways of the host by attracting TfR to the phagosome.
Subject(s)
Chlamydophila pneumoniae/growth & development , Epithelial Cells/microbiology , Iron Deficiencies , Chlamydia trachomatis/drug effects , Chlamydia trachomatis/growth & development , Chlamydia trachomatis/pathogenicity , Chlamydophila Infections/etiology , Chlamydophila pneumoniae/pathogenicity , Endocytosis , Epithelial Cells/pathology , Humans , Iron Chelating Agents/pharmacology , Species Specificity , Tumor Cells, CulturedABSTRACT
Helicobacter pylori has been identified as the major aetiological agent in the development of chronic gastritis and duodenal ulcer, and it plays a role in the development of gastric carcinoma. Attachment of H. pylori to gastric epithelial cells leads to nuclear and cytoskeletal responses in host cells. Here, we show that Rho GTPases Rac1 and Cdc42 were activated during infection of gastric epithelial cells with either the wild-type H. pylori or the mutant strain cagA. In contrast, no activation of Rho GTPases was observed when H. pylori mutant strains (virB7 and PAI) were used that lack functional type IV secretion apparatus. We demonstrated that H. pylori-induced activation of Rac1 and Cdc42 led to the activation of p21-activated kinase 1 (PAK1) mediating nuclear responses, whereas the mutant strain PAI had no effect on PAK1 activity. Activation of Rac1, Cdc42 and PAK1 represented a very early event in colonization of gastric epithelial cells by H. pylori. Rac1 and Cdc42 were recruited to the sites of bacterial attachment and are therefore probably involved in the regulation of local and overall cytoskeleton rearrangement in host cells. Finally, actin rearrangement and epithelial cell motility in H. pylori infection depended on the presence of a functional type IV secretion system encoded by the cag pathogenicity island (PAI).
Subject(s)
Antigens, Bacterial , Helicobacter Infections/metabolism , Helicobacter pylori/metabolism , Helicobacter pylori/pathogenicity , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , Bacterial Adhesion/physiology , Bacterial Proteins/genetics , Cell Movement , Cells, Cultured , Enzyme Activation , Epithelial Cells/microbiology , Gastric Mucosa/cytology , Gastric Mucosa/microbiology , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Humans , Protein Serine-Threonine Kinases/metabolism , p21-Activated KinasesABSTRACT
Neisseria gonorrhoeae express opacity-associated (Opa) protein adhesins that mediate binding to various members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM; previously CD66) receptor family. Although human umbilical vein endothelial cells express little CEACAM receptor in vitro, we found neisserial infection to induce expression of CEACAM1, CEACAM1-3L, and CECAM1-4L splice variants. This mediates an increased Opa(52)-dependent binding of gonococci by these cells. The induced receptor expression did not require bacterial Opa expression, but it was more rapid with adherent bacteria. Because the time course of induction was similar to that seen for induced proinflammatory cytokines, we tested whether CEACAM1 expression could be controlled by a similar mechanism. Gonococcal infection activated a nuclear factor-kappaB (NF-kappaB) heterodimer consisting of p50 and p65, and inhibitors that prevent the nuclear translocation of activated NF-kappaB complex inhibited CEACAM1 transcript expression. Each of these effects could be mimicked by using culture filtrates or purified lipopolysaccharide instead of intact bacteria. Together, our results support a model whereby the outer membrane "blebs" that are actively released by gonococci trigger a Toll-like receptor-4-dependent activation of NF-kappaB, which up-regulates the expression of CEACAM1 to allow Opa(52)-mediated neisserial binding. The regulation of CEACAM1 expression by NF-kappaB also implies a broader role for this receptor in the general inflammatory response to infection.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation/biosynthesis , Bacterial Adhesion , Drosophila Proteins , Endothelium, Vascular/microbiology , NF-kappa B/metabolism , Neisseria gonorrhoeae/pathogenicity , Antigens, Bacterial/physiology , Antigens, CD/genetics , Antigens, Differentiation/genetics , Bacterial Outer Membrane Proteins/physiology , Cell Adhesion Molecules , Cells, Cultured , Cytokines/biosynthesis , Cytokines/genetics , Endothelium, Vascular/metabolism , Humans , Immediate-Early Proteins/metabolism , Kinetics , Membrane Glycoproteins/physiology , Models, Biological , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , RNA, Messenger/biosynthesis , Receptors, Cell Surface/physiology , Toll-Like Receptor 4 , Toll-Like Receptors , Transcriptional Activation , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Helicobacter pylori infection of the stomach epithelium is characterized by an infiltration of polymorphonuclear and mononuclear cells. These immune cells contribute to mucosal damage which may eventually lead to gastritis, peptic ulcer, gastric cancer, and/or MALT-associated gastric lymphoma. Here we show that H. pylori inhibits its own uptake, as well as in trans the phagocytosis of Neisseria gonorrhoeae, by human and murine macrophages. This antiphagocytic activity is dependent on the presence of the cag pathogenicity island in the H. pylori genome. We demonstrate that H. pylori also expresses its antiphagocytic activity towards the myelomonocytic cell line JOSKM, thus providing a potent model for the study of the interaction between H. pylori and phagocytes. Our data were obtained using laser confocal microscopy and flow cytometry after quenching the fluorescence of labeled extracellular bacteria. The antiphagocytic activity of H. pylori may explain the persistence of H. pylori and its pathological consequences. The use of cell lines and flow cytometry will hopefully facilitate progress in our understanding of the immune escape of these persistent bacteria.
Subject(s)
Flow Cytometry , Helicobacter pylori/immunology , Macrophages/immunology , Phagocytosis , Animals , Cell Line , Humans , Mice , Microscopy, ConfocalABSTRACT
An important attribute that contributes to the virulence of Neisseria gonorrhoeae is its phenotypic variability, which is based on recombination within complex gene families in the genome. In this study we report on the in vivo amplification of large segments of the genome and the existence of circular DNA intermediates in the cell, which might help to explain the evolution of these gene families and provide possible clues as to how genetic variability is maintained. Using an inserted chromosomal marker (cat) in the N. gonorrhoeae MS11 genome that confers low-level resistance to chloramphenicol (Cm), we isolated variants that express resistance to high levels of Cm. Genetic analysis revealed that virtually all variants harboured single or multiple tandem amplifications of the respective genome segments carrying the cat insert. This process occurred independently of both the location of the cat insertion site and of the presence of a functional recA gene. Analysis of the genetically well characterised pilC region revealed a head-to-tail orientation of the amplified segments, with the junctions being located within direct repeats. Identical junctions were detected in extra-chromosomal circular DNA molecules isolated from non-selected wild-type and recA strains, suggesting that both types of structure arise by related processes. The existence of DNA circles was shown by their banding behaviour in caesium chloride/ethidium bromide density centrifugation and their resistance to digestion by exonuclease. The possible roles of such circles in processes such as pilin gene recombination, chromosomal gene amplification and genetic transformation are discussed.
Subject(s)
DNA, Circular , Neisseria gonorrhoeae/genetics , Recombination, Genetic , Blotting, Southern , Centrifugation, Density Gradient , Cesium/pharmacology , Chloramphenicol/pharmacology , Chlorides/pharmacology , Drug Resistance/genetics , Genetic Markers , Genetic Variation , Models, Genetic , Phenotype , Plasmids/metabolism , Polymerase Chain Reaction , Protein Synthesis Inhibitors/pharmacology , Sequence Analysis, DNA , Tandem Repeat SequencesABSTRACT
PrimeArray is a Windows program that computes oligonuceotide primer pairs for genome-scale gene amplification by the Polymerase Chain Reaction (PCR). The program supports the automated extraction of coding sequences (CDS) from various input-file formats and allows highly automated primer pair-optimization.
Subject(s)
DNA Primers/genetics , Oligonucleotide Array Sequence Analysis/methods , Software , Base Sequence , Computational Biology , Computer-Aided Design , Drug Design , Helicobacter pylori/genetics , Oligonucleotide Array Sequence Analysis/statistics & numerical data , Polymerase Chain ReactionABSTRACT
Protection in the murine model of Helicobacter pylori infection may be mediated by CD4+ T cells, but the mechanism remains unclear. To better understand how protection occurs in this model, we generated and characterized H. pylori urease-specific CD4+ T cells from BALB/c mice immunized with Salmonella enterica serovar Typhimurium expressing H. pylori urease (subunits A and B). The CD4+ T cells were found to be specific for subunit A (UreA). Upon antigen-specific stimulation, expression of interleukin 4 (IL-4), IL-10, gamma interferon (IFN-gamma), and tumor necrosis factor alpha was induced. Immunocytochemical analysis showed that the majority of cells produced IFN-gamma and IL-10. Adoptive transfer of the UreA-specific CD4+ T cells into naive syngeneic recipients led to a threefold reduction in the number of bacteria in the recipient group when compared to that in the nonrecipient group. Stomach colonization was also reduced significantly after transfer of these cells into patently infected mice. Adoptive transfer of UreA-specific CD4+ T cells into IL-4 receptor alpha chain-deficient BALB/c mice indicated that IL-4 and IL-13 were not critical in the control of bacterial load. In addition, synthetic peptides were used to identify three functional T-cell epitopes present in subunit A which were recognized by the UreA-specific T cells. Analysis of H. pylori-specific cellular immune responses in recipient challenged and nonrecipient infected mice indicated a strong local restriction of the response in infected animals. The implications of these findings for the mechanism of protection and the development of peptide-based vaccination are discussed.