RESUMO
Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.
Assuntos
Gastrite/genética , Inativação Gênica/fisiologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/genética , Urease/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Doença Crônica , Mucosa Gástrica/microbiologia , Gastrite/microbiologia , Expressão Gênica/genética , Camundongos , Neoplasias Gástricas/genéticaRESUMO
Helicobacter pylori lipopolysaccharide promotes chronic gastric colonisation through O-antigen host mimicry and resistance to mucosal antimicrobial peptides mediated primarily by modifications of the lipid A. The structural organisation of the core and O-antigen domains of H. pylori lipopolysaccharide remains unclear, as the O-antigen attachment site has still to be identified experimentally. Here, structural investigations of lipopolysaccharides purified from two wild-type strains and the O-antigen ligase mutant revealed that the H. pylori core-oligosaccharide domain is a short conserved hexasaccharide (Glc-Gal-DD-Hep-LD-Hep-LD-Hep-KDO) decorated with the O-antigen domain encompassing a conserved trisaccharide (-DD-Hep-Fuc-GlcNAc-) and variable glucan, heptan and Lewis antigens. Furthermore, the putative heptosyltransferase HP1284 was found to be required for the transfer of the third heptose residue to the core-oligosaccharide. Interestingly, mutation of HP1284 did not affect the ligation of the O-antigen and resulted in the attachment of the O-antigen onto an incomplete core-oligosaccharide missing the third heptose and the adjoining Glc-Gal residues. Mutants deficient in either HP1284 or O-antigen ligase displayed a moderate increase in susceptibility to polymyxin B but were unable to colonise the mouse gastric mucosa. Finally, mapping mutagenesis and colonisation data of previous studies onto the redefined organisation of H. pylori lipopolysaccharide revealed that only the conserved motifs were essential for colonisation. In conclusion, H. pylori lipopolysaccharide is missing the canonical inner and outer core organisation. Instead it displays a short core and a longer O-antigen encompassing residues previously assigned as the outer core domain. The redefinition of H. pylori lipopolysaccharide domains warrants future studies to dissect the role of each domain in host-pathogen interactions. Also enzymes involved in the assembly of the conserved core structure, such as HP1284, could be attractive targets for the design of new therapeutic agents for managing persistent H. pylori infection causing peptic ulcers and gastric cancer.
Assuntos
Helicobacter pylori/química , Helicobacter pylori/patogenicidade , Lipopolissacarídeos/química , Antígenos O/química , Animais , Western Blotting , Cromatografia Gasosa , Modelos Animais de Doenças , Infecções por Helicobacter/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Ressonância Magnética Nuclear Biomolecular , Oligossacarídeos/química , Domínios Proteicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.
Assuntos
Proteínas de Bactérias/fisiologia , Helicobacter pylori/enzimologia , Recombinases/fisiologia , Recombinação Genética/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Segregação de Cromossomos/genética , Ciprofloxacina/farmacologia , DNA Topoisomerase IV/genética , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Mutação Puntual , Recombinases/química , Recombinases/genética , Recombinação Genética/genéticaRESUMO
Helicobacter pylori genomes typically contain 8 or 9 genes that code for secreted and highly disulfide-bridged proteins designated Helicobacter cysteine-rich proteins (Hcp). Here we show that HcpA (hp0211) but not HcpC (hp1098) triggers the differentiation of human myeloid Thp1 monocytes into macrophages. Small amounts of HcpA cause the transition of round-shaped monocytes into cells with star-like morphologies, adherence to the culture dish surface, phagocytosis of opsonized fluorescent microspheres, and expression of the surface marker protein CD11b, all of which are indicative of a macrophage-like phenotype. We conclude that HcpA acts as a bacterial immune modulator similar to a eukaryotic cytokine.
Assuntos
Proteínas de Bactérias/metabolismo , Diferenciação Celular , Helicobacter pylori/metabolismo , Macrófagos/citologia , Monócitos/citologia , beta-Lactamases/metabolismo , Adesão Celular , Humanos , Macrófagos/metabolismo , Microscopia Eletrônica de Varredura , Monócitos/metabolismo , Fagocitose , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: According to the WHO, more than 1 billion people worldwide are overweight and at risk of developing chronic illnesses, including cardiovascular disease, type 2 diabetes, hypertension and stroke. Current therapies show limited efficacy and are often associated with unpleasant side-effect profiles, hence there is a medical need for new therapeutic interventions in the field of obesity. Gastric inhibitory peptide (GIP, also known as glucose-dependent insulinotropic polypeptide) has recently been postulated to link over-nutrition with obesity. In fact GIP receptor-deficient mice (GIPR(-/-)) were shown to be completely protected from diet-induced obesity. Thus, disrupting GIP signaling represents a promising novel therapeutic strategy for the treatment of obesity. METHODOLOGY/PRINCIPAL FINDINGS: In order to block GIP signaling we chose an active vaccination approach using GIP peptides covalently attached to virus-like particles (VLP-GIP). Vaccination of mice with VLP-GIP induced high titers of specific antibodies and efficiently reduced body weight gain in animals fed a high fat diet. The reduction in body weight gain could be attributed to reduced accumulation of fat. Moreover, increased weight loss was observed in obese mice vaccinated with VLP-GIP. Importantly, despite the incretin action of GIP, VLP-GIP-treated mice did not show signs of glucose intolerance. CONCLUSIONS/SIGNIFICANCE: This study shows that vaccination against GIP was safe and effective. Thus active vaccination may represent a novel, long-lasting treatment for obesity. However further preclinical safety/toxicology studies will be required before the therapeutic concept can be addressed in humans.
Assuntos
Polipeptídeo Inibidor Gástrico/química , Obesidade/metabolismo , Obesidade/terapia , Vacinas/química , Células 3T3 , Animais , Peso Corporal , Células CHO , Cricetinae , Cricetulus , Polipeptídeo Inibidor Gástrico/imunologia , Homeostase , Humanos , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de SinaisRESUMO
BACKGROUND: In mice, highly repetitive antigens, such as those present on bacterial or viral surfaces, efficiently cross-link B-cell receptors and therefore induce strong IgG responses. In this study we covalently coupled a synthetic 16-amino-acid sequence of the allergen Der p 1 to a virus-like particle derived from the bacteriophage Qbeta (Qbeta-Der p 1). OBJECTIVE: We evaluated the safety and immunogenicity of Qbeta-Der p 1 in human subjects and compared different doses and routes of immunization. METHODS: In a phase I trial 24 healthy volunteers were randomly assigned to one of 4 treatment groups. Group 1 received 50 microg of Qbeta-Der p 1 intramuscularly, group 2 received 50 microg of Qbeta-Der p 1 subcutaneously, group 3 received 10 microg of Qbeta-Der p 1 intramuscularly, and group 4 received 10 microg of Qbeta-Der p 1 subcutaneously. Boosting immunizations with 10 microg were given after 1 and 3 months. Antibody titers were measured after 1, 3, 4, 6, 12, and 18 months. RESULTS: The vaccine Qbeta-Der p 1 was well tolerated. Significant IgG responses were observed 4 weeks after a single injection. Individuals receiving 50 microg of the vaccine had significantly higher IgG titers than those vaccinated with 10 microg. However, the route of immunization (subcutaneous vs intramuscular) had no effect. In the 50-microg dose group, strong antibody responses against Der p 1 with average titers of 1:2000 were obtained. CONCLUSION: Vaccination with a peptide antigen covalently coupled to highly repetitive virus-like particles represents an adjuvant-free means of rapidly inducing high antibody titers in human subjects. CLINICAL IMPLICATIONS: Allergens coupled to virus-like particles can be used to enhance the efficiency of allergen-specific immunotherapy.
Assuntos
Allolevivirus/imunologia , Anticorpos Antivirais/biossíntese , Antígenos de Dermatophagoides/imunologia , Fragmentos de Peptídeos/imunologia , Pyroglyphidae/imunologia , Vacinas Virais/imunologia , Vírion/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/administração & dosagem , Proteínas de Artrópodes , Cisteína Endopeptidases , Feminino , Antígenos do Núcleo do Vírus da Hepatite B/administração & dosagem , Antígenos do Núcleo do Vírus da Hepatite B/efeitos adversos , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/efeitos adversos , Vacinas contra Hepatite B/imunologia , Humanos , Imunoglobulina G/biossíntese , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/efeitos adversos , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/efeitos adversos , Vacinas Combinadas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversosRESUMO
The factors limiting neonatal and infant IgG Ab responses to T-dependent Ags are only partly known. In this study, we assess how these B cell responses are influenced by the postnatal development of the spleen and lymph node microarchitecture. When BALB/c mice were immunized with alum-adsorbed tetanus toxoid at various stages of their immune development, a major functional maturation step for induction of serum IgG, Ab-secreting cells, and germinal center (GC) responses was identified between the second and the third week of life. This correlated with the development of the follicular dendritic cell (FDC) network, as mature FDC clusters only appeared at 2 wk of age. Adoptive transfer of neonatal splenocytes into adult SCID mice rapidly induced B cell follicles and FDC precursor differentiation into mature FDC, indicating effective recruitment and signaling capacity of neonatal B cells. In contrast, adoptive transfer of adult splenocytes into neonatal SCID mice induced primary B cell follicles without any differentiation of mature FDC and failed to correct limitations of tetanus toxoid-induced GC. Thus, unresponsiveness to lymphoid-mediated signals at the level of neonatal FDC precursors delays FDC maturation and GC induction, thus limiting primary Ab-secreting cell responses to T-dependent Ags in early postnatal life.
Assuntos
Animais Recém-Nascidos/imunologia , Células Produtoras de Anticorpos/metabolismo , Células Dendríticas Foliculares/imunologia , Centro Germinativo/imunologia , Tolerância Imunológica/imunologia , Subpopulações de Linfócitos/imunologia , Transdução de Sinais/imunologia , Células-Tronco/imunologia , Envelhecimento/imunologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Anticorpos Antibacterianos/biossíntese , Células Produtoras de Anticorpos/citologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Quimiocina CXCL13 , Quimiocinas CXC/fisiologia , Células Dendríticas Foliculares/citologia , Feminino , Centro Germinativo/citologia , Haptenos/imunologia , Subpopulações de Linfócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Ovalbumina/imunologia , Receptores CXCR5 , Receptores de Quimiocinas , Receptores de Citocinas/fisiologia , Células-Tronco/citologia , Toxoide Tetânico/imunologia , Trinitrobenzenos/imunologiaRESUMO
The effect of aging on host resistance to systemic candidosis was assessed by monitoring the course of infection in 16-month-old CBA/CaH mice (aged non-immune) and in a comparable group that had been infected with a sublethal dose of Candida albicans at 6 weeks of age (aged immune). Aged non-immune mice showed rapid progression of the disease, with a marked increase in the number of mycelia in the brain and kidney, and early morbidity. Foci of myocardial necrosis were evident, but inflammatory cells were sparse. The histological picture in the aged immune mice was similar to that in the aged non-immune group, although fewer mycelial aggregates were seen. Both groups of aged mice showed a significantly lower fungal burden in the brain on day 1 of infection, but on day 4, colony counts increased significantly in the aged non-immune mice. Comparison of cytokine gene expression in the infected brains showed that the relative amount of interferon-gamma and tumour necrosis factor-alpha cDNA were similar in all three groups. Interleukin-6 was elevated in both infected non-immune and uninfected aged mice. Aged immune mice showed no morbidity after challenge, and both colonisation and tissue damage were reduced in comparison with the aged non-immune animals.