RESUMO
Duodenal confocal laser endomicroscopy (CLE) was carried out in six patients to compare the findings with histology. The visibility and quality of the endomicroscopy images were quantified using the following score: 0 = none; 1 = poor; 2 = fair; 3 = good. Four patients had a normal duodenal mucosa, whereas two patients in whom CLE indicated villous atrophy showed histologic features typical of celiac disease. Histology and CLE images were similar in both normal and celiac disease patients; patients with celiac disease had an average score of 3 for epithelial architecture, 3 for goblet cells, 3 for vessels, 1 for inflammatory infiltrate, and 2 for crypt visibility.
Assuntos
Doença Celíaca/patologia , Endoscópios Gastrointestinais , Microscopia Confocal/métodos , Adulto , Idoso , Estudos de Casos e Controles , Doença Celíaca/diagnóstico , Endoscopia Gastrointestinal/métodos , Feminino , Humanos , Imuno-Histoquímica , Mucosa Intestinal/patologia , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
The theoretical bases of medical knowledge exert a strong influence on both clinical practice and representations of living and health. In this perspective, reduction and emergence notions play a major role. Microreduction is the predominant analytical strategy used today in biology, as it is usually considered that essential life mechanisms can be reduced to molecular processes. Likewise, macroreduction proposes that parts can be defined in terms of their belonging to wholes, as it is usually assumed, for instance, in genetic epidemiology. With regard to emergence, this notion, which focuses on properties of a whole that cannot be deduced from properties of its parts, is consistent with both nature of living and evolution theory. The apparent success of reduction like analytical modality has generated in scientific community and public opinion an ideological reductionism, which corresponds, ontologically, to both physicalism (things can be entirely understood in terms of their parts), and atomism (things go their own way, independently of other things). Genetic reductionism has generated new cosmological representations of living, where past, present and future of living beings could potentially be deduced from fallacious, simple views of genome sequences. These views may lead to quantitative or qualitative definitions of standard patterns and hierarchies. In practical terms, research activity should integrate limits, strains as well as reductionism advantages. Biologists should also consider risks associated with an ideological, unrestricted reductionism, applied to any existence aspect, a notion with questionable legitimacy and with potential ethical, philosophical, and political involvements that go beyond the simple selection of a research strategy.
Assuntos
Pesquisa Biomédica/normas , FilosofiaRESUMO
The development of safe and potent mucosal adjuvants remains a major objective in vaccinology. The potential usefulness of filamentous haemagglutinin (FHA) of Bordetella pertussis as an adjuvant was assessed in a mouse model. The glutathione-S-transferase of Schistosoma mansoni (Sm28GST) was used for intranasal administration, while the gut-resistant keyhole limpet haemocyanin (KLH) was administrated by the oral route. For both antigens, coadministration with FHA increased antigen-specific immunoglobulin titres. This adjuvant effect did not require chemical cross-linking or direct interaction between FHA and the antigen tested. FHA also behaved as an adjuvant by the subcutaneous route, indicating that its adjuvanticity is not restricted to binding to mucosal surfaces. The FHA-induced adjuvanticity was also observed in mice with high anti-FHA antibody titres as a result of antipertussis vaccination, indicating that pre-existing anti-FHA antibodies do not impair FHA adjuvanticity. No mRNA coding for proinflammatory cytokines was induced in the lungs after intranasal FHA administration. However, an increase in the levels of mRNAs coding for B7-1, transforming growth factor (TGF)-beta and major histocompatibility complex (MHC)-II was detected in the lungs after FHA administration. Although the molecular mechanisms of the FHA-induced adjuvanticity remain to be elucidated, the data presented here indicate that this adhesin, already assessed for human use as a pertussis vaccine constituent, represents a promising adjuvant to improve the humoral immune response when given by mucosal routes.
Assuntos
Adesinas Bacterianas/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Hemaglutininas/administração & dosagem , Fatores de Virulência de Bordetella/administração & dosagem , Adesinas Bacterianas/química , Adesinas Bacterianas/farmacologia , Administração Intranasal , Animais , Antígeno B7-1/genética , Feminino , Genes MHC da Classe II , Glutationa Transferase/imunologia , Hemaglutininas/química , Hemaglutininas/farmacologia , Hemocianinas/imunologia , Camundongos , Schistosoma mansoni/imunologia , Fator de Crescimento Transformador beta/genética , Fatores de Virulência de Bordetella/química , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Tuberculosis remains the world's leading cause of death due to a single infectious agent, Mycobacterium tuberculosis, with 3 million deaths and 10 million new cases per year. The infection initiates in the lungs and can then spread rapidly to other tissues. The availability of the entire M. tuberculosis genome sequence and advances in gene disruption technologies have led to the identification of several mycobacterial determinants involved in virulence. However, no virulence factor specifically involved in the extrapulmonary dissemination of M. tuberculosis has been identified to date. Here we show that the disruption of the M. tuberculosis or Mycobacterium bovis Bacille Calmette-Guérin (BCG) hbhA gene encoding the heparin-binding haemagglutinin adhesin (HBHA) markedly affects mycobacterial interactions with epithelial cells, but not with macrophage-like cells. When nasally administered to mice, the mutant strains were severely impaired in spleen colonization, but not in lung colonization. Coating wild-type mycobacteria with anti-HBHA antibodies also impaired dissemination after intranasal infection. These results provide evidence that adhesins such as HBHA are required for extrapulmonary dissemination, and that interactions with non-phagocytic cells have an important role in the pathogenesis of tuberculosis. They also suggest that antibody responses to HBHA may add to immune protection against tuberculosis.
Assuntos
Hemaglutininas/fisiologia , Pulmão/microbiologia , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Aderência Bacteriana , Linhagem Celular , Células Epiteliais/microbiologia , Marcação de Genes , Hemaglutininas/genética , Hemaglutininas/imunologia , Humanos , Lectinas , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , Mycobacterium tuberculosis/imunologia , Baço/microbiologia , VirulênciaRESUMO
A monoclonal antibody (mAbKT4), produced against the Pichia anomala ATCC 96603 killer toxin (PaKT) was used to detect the toxin (WmKT) produced by Williopsis saturnus var. mrakii MUCL 41968 which inhibits the growth of a PaKT-sensitive P. anomala strain MUCL 41969. Immunofluorescence studies revealed that mAbKT4 specifically labels the surface of P. anomala and W. saturnus var. mrakii, suggesting that both taxa secrete a killer toxin bearing a common epitope. Immunoblot analyses of concentrated supernatants from P. anomala and W. saturnus var. mrakii cultures showed that in both taxa mAbKT4 reacts with high molecular weight secreted proteins ranging 85-200 kDa. However, immunoblot experiments showed that the molecular weights of PaKT and WmKT are quite different, indicating that the two toxins are related but not identical molecules.
Assuntos
Anticorpos Monoclonais/imunologia , Micotoxinas/imunologia , Pichia/imunologia , Animais , Reações Cruzadas , Imunofluorescência , Immunoblotting , Fatores Matadores de Levedura , Camundongos , Micotoxinas/análiseRESUMO
Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.
Assuntos
Mycobacterium smegmatis/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Laminina/imunologia , Anticorpos Antibacterianos/imunologia , Aderência Bacteriana , Compartimento Celular , Clonagem Molecular , Reações Cruzadas , Epitopos , Escherichia coli/genética , Genes Bacterianos , Glicosilação , Histonas/genética , Histonas/imunologia , Histonas/isolamento & purificação , Macrófagos/microbiologia , Microscopia Imunoeletrônica , Infecções por Mycobacterium/etiologia , Alvéolos Pulmonares/microbiologia , Receptores de Laminina/genética , Receptores de Laminina/isolamento & purificaçãoRESUMO
The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.
Assuntos
Hemaglutininas/metabolismo , Heparina/metabolismo , Mycobacterium/metabolismo , Sequência de Bases , Sítios de Ligação , Primers do DNA , Sulfato de Dextrana/química , Hemaglutininas/química , Heparitina Sulfato/metabolismo , Humanos , Hidrólise , Lectinas , Pulmão/citologia , Pulmão/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Deleção de Sequência , Ressonância de Plasmônio de SuperfícieRESUMO
In gram-negative bacteria, high-affinity iron uptake requires the TonB/ExbB/ExbD envelope complex to release iron chelates from their specific outer membrane receptors into the periplasm. Based on sequence similarities, the Bordetella pertussis tonB exbB exbD locus was identified on a cloned DNA fragment. The tight organization of the three genes suggests that they are cotranscribed. A putative Fur-binding sequence located upstream from tonB was detected in a Fur titration assay, indicating that the tonB exbB exbD operon may be Fur-repressed in high-iron growth conditions. Putative structural genes of the beta-subunit of the histone-like protein HU and of a new two-component regulatory system were identified upstream from tonB and downstream from exbD, respectively. A B. pertussis DeltatonB exbB::Km(r) mutant was constructed by allelic exchange and characterized. The mutant was impaired for growth in low-iron medium in vitro and could not use ferrichrome, desferal, or hemin as iron sources. Levels of production of the major bacterial toxins and adhesins were similar in the TonB(+)/TonB(-) pair. The DeltatonB exbB mutant was still responsive to chemical modulators of virulence; thus, the BvgA/BvgS two-component system is not TonB dependent. Nevertheless, in vivo in the mouse respiratory infection model, the colonization ability of the mutant was reduced compared to the parental strain.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Bactérias/fisiologia , Bordetella pertussis/imunologia , Bordetella pertussis/patogenicidade , Proteínas de Escherichia coli , Proteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/imunologia , Escherichia coli/metabolismo , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Proteínas Fúngicas/imunologia , Immunoblotting , Ferro/farmacocinética , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Óperon , Plasmídeos , Homologia de Sequência de Aminoácidos , Sideróforos/imunologia , Fatores de Tempo , Fatores de Transcrição/imunologia , VirulênciaRESUMO
Intranasal administration of live attenuated Bordetella pertussis, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of B. pertussis. By comparing the immune responses induced after infection with wild-type virulent B. pertussis with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of B. pertussis-infected mice. The intranasal infection with either the attenuated or the virulent B. pertussis strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuated B. pertussis could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/biossíntese , Bordetella pertussis/imunologia , Genitália Feminina/imunologia , Hemaglutininas/imunologia , Vacinas Sintéticas/imunologia , Fatores de Virulência de Bordetella , Animais , Feminino , Imunidade nas Mucosas , Imunização , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Although it generally is accepted that the interaction of Mycobacterium tuberculosis with alveolar macrophages is a key step in the pathogenesis of tuberculosis, interactions with other cell types, especially epithelial cells, also may be important. In this study we describe the molecular characterization of a mycobacterial heparin-binding hemagglutinin (HBHA), a protein that functions as an adhesin for epithelial cells. The structural gene was cloned from M. tuberculosis and bacillus Calmette-Guérin, and the sequence was found to be identical between the two species. The calculated Mr was smaller than the observed Mr when analyzed by SDS/PAGE. This difference can be attributed to the Lys/Pro-rich repeats that occur at the C-terminal end of the protein and to a putative carbohydrate moiety. Glycosylation of HBHA appears to protect the protein from proteolytic degradation, which results in the removal of the C-terminal Lys/Pro-rich region responsible for binding of HBHA to sulfated carbohydrates. Evidence suggests that glycosylation is also important for HBHA-mediated hemagglutination and for certain immunologic properties of the protein. Finally, the absence of a signal peptide in the coding region of HBHA raises the possibility that this protein is not secreted via the general secretion pathway.
Assuntos
Adesinas Bacterianas/genética , Hemaglutininas/genética , Mycobacterium bovis/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Escherichia coli/genética , Genes Bacterianos , Lectinas , Dados de Sequência MolecularRESUMO
To identify immunologically important domains on filamentous hemagglutinin (FHA), a Bordetella pertussis protein included in new acellular pertussis vaccines (ACPVs), a series of monoclonal antibodies, sera from infants vaccinated with ACPVs or whole cell pertussis vaccine (WCPV), and sera from patients with pertussis were analyzed by immunoblots containing FHA fragments and recombinant FHA proteins. Immunodominant domains located at the COOH-terminus of FHA (type I domain) and near the NH2-terminus (type II domain) were defined by the reactivity with monoclonal antibodies. The sera from patients with pertussis and sera from infants vaccinated with WCPV or with 6 different investigational ACPVs specifically recognized well-defined regions within the type I and type II domains. Identification of these prominent immunologic epitopes on FHA should be useful for the construction of more well-defined pertussis vaccines and for the interpretation of human serologic responses, which may correlate with efficacy of pertussis vaccines.
Assuntos
Adesinas Bacterianas/imunologia , Bordetella pertussis/imunologia , Hemaglutininas/imunologia , Epitopos Imunodominantes/análise , Vacina contra Coqueluche/imunologia , Fatores de Virulência de Bordetella , Sequência de Aminoácidos , Anticorpos Antibacterianos , Anticorpos Monoclonais , Criança , Humanos , Soros Imunes , Lactente , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Coqueluche/imunologiaRESUMO
Adherence to mammalian host tissues is an important virulence trait in microbial pathogenesis, yet little is known about the adherence mechanisms of mycobacteria. Here, we show that binding of mycobacteria to epithelial cells but not to macrophages can be specifically inhibited by sulfated carbohydrates. Using heparin-Sepharose chromatography, a 28-kD heparin-binding protein was purified from culture supernatants and cell extracts of Mycobacterium bovis and Mycobacterium tuberculosis. This protein, designated heparin-binding hemagglutinin (HBHA), promotes the agglutination of rabbit erythrocytes, which is specifically inhibited by sulfated carbohydrates. HBHA also induce mycobacterial aggregation, suggesting that it can mediate bacteria-bacteria interactions as well. Hemagglutination, mycobacterial aggregation, as well as attachment to epithelial cells are specifically inhibited in the presence of anti-HBHA antibodies. Immunoelectron microscopy using anti-HBHA monoclonal antibodies revealed that the protein is surface exposed, consistent with a role in adherence. Immunoblot analyses using antigen-specific antibodies indicated that HBHA is different from the fibronectin-binding proteins of the antigen 85 complex and p55, and comparison of the NH2-terminal amino acid sequence of purified HBHA with the protein sequence data bases did not reveal any significant similarity with other known proteins. Sera from tuberculosis patients but not from healthy individuals were found to recognize HBHA, indicating its immunogenicity in humans during mycobacterial infections. Identification of putative mycobacterial adhesins, such as the one described in this report, may provide the basis for the development of new therapeutic and prophylactic strategies against mycobacterial diseases.
Assuntos
Hemaglutininas/metabolismo , Heparina/metabolismo , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Animais , Aderência Bacteriana , Adesão Celular/efeitos dos fármacos , Agregação Celular , Galinhas , Células Epiteliais , Epitélio/microbiologia , Hemaglutininas/química , Humanos , Lectinas , Peso Molecular , Coelhos , Tuberculose/imunologiaRESUMO
The 220-kDa Bordetella pertussis filamentous hemagglutinin (FHA) is the major exported protein found in culture supernatants. The structural gene of FHA has a coding potential for a 367-kDa protein, and the mature form constitutes the N-terminal 60% of the 367-kDa precursor. The C-terminal domain of the precursor was found to be important for the high-level secretion of full-length FHA but not of truncated analogs (80 kDa or less). The secretion of full-length and truncated FHA polypeptides requires the presence of the approximately 100-amino-acid N-terminal domain and the outer membrane protein FhaC, homologous to the N-terminal domains of the Serratia marcescens and Proteus mirabilis hemolysins and their accessory proteins, respectively. By analogy to these hemolysins, it is likely that the N-terminal domain of the FHA precursor interacts, directly or indirectly, with the accessory protein during FHA biogenesis. However, immunogenicity and antigenicity studies suggest that the N-terminal domain of FHA is masked by its C-terminal domain and therefore should not be available for its interactions with FhaC. These observations suggest a model in which the C-terminal domain of the FHA precursor may play a role as an intramolecular chaperone to prevent premature folding of the protein. Both heparin binding and hemagglutination are expressed by the N-terminal half of FHA, indicating that this domain contains important functional regions of the molecule.
Assuntos
Adesinas Bacterianas/metabolismo , Bordetella pertussis/metabolismo , Hemaglutininas/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Virulência de Bordetella , Adesinas Bacterianas/genética , Animais , Antígenos de Bactérias , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Bordetella pertussis/genética , Genes Bacterianos , Teste de Complementação Genética , Testes de Hemaglutinação , Hemaglutininas/genética , Modelos Biológicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/genética , Ratos , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secretion signals are localized in the N-terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8-9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44. Regardless of the OmpA signal peptide-Fha44 fusion point, the E. coli-secreted Fha44 had the same M(r) as that secreted by B. pertussis, indicating that the N-terminal proteolytic maturation does not require a B. pertussis-specific factor. Similar to FHA, the B. pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N-terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB. The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias , Bordetella pertussis/metabolismo , Proteínas de Escherichia coli , Proteínas de Fímbrias , Processamento de Proteína Pós-Traducional/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Espaço Extracelular/enzimologia , Espaço Extracelular/metabolismo , Hemaglutininas/química , Immunoblotting , Óperon Lac/genética , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Sinais Direcionadores de Proteínas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The lengthy and cumbersome protocol used to establish the growth kinetics characteristics of anchorage-dependent cells (ADC's)in situ (i.e. while the cells adhere on their microsupport) by Aperture Impedance Pulse Spectroscopy (AIPS) can be replaced by an accelerated procedure. This we have named Turbo AIPS whereby the same results can be obtained without actually performing the manipulations leading to the determination of the biomass.
RESUMO
Filamentous hemagglutinin (FHA) is a major adhesin present on the surface of the gram-negative respiratory pathogen Bordetella pertussis. A number of binding mechanisms have been described for the interaction of FHA with eukaryotic cells. We have focused on its function as a sulfated polysaccharide-binding protein and on identifying potential receptors for FHA on the epithelial cell surface. Using a thin-layer overlay technique, we found that FHA binds specifically to sulfated glycolipids but not to gangliosides or other neutral glycolipids. These results suggest that epithelial cell surface sulfated glycolipids function as receptors for FHA. Further studies demonstrated that a Chinese hamster ovary (CHO) cell strain deficient in glycosaminoglycan expression exhibits greatly diminished attachment to FHA. By FHA-Affi-Gel chromatography, a putative receptor for FHA that has characteristics consistent with a heparan sulfate proteoglycan was isolated from epithelial cell extracts. In addition, by using recombinant FHA fusion proteins, a specific glycosaminoglycan-binding domain located near the N terminus of the FHA molecule was identified. Our results indicate that the B. pertussis adhesin FHA may utilize sulfated glycolipids and proteoglycans commonly found on the surface of human cells and tissues to initiate infection.
Assuntos
Adesinas Bacterianas/metabolismo , Bordetella pertussis/patogenicidade , Hemaglutininas/metabolismo , Heparitina Sulfato/metabolismo , Fatores de Virulência de Bordetella , Adesinas Bacterianas/química , Animais , Sequência de Bases , Sítios de Ligação , Bordetella , Células CHO , Cricetinae , Primers do DNA/química , Genes Bacterianos , Glicolipídeos/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Mapeamento por Restrição , Sulfoglicoesfingolipídeos/metabolismoRESUMO
Filamentous hemagglutinin (FHA) is a major adhesin produced by Bordetella pertussis, the etiologic agent of whooping cough. FHA has been shown to be surface associated but is also secreted by virulent bacteria. Microscopic observations of lungs of mice infected with B. pertussis showed that the bacteria grow as clusters within the alveolar lumen. When B. pertussis was cultivated in vitro with chemically defined medium, bacteria grew as aggregates, mimicking growth observed in vivo. This aggregation was abolished by the addition of cyclodextrin (CDX) to the growth medium and depended on the production of FHA, because a mutant lacking the FHA structural gene failed to form aggregates in a CDX-free medium. Western blot (immunoblot) analyses revealed that, in the absence of CDX, FHA was attached to the bacterial surface and was not efficiently released into the growth medium. Hydrophobic chromatography of FHA showed that CDX drastically reduced the hydrophobicity of FHA, suggesting a direct binding of CDX to FHA, which was further supported by the partial protection of FHA from trypsin digestion in the presence of CDX. In addition, free FHA can interact in a CDX-inhibitable manner with solid phase-immobilized FHA. It can therefore be postulated that the B. pertussis aggregates are most likely due to direct FHA-FHA interaction.
Assuntos
Adesinas Bacterianas , Aglutinação , Bordetella pertussis/crescimento & desenvolvimento , Hemaglutininas/fisiologia , Fatores de Virulência de Bordetella , Animais , Aderência Bacteriana , Ciclodextrinas/farmacologia , Feminino , Pulmão/microbiologia , Camundongos , Tripsina/farmacologiaRESUMO
Control of virulence factor expression in Bordetella pertussis is mediated by the products of the bvg operon. The BvgS membrane protein responds to certain environmental cues by activating the BvgA protein, which in turn modulates the expression of the target virulence factor genes. The BvgA and BvgS proteins are members of a large family of sensory transduction proteins called the two-component systems. We show that BvgA fusion proteins can activate transcription of a reporter gene containing the bvg promoter in Escherichia coli, and that this activity correlates with its ability to interact specifically with a recognition sequence in cognate promoters. Using homologies between BvgA and other bacterial response regulators as a guide, two BvgA truncation mutants were constructed and their transactivation and DNA-binding capacities were examined. We discovered that (1) DNA-binding activity is localized to the C-terminal half of BvgA, (2) sequence-specific DNA-binding is necessary, but not sufficient for transactivation, and (3) DNA-binding requires the last 20 amino acid residues at its carboxy terminus. A BvgA fusion protein lacking the receiver domain is inactive in transcriptional activation, but retains sequence-specific DNA-binding activity and forms multimeric complexes. We show that BvgA is able to utilize acetyl phosphate as a phosphoryl group donor and the instability of the covalent linkage at extremes of pH is consistent with an acyl phosphate group. Furthermore, the in vitro phosphorylated form of BvgA exhibits an enhanced capacity for binding DNA target sites, while a dephosphorylated form exhibits a limited capacity to bind these sites. We discuss the implications that these observations have on the mechanism by which BvgA is activated to a transcriptionally competent state.