RESUMO
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.
Assuntos
Adenocarcinoma , Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Humanos , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/patologia , Helicobacter pylori/metabolismo , Fator Trefoil-1/genética , Fator Trefoil-1/metabolismo , Fator Trefoil-1/farmacologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Adenocarcinoma/genética , Citocinas/metabolismoRESUMO
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.
Assuntos
Citocinas/biossíntese , NF-kappa B/metabolismo , Neisseria gonorrhoeae/fisiologia , Fator de Transcrição AP-1/metabolismo , Sequência de Bases , Linhagem Celular , Citocinas/genética , Epitélio/microbiologia , Humanos , Interleucina-6/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Ativação TranscricionalRESUMO
Neisseria gonorrhoeae (Ngo), the etiologic agent of gonorrhea, induce a number of proinflammatory cytokines by contact to epithelial cells. Cytokine genes and a variety of other immune response genes are activated as a result of the regulatory function of immediate early response transcription factors including activator protein 1 (AP-1). Since it is established that phosphorylation of c-Jun, the central component of AP-1, by the stress-activated c-Jun NH2-terminal kinase (JNK) increases the transcriptional activity of AP-1, we studied whether Ngo could induce stress response pathways involving JNK. We found that virulent Ngo strains induce phosphorylation and activation of JNK but not of p38 kinase. Analysis of a nonpathogenic Ngo strain revealed only weak JNK activation. In respect to the molecular components upstream of the JNK signaling cascade, we show that a dominant negative mutant of MAP kinase kinase 4 (MKK4) represses transcription of an AP-1-dependent reporter gene. Regarding upstream stress response factors involved in Ngo-induced MKK4/JNK/AP-1 activation, we identified p21-activated kinase (PAK) but not MAPK/ERK kinase kinase (MEKK1). Inhibition of small GTPases including Rac1 and Cdc42 by Toxin B prevented JNK and AP-1 activation. Our results indicate that Ngo induce the activation of proinflammatory cytokines via a cascade of cellular stress response kinases involving PAK, which directs the signal from the Rho family of small GTPases to JNK/AP-1 activation.
Assuntos
Proteínas de Bactérias , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citocinas/metabolismo , MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Neisseria gonorrhoeae/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição AP-1/metabolismo , Toxinas Bacterianas/farmacologia , Ativação Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases Ativadas por p21RESUMO
A characteristic of human pathogenic Neisseriae is the production and secretion of an immunoglobulin (Ig)A1-specific serine protease (IgA1 protease) that cleaves preferentially human IgA1 and other target proteins. Here we show a novel function for native IgA1 protease, i.e., the induction of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8 from peripheral blood mononuclear cells. The capacity of IgA1 protease to elicit such cytokine responses in monocytes was enhanced in the presence of T lymphocytes. IgA1 protease did not induce the regulatory cytokine IL-10, which was, however, found in response to lipopolysaccharide and phytohemagglutinin. The immunomodulatory effects caused by IgA1 protease require a native form of the enzyme, and denaturation abolished cytokine induction. However, the proteolytic activity is not required for the cytokine induction by IgA1 protease. Our results indicate that IgA1 protease exhibits important immunostimulatory properties and may contribute substantially to the pathogenesis of neisserial infections by inducing large amounts of TNF-alpha and other proinflammatory cytokines. In particular, IgA1 protease may represent a key virulence determinant of bacterial meningitis.
Assuntos
Citocinas/imunologia , Monócitos/efeitos dos fármacos , Neisseria gonorrhoeae/enzimologia , Serina Endopeptidases/farmacologia , Adjuvantes Imunológicos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Imunoglobulina A/metabolismo , Inflamação/etiologia , Inflamação/imunologia , Interferon gama/metabolismo , Interleucinas/metabolismo , Lipopolissacarídeos/farmacologia , Neisseria gonorrhoeae/patogenicidade , Fito-Hemaglutininas/farmacologia , Serina Endopeptidases/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/imunologia , VirulênciaRESUMO
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.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Salmonella typhi/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Antígenos de Bactérias/imunologia , Testes Respiratórios , Linfócitos T CD4-Positivos/imunologia , Método Duplo-Cego , Gastrite/imunologia , Gastrite/microbiologia , Gastrite/patologia , Infecções por Helicobacter/complicações , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/patologia , Helicobacter pylori/enzimologia , Helicobacter pylori/isolamento & purificação , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Vacinas contra Salmonella/imunologia , Urease/imunologia , Vacinação/efeitos adversos , Vacinas Sintéticas/imunologiaRESUMO
In contrast to textbook ideas of pure cultures and defined strains, genetic variation is a fact of life in the microbial world. It not only allows pathogens to establish themselves in their chosen host, but also allows them to resist that host's subsequent attempts to evict them. Here we review some of the mechanisms that bring about this variation, and some of the functional consequences that result from it.
Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Variação Genética/genética , Adaptação Biológica , Antígenos de Bactérias/genética , Bactérias/patogenicidade , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/genética , Fímbrias Bacterianas , Conversão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Lipopolissacarídeos , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , VirulênciaRESUMO
Neisseria gonorrhoeae is a highly adapted human pathogen that utilises multiple adhesins to interact with a variety of host cell receptors. Recently, substantial progress has been made in unravelling the signalling events induced by N. gonorrhoae that can lead to cytoskeletal reorganisation, invasion or phagocytic uptake, intraphagosomal accommodation, nuclear signalling, cytokine/chemokine release and apoptosis.
Assuntos
Células Epiteliais/fisiologia , Proteínas de Fímbrias , Neisseria gonorrhoeae/patogenicidade , Animais , Antígenos de Bactérias/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Apoptose , Proteínas de Bactérias/metabolismo , Células CHO , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Cricetinae , Citocinas/biossíntese , Células Epiteliais/microbiologia , Células HeLa , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Mucosa/microbiologia , Mucosa/fisiologia , NF-kappa B/metabolismo , Neisseria gonorrhoeae/química , Porinas/metabolismo , Transdução de Sinais , Especificidade da Espécie , Fator de Transcrição AP-1/metabolismo , VirulênciaRESUMO
Pathogenic Neisseria spp. possess a repertoire of phase-variable Opa proteins that mediate various pathogen--host cell interactions, including bacterial engulfment by epithelial cells and opsonin-independent phagocytosis by professional phagocytes. Recent studies have identified cellular targets recognized by defined Opa proteins and have begun to reveal host signalling events involved in mediating these Opa-dependent cellular processes.
Assuntos
Antígenos de Bactérias/fisiologia , Neisseria gonorrhoeae/fisiologia , Neisseria meningitidis/fisiologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Moléculas de Adesão Celular , Células Epiteliais/microbiologia , Proteoglicanas de Heparan Sulfato/metabolismo , HumanosRESUMO
Extracellular transport of Neisseria IgA proteases across the bacterial outer membrane is accomplished by the translocation function contained within the C-terminal Iga beta domain of IgA protease precursor proteins. Recently, we reported that Iga beta from N. gonorrhoeae MS11 (Val1097 to Phe1505), fused to a periplasmic passenger protein, facilitated its transport across the outer membrane, leading to surface exposure of the passenger. In the present work we show, by systematic N-terminal truncation of Iga beta, that the functional and structural unit, termed Iga beta-core, corresponds to the C-terminal approximately 274 amino acid residues (Ser1231 to Phe1505). This minimal region retains all the essential features necessary for the translocation of an N-terminally attached passenger across the outer membrane of Escherichia coli, and for its own correct integration into the outer membrane, even in the absence of a passenger protein. The membrane-integrated Iga beta-core constitutes a conserved entity found in the C-terminal regions of Iga beta domains of different N. gonorrhoeae, N. meningitidis and Haemophilus influenzae strains. In contrast, the surface-exposed N termini of the Iga beta domains vary in size and sequence. Based on secondary structure predictions, the key structural feature of the core is a beta-barrel (amphipathic, antiparallel transmembrane beta-strands, interspersed by hairpin turns and loops) which is common to many integral outer membrane proteins of Gram-negative bacteria. We propose that the core has been conserved in evolution, to provide a selective outer membrane export channel for covalently attached polypeptides.
Assuntos
Genes Bacterianos , Neisseria gonorrhoeae/enzimologia , Neisseria meningitidis/enzimologia , Peptídeo Hidrolases/metabolismo , Precursores de Proteínas/metabolismo , Serina Endopeptidases , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/metabolismo , Clonagem Molecular , Primers do DNA , DNA Bacteriano/análise , DNA Bacteriano/metabolismo , Escherichia coli , Dados de Sequência Molecular , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genética , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/química , Plasmídeos , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
As outlined in this review, various experimental techniques have been employed in an attempt to understand neisserial pathogenesis. In vitro genetic analysis has been used to study the genetic basis for the structural variability of cell surface components. Transformed or primary epithelial cell cultures have provided the simplest model to analyze bacterial adherence and invasion, while the infection of polarized epithelial monolayers, fallopian tube and nasopharyngeal organ cultures, and ureteral tissue have each been used to more closely represent the events which occur in vivo. Finally, the in vivo infection of human volunteers with N. gonorrhoeae has provided a powerful means to confirm and expand the results obtained in vitro. By these various approaches, a number of neisserial adhesins (i.e. pilli, Opa, Opc and P36) and additional putative virulence determinants which affect bacterial adherence and invasion into host cells (i.e. LOS, capsule, PorB) have been identified. Clearly, neisserial surface variation serves as an adaptive mechanism which can modulate tissue tropism, immune evasion and survival in the changing host environment. Important progress has been made in recent years with respect to the host cellular receptors and subsequent signal transduction processes which are involved in neisserial adherence, invasion and transcytosis. This has led to the identification of (i) CD46 as a receptor for pilus which allows adherence to epithelial and endothelial cells, (ii) HSPGs, in cooperation with vitronectin and fibronectin, as receptors for a particular subset of Opa proteins and Opc, which may both mediate invasion into most epithelial and endothelial cells, and (iii) CD66 as the receptors for most Opa variants, potentially being involved in cellular interactions including adherence, invasion and transcytosis with epithelial, endothelial and phagocytic cells. As most of these data have been obtained using transformed cell lines growing in vitro, attempts must be made to translate these basic observations into a more natural situation. It can be expected that the successful ongoing integration of laboratory findings from the various infection models with human volunteer studies will further increase our understanding of the biology of neisserial infection. Perhaps the most difficult but also most rewarding challenge for the future will be to use volunteer studies to identify and understand the role of host factors which are important for the infectious process. Hopefully, insights gained from each of these studies will reveal new and useful strategies for the preventive and/or therapeutic intervention into infection and disease by these fascinating microbes.
Assuntos
Neisseria gonorrhoeae/patogenicidade , Neisseria meningitidis/patogenicidade , Animais , HumanosRESUMO
The value of experimental culture models using epithelial cells often depends on the degree of polarization and other critical features observed in natural tissues, including the formation of tight junctions, desmosomes and membrane interdigitations. However, growth of normal epithelial cells as monolayers on artificial supports also leads to partial loss of the original characteristics of epithelial cells, and the quality of the monolayer is strongly influenced by the physicochemical properties of the support. In addition, not all normal epithelial cell types are able to adhere and to grow well on artificial substrata. In order to circumvent the drawbacks of two-dimensional cultures we established an in vitro model that closely resembles the in vivo situation of the intact epithelium. Human epithelial cells from nasopharynx (HNPEC) were used to prepare multicellular epithelial vesicles consisting of both non-ciliated and ciliated mucosal cells. Electron microscopy investigations showed that the morphological appearance of the epithelial cells was similar to that in situ. HNPEC vesicle cultures maintain a geometrically intact organization of individual cells that is not achieved using conventional culture conditions. HNPEC vesicles are more in vivo-like than two-dimensional cultures and therefore represent a suitable model for a variety of research purposes including studies on the pathogenesis of invasive microorganisms.
Assuntos
Polaridade Celular , Células Cultivadas/citologia , Nasofaringe/citologia , Adesão Celular , Criança , Cílios/ultraestrutura , Meios de Cultura Livres de Soro , Células Epiteliais , Epitélio/ultraestrutura , Humanos , Junções Intercelulares/ultraestrutura , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Mucosa/citologia , Mucosa/ultraestrutura , Nasofaringe/ultraestruturaRESUMO
A novel plasmid expression system has been constructed that combines two useful functions: it facilitates single-step affinity purification of cytoplasmically overproduced fusion proteins and the in vitro processing of fusions with IgA protease (Igase). The significant features directing the high expression rate of pEV41-based gene fusions in Escherichia coli are the lambda pL promoter for temperature-regulated transcription and the translation initiation region of the bacteriophage MS2 polymerase gene including a downstream box (db) within the first few codons of the open reading frame. Fusion proteins generated with this system contain a short N-terminal carrier peptide allowing convenient affinity purification by means of the His6 peptide. As exemplified by the production of the variable heavy (VH) and light (VL)-chain domains of a monoclonal antibody, the fusion proteins can be specifically processed with Igase either in purified form or simply by incubation with the culture medium of recombinant E. coli [pJP10] cells. Chemical cross-linking of processed VH and VL domains resulted in a recombinant antibody Fv fragment that can specifically bind to its antigen.
Assuntos
Anticorpos Monoclonais/biossíntese , Plasmídeos/genética , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Serina Endopeptidases , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Sequência de Bases , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Histidina , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Dados de Sequência Molecular , Níquel , Oligonucleotídeos , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
One of the most efficient systems for the high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulated transcription unit supplying the specific T7 RNA polymerase. Various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase are in use. Most of them show high levels of expression in non-induced cells, low factor of induction or impaired growth of host cells. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is suppressed by the use of the genes for the Lac repressor and T7 lysozyme, integrated on the expression vector. T7 lysozyme expression is probably down-regulated in the induced expression system by antisense RNA. This overcomes the inhibitory effect of T7 lysozyme on T7 RNA polymerase as shown by SDS PAGE and flow cytometry analysis of expressed GFP. The main features of the expression vector compared with other systems are low background, high factor of induction and unaffected growth of non-induced cells.
Assuntos
Bacteriófago T7/genética , Regulação da Expressão Gênica , Muramidase/genética , Clonagem Molecular/métodos , DNA Recombinante/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Citometria de Fluxo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage lambda, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to lambda DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42 degrees C without selective pressure results in loss of pfd plasmids.
Assuntos
Clonagem Molecular , Colífagos/genética , DNA Viral/genética , Genes Virais , Plasmídeos , Bacteriófago lambda/genética , Sequência de Bases , Mapeamento Cromossômico , Enzimas de Restrição do DNA , Elementos de DNA Transponíveis , Resistência Microbiana a Medicamentos , Escherichia coli/genéticaRESUMO
In Neisseria gonorrhoea (Ngo), the processes of type-4 pilus biogenesis and DNA transformation are functionally linked and play a pivotal role in the life style of this strictly human pathogen. The assembly of pili from its main subunit pilin (PilE) is a prerequisite for gonococcal infection since it allows the first contact to epithelial cells in conjunction with the pilus tip-associated PilC protein. While the components of the pilus and its assembly machinery are either directly or indirectly involved in the transport of DNA across the outer membrane, other factors unrelated to pilus biogenesis appear to facilitate further DNA transfer across the murein layer (ComL, Tpc) and the inner membrane (ComA) before the transforming DNA is rescued in the recipient bacterial chromosome in a RecA-dependent manner. Interestingly, PilE is essential for the first step of transformation, i.e., DNA uptake, and is itself also subject to transformation-mediated phase and antigenic variation. This short-term adaptive mechanism allows Ngo to cope with changing micro-environments in the host as well as to escape the immune response during the course of infection. Given the fact that Ngo has no ecological niche other than man, horizontal genetic exchange is essential for a successful co-evolution with the host. Horizontal exchange gives rise to heterogeneous populations harboring clones which better withstand selective forces within the host. Such extended horizontal exchange is reflected by a high genome plasticity, the existence of mosaic genes and a low linkage disequilibrium of genetic loci within the neisserial population. This led to the concept that rather than regarding individual Neisseria species as independent traits, they comprise a collective of species interconnected via horizontal exchange and relying on a common gene pool.
Assuntos
Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Transformação Bacteriana , Variação Antigênica , Proteínas da Membrana Bacteriana Externa/metabolismo , Evolução Biológica , DNA Bacteriano/metabolismo , Proteínas de Fímbrias , Fímbrias Bacterianas/imunologia , Humanos , Modelos Genéticos , Neisseria gonorrhoeae/imunologiaRESUMO
The Iga beta autotransporter function of IgA1 protease from Neisseria gonorrhoeae was assessed in Escherichia coli using the Vibrio cholerae toxin B subunit (CtxB) as a heterologous passenger. N-terminal fusions with Iga beta of native CtxB or mutant CtxB protein containing no cysteines were constructed and analysed in isogenic E. coli mutants carrying defects in either or both the ompT (outer membrane protease T) and dsbA (periplasmic disulfide oxidoreductase) determinants. While export of the cystein-less CtxB passenger was independent of the dsbA genotype, the native CtxB passenger was properly translocated across the outer membrane only in the dsbA mutant background. This effect was consistent in the presence and in the absence of the OmpT protease which rather determined the release of surface-bound CtxB into the medium. Therefore, in agreement with previous observations Iga beta-dependent protein secretion requires an unfolded conformation of the passenger domain and can be blocked by disulfide loop formation in the presence of DsbA. Since DsbA acts in the periplasm, this provides evidence for a periplasmic intermediate in the Iga beta-mediated export pathway. E. coli (dsbA ompT) is highly suitable as a strain for the surface display of recombinant proteins via Iga beta, whether or not they contain cysteine residues.
Assuntos
Cisteína/metabolismo , Escherichia coli/metabolismo , Isomerases/metabolismo , Oxirredutases/metabolismo , Proteínas Periplásmicas , Serina Endopeptidases/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Toxina da Cólera/genética , Toxina da Cólera/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Isomerases/genética , Dados de Sequência Molecular , Mutação , Oxirredutases/genética , Isomerases de Dissulfetos de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/genética , Vibrio cholerae/genéticaRESUMO
A series of Tn1721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site (res), a suicide replication origin (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) carry a promoterless alkaline phosphatase-encoding gene (phoA') useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10(-2) events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis, i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.
Assuntos
Análise Mutacional de DNA/métodos , Elementos de DNA Transponíveis/genética , DNA Bacteriano/análise , Genes Bacterianos , Mutagênese Insercional/métodos , Sequência de Bases , Clonagem Molecular , Reparo do DNA , Escherichia coli/genética , Vetores Genéticos , Dados de Sequência Molecular , Neisseria gonorrhoeae/genética , Oligonucleotídeos , Regiões Promotoras GenéticasRESUMO
A new collection of mini-transposons (mini-Tn) of the previously described TnMax series [Haas et al, Gene 130 (1993a) 23-31] has been constructed. The transposons (Tn) bear genes conferring resistance to either chloramphenicol (Cm) or kanamycin (Km). Each member of the new series (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and reverse (M13-RP1) sequencing primers close to the inverted repeats (IR), facilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site. Furthermore, the mini-Tn possess the infrequently occurring NotI sites, allowing the localization of genes on macro-restriction maps of bacterial species. Some derivatives contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the generation of in vivo gene- and operon fusions to study gene regulation, protein export, or to determine the topology of proteins in bacterial membranes. A set of conjugative minimal plasmid vectors (pMin1, pMin2) are used to select for TnMax insertions into the cloned insert, rather than the vector sequences. Due to the small size of the mini-Tn, and a simple and efficient mutagenesis procedure, the TnMax system is a useful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species which cannot be targeted by direct transposition.
Assuntos
Elementos de DNA Transponíveis , Vetores Genéticos , Mutagênese , Proteínas Recombinantes de Fusão , Sequência de Bases , Conjugação Genética , Genes Bacterianos , Dados de Sequência MolecularRESUMO
Lysosomal/phagosomal membranes of mammalian cells are coated by highly conserved glycoproteins (lamps) that are thought to protect the membranes from degradation. Interestingly, we identified an amino acid sequence in human lamp-1 characteristic of a cleavage site for the Neisseria gonorrhoeae IgA1 protease. Furthermore, gonococci are detected in h-lamp-1-positive vacuoles after their uptake by professional phagocytes and epithelial cells. Here we demonstrate cleavage of glycosylated h-lamp-1 by the secreted gonococcal IgA1 protease. The cleavage was observed with h-lamp-1 purified from epithelial cells but not from professional phagocytes. The biological role of lamp-1 cleavage by the gonococcal protease is discussed.
Assuntos
Antígenos CD/metabolismo , Proteínas de Bactérias/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Neisseria gonorrhoeae/enzimologia , Fagossomos/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Sequência Consenso , Células Epiteliais , Células HL-60 , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Lisossomal , Camundongos , Dados de Sequência Molecular , Neisseria gonorrhoeae/crescimento & desenvolvimento , Coelhos , Células Tumorais CultivadasRESUMO
The modulation of programmed cell death is a common theme in the patho-physiology of inflammation and infectious disease. The synthesis and secretion of an IgA1 protease is strictly associated with virulence of the Neisseria species. Here, we report on the inhibition of tumor necrosis factor alpha (TNFalpha)-mediated apoptosis of the human myelo-monocytic cell line U937 by highly purified IgA1 protease. Apoptosis was verified by the cell surface exposure of phosphatidyl serine and by terminal transferase mediated end-labeling of fragmented DNA. Interestingly, IgA1 protease specifically cleaved the TNF receptor II (TNF-RII) on the surface of intact cells whereas TNF-RI was not affected by the enzyme. Therefore, inhibition of TNFalpha-mediated apoptosis might be correlated to specific cleavage of the TNF-RII by neisserial IgA1 protease.