RESUMO
Many bacterial pathogens exploit mammalian cell functions in order to promote their adherence to or uptake by host cells. Recent work has led to the identification of some of the bacterial and mammalian proteins involved in these processes. Although specific mechanisms differ among pathogens, a common aspect appears to be regulation of signaling pathways that control the actin cytoskeleton.
Assuntos
Bactérias/patogenicidade , Células/microbiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Modelos BiológicosRESUMO
Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. Inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 3-kinase p85alpha-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3, 4)P2 and PI(3,4,5)P3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein InlB, host cell tyrosine phosphorylation, and association of p85alpha with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.
Assuntos
Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Androstadienos/farmacologia , Animais , Proteínas de Bactérias/fisiologia , Linhagem Celular , Cromonas/farmacologia , Citocalasina D/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Genisteína , Humanos , Isoflavonas/farmacologia , Proteínas de Membrana/fisiologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotirosina/metabolismo , Células Tumorais Cultivadas , WortmaninaRESUMO
Listeria monocytogenes is a pathogenic bacterium that induces its own uptake into mammalian cells, and spreads from one cell to another by an actin-based motility process. Entry into host cells involves the bacterial surface proteins InlA (internalin) and InlB. The receptor for InlA is the cell adhesion molecule E-cadherin. InlB-mediated entry requires activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. Actin-based movement of L. monocytogenes is mediated by the bacterial surface protein ActA. The N-terminal region of this protein is necessary and sufficient for polymerization of host cell actin. Other host proteins involved in bacterial motility include profilin, Vasodilator-Stimulated Phosphoprotein (VASP), the Arp2/Arp3 complex, and cofilin. Studies of entry and intracellular movement of L. monocytogenes could lead to a better understanding of receptor-ligand signaling and dynamics of actin polymerization in mammalian cells.
Assuntos
Actinas/fisiologia , Proteínas de Bactérias/fisiologia , Listeria monocytogenes/fisiologia , Animais , Humanos , MamíferosRESUMO
In the Gram-positive bacterium Bacillus subtilis, the developmental process of spore formation occurs in response to nutrient deprivation and requires the generation of two different cell types with distinct programs of gene expression. Entry into sporulation is regulated primarily by activation (phosphorylation) of the transcription factor encoded by spo0A. The phosphorylation state of Spo0A is controlled by a multi-component phospho-transfer pathway and by at least one phosphatase. Recent experiments indicate that several intracellular conditions that decrease the fidelity of chromosome transmission inhibit production of Spo0A approximately P. These conditions include inhibition of DNA replication, DNA damage, and some alterations in the chromosome partitioning and cell division machinery. Coupling accumulation of a critical level of Spo0A approximately P to these conditions seems to serve as a developmental checkpoint to ensure that cells do not attempt to sporulate unless they are able to provide an intact, undamaged chromosome for each of the two cell types needed for sporulation.
Assuntos
Bacillus subtilis/fisiologia , DNA Bacteriano/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Cromossomos Bacterianos , Dano ao DNA , Replicação do DNA , DNA Bacteriano/genética , Genes Bacterianos , Dados de Sequência Molecular , Fosforilação , Pseudomonas putida , Transdução de Sinais , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Endospore formation in the bacterium Bacillus subtilis involves generation of two cell types, each with different developmental fates. Each cell type contains an active chromosome, and treatments that inhibit DNA synthesis at the beginning of development inhibit spore formation. We describe experiments demonstrating that gene expression early during sporulation is coupled to DNA synthesis. Expression of several genes that are induced early during sporulation, before the formation of two cell types, is inhibited when DNA synthesis is inhibited. Genes that are affected require the transcription factor encoded by spo0A for normal induction. Spo0A protein is normally activated early in development by a multicomponent phosphorylation pathway, or phospho-relay. Altered function mutations in spo0A that bypass the need for the phospho-relay allow early sporulation gene expression, even when DNA synthesis is inhibited. These results indicate that inhibition of DNA synthesis prevents activation of the Spo0A transcription factor by inhibiting a step in the phospho-relay. It seems likely that coupling early developmental gene expression to DNA synthesis is a general mechanism to prevent inappropriate or unnecessary gene expression.
Assuntos
Bacillus subtilis/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Esporos Bacterianos , Replicação do DNA , Genes Bacterianos , RNA Mensageiro/genética , Recombinases Rec A/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The ski4::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but that are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). ski4::Tn917lac caused a small defect in sporulation, but in combination with a null mutation in kinA, it caused a much more severe defect. The insertion mutation was in an 87-amino-acid open reading frame (orf87 bofA) that controls the activation of a sigma factor, sigma K, at intermediate times during sporulation. The ski4 mutation caused the premature expression of cotA, a gene controlled by sigma K. An independent mutation that causes the premature activation of sigma K also caused a synthetic (synergistic) sporulation phenotype in combination with a null mutation in kinA, indicating that the defect was due to altered timing of gene expression directed by sigma K. Expression of ski4 was shown to be controlled by the sporulation-specific sigma factor sigma E.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Mutação/genética , Esporos Bacterianos/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Elementos de DNA Transponíveis , Genes Bacterianos/genética , Dados de Sequência Molecular , Mutagênese Insercional , Fenótipo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência do Ácido Nucleico , Fator sigma/biossíntese , Fatores de Tempo , Transdução Genética , Transformação BacterianaRESUMO
Spore formation in Bacillus subtilis requires the generation of two distinct cell types, each with an active chromosome that becomes committed to a defined program of gene expression. Here we show that a developmental checkpoint couples the initiation of sporulation, and the subsequent formation of these two cell types, to DNA replication early during development. Inhibiting the initiation of chromosomal replication prevents the onset of sporulation and inhibits expression of several genes that are normally induced early during development. This defect in gene expression is due to inhibition of the multi-component phosphorylation pathway needed to activate the developmental transcription factor encoded by spo0A. The target affected by inhibiting the initiation of replication is neither Spo0A nor the major kinase, KinA, needed for production of Spo0A approximately P. Rather, the target appears to be one of the proteins that transfers phosphate from the kinase to the Spo0A transcription factor. The signal that couples activity of the phosphorelay to the initiation of DNA replication is different from the previously described DNA damage signal that inhibits the phosphorelay during SOS induction in a recA-dependent response. Thus, DNA replication as well as DNA damage signals control production of Spo0A approximately P and initiation of sporulation.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Replicação do DNA , DNA Bacteriano/biossíntese , Regulação Bacteriana da Expressão Gênica , Fator sigma , Esporos Bacterianos/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA/fisiologia , Fosforilação , Recombinases Rec A/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The Gram-positive pathogen Listeria monocytogenes induces its own internalization into some non-phagocytic mammalian cells by stimulating host tyrosine phosphorylation, phosphoinositide (PI) 3-kinase activity, and rearrangements in the actin cytoskeleton. Entry into many cultured cell lines is mediated by the bacterial protein InlB. Here we investigate the role of InlB in regulating mammalian signal transduction and cytoskeletal structure. Treatment of Vero cells with purified InlB caused rapid and transient increases in the lipid products of the PI 3-kinase p85-p110, tyrosine phosphorylation of the mammalian adaptor proteins Gab1, Cbl, and Shc, and association of these proteins with p85. InlB also stimulated large scale changes in the actin cytoskeleton (membrane ruffling), which were PI 3-kinase-dependent. These results identify InlB as the first reported non-mammalian agonist of PI 3-kinase and demonstrate similarities in the signal transduction events elicited by this bacterial protein and known agonists such as epidermal growth factor.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ubiquitina-Proteína Ligases , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/ultraestrutura , Chlorocebus aethiops , Citoesqueleto , Receptores ErbB/metabolismo , Proteínas de Membrana/isolamento & purificação , Fosfatidilinositóis/análise , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Células VeroRESUMO
The ski22::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). The ski22::Tn917lac insertion mutation was in ald, the gene encoding alanine dehydrogenase. Alanine dehydrogenase catalyzes the deamination of alanine to pyruvate and ammonia and is needed for growth when alanine is the sole carbon or nitrogen source. The sporulation defect caused by null mutations in ald was partly relieved by the addition of pyruvate at a high concentration, indicating that the normal role of alanine dehydrogenase in sporulation might be to generate pyruvate to provide an energy source for sporulation. The spoVN::Tn917 mutation was also found to be an allele of ald. Transcription of ald was induced very early during sporulation and by the addition of exogenous alanine during growth. Expression of ald was normal in all of the regulatory mutants tested, including spo0A, spo0K, comA, sigB, and sigD mutants. The only gene in which mutations affected expression of ald was ald itself. This regulation is probably related to the metabolism of alanine.
Assuntos
Aminoácido Oxirredutases/metabolismo , Bacillus subtilis/fisiologia , Genes Bacterianos , Alanina Desidrogenase , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Bacteriano/análise , DNA Bacteriano/química , Escherichia coli , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Cinética , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Esporos Bacterianos/fisiologia , Transcrição Gênica , beta-Galactosidase/biossíntese , beta-Galactosidase/metabolismoRESUMO
The spo0J gene of Bacillus subtilis is required for the initiation of sporulation. We show that the sporulation defect caused by null mutations in spo0J is suppressed by a null mutation in the gene located directly upstream from spo0J, soj (suppressor of spo0J). These results indicate that Soj inhibits the initiation of sporulation and that Spo0J antagonizes that inhibition. Further genetic experiments indicated that Soj ultimately affects sporulation by inhibiting the activation (phosphorylation) of the developmental transcription factor encoded by spo0A. In addition, the temperature-sensitive sporulation phenotype caused by the ftsA279 (spoIIN279) mutation was partly suppressed by the soj null mutation, indicating that FtsA might also affect the activity of Soj. Soj and Spo0J are known to be similar in sequence to a family of proteins involved in plasmid partitioning, including ParA and ParB of prophage P1, SopA and SopB of F, and IncC and KorB of RK2, spo0J was found to be required for normal chromosome partitioning as well as for sporulation. spo0J null mutants produced a significant proportion of anucleate cells during vegetative growth. The dual functions of Spo0J could provide a mechanism for regulating the initiation of sporulation in response to activity of the chromosome partition machinery.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/fisiologia , Cromossomos Bacterianos/fisiologia , Genes Bacterianos , Genes Supressores , Óperon , Bacillus subtilis/citologia , Expressão Gênica , Genótipo , Cinética , Mutação , Fases de Leitura Aberta , Fenótipo , Mapeamento por Restrição , Esporos Bacterianos/citologia , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologia , Temperatura , Fatores de TempoRESUMO
The Listeria monocytogenes surface protein InlB promotes bacterial entry into mammalian cells. Here, we identify a cellular surface receptor required for InlB-mediated entry. Treatment of mammalian cells with InlB protein or infection with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase (RTK) for which the only known ligand is Hepatocyte Growth Factor (HGF). Like HGF, InlB binds to the extracellular domain of Met and induces "scattering" of epithelial cells. Experiments with Met-positive and Met-deficient cell lines demonstrate that Met is required for InlB-dependent entry of L. monocytogenes. InlB is a novel Met agonist that induces bacterial entry through exploitation of a host RTK pathway.
Assuntos
Proteínas Aviárias , Proteínas de Bactérias/metabolismo , Endocitose , Listeria monocytogenes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Ubiquitina-Proteína Ligases , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/microbiologia , Deleção de Genes , Hepatócitos , Humanos , Rim , Cinética , Ligantes , Listeria monocytogenes/genética , Listeria monocytogenes/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/genética , Transdução de SinaisRESUMO
We fused obg, encoding an essential GTP-binding protein in Bacillus subtilis, to the LacI-repressible, IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible promoter Pspac. Depletion of Obg, following removal of IPTG, caused a defect in sporulation and in expression of sporulation genes that are activated by Spo0A approximately P. These defects were significantly relieved by a mutation in spo0A (rvtA11) that bypasses the normal phosphorylation pathway, indicating that Obg might normally be required, either directly or indirectly, to stimulate activity of the phosphorelay that activates Spo0A.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Fator sigma , Esporos Bacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , RNA Mensageiro/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Spore formation in Bacillus subtilis is a dramatic response to environmental signals that is controlled in part by a two-component regulatory system composed of a histidine protein kinase (SpoIIJ) and a transcriptional regulator (Spo0A). The spo0K locus plays an important but undefined role in the initiation of sporulation and in the development of genetic competence. spoIIJ spo0K double mutants had a more severe defect in sporulation than either single mutant. Overproduction of the spoIIJ gene product resulted in the suppression of the sporulation defect, but not the competence defect, caused by mutations in the spo0K locus. On the basis of the phenotype of the spoIIJ spo0K double mutant and the effect of overproduction of the spoIIJ gene product, a transposon insertion in the spo0K locus was isolated. The spo0K locus was cloned and sequenced. spo0K proved to be an operon of five genes that is homologous to the oligopeptide permease (opp) operon of Salmonella typhimurium and related to a large family of membrane transport systems. The requirement for the transport system encoded by spo0K in the development of competence was somewhat different than its requirement in the system encoded by spo0K in the development of competence was somewhat different than its requirement in the initiation of sporulation. Disruption of the last open reading frame in the spo0K operon caused a defect in competence but had little or no effect on sporulation. We hypothesize that the transport system encoded by spo0K may have a role in sensing extracellular peptide factors that we have shown are required for efficient sporulation and perhaps in sensing similar factors that may be necessary for genetic competence.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias , Proteínas de Membrana Transportadoras/genética , Sequência de Aminoácidos , Bacillus subtilis/fisiologia , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Óperon/genética , Plasmídeos , Mapeamento por Restrição , Salmonella typhimurium/genética , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Esporos BacterianosRESUMO
Early during endospore formation in the bacterium Bacillus subtilis, two distinct cell types are formed. The initiation of this developmental pathway requires several physiological conditions (e.g., nutrient deprivation) and is controlled by the Spo0A transcription factor. We have found that in a culture of sporulating cells, there are two subpopulations, one that has initiated the developmental program and activated the expression of early developmental genes and one in which early developmental gene expression remains uninduced. We measured the expression of developmental (spo) genes in single cells of B. subtilis by using spo-lacZ fusions. Cells containing a spo-lacZ fusion were stained with a dye that fluoresces upon hydrolysis by beta-galactosidase, and the fluorescence in individual cells was measured with a flow cytometer. For Spo+ cells, we found that the proportion of the population expressing early developmental genes correlates well with the fraction of the population that eventually produces spores. In addition, mutations that cause a decrease in the amount of activated (phosphorylated) Spo0A transcription factor cause a decrease in the size of the subpopulation expressing early developmental genes that are directly activated by Spo0A approximately P. Again, the size of the subpopulation correlates well with the fraction of cells that produce spores. These results indicate that a threshold level of activated Spo0A (Spo0A approximately P) or of a component of the phosphorylation pathway must accumulate to induce sporulation gene expression and that most of the cells that are able to induce the expression of early genes that are directly activated by Spo0A approximately P go on to produce mature spores.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fator sigma , Esporos Bacterianos/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/biossíntese , Variação Genética , Óperon Lac , Morfogênese , Mutação , Proteínas Quinases/genética , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Expression of genes early during sporulation in Bacillus subtilis requires the activity of the transcription factor encoded by spo0A. The active, phosphorylated form of Spo0A is produced through the action of a multicomponent pathway, the phosphorelay. A mutant defective in the first three enzymes of the Krebs citric acid cycle was unable to express early sporulation genes, apparently because of a failure to activate the phosphorelay. Cells that produce an altered Spo0A protein that can be phosphorylated by an alternative pathway were not dependent on Krebs cycle function for early sporulation gene expression. These findings suggest that Krebs cycle enzymes transmit a signal to activate the phosphorelay and that B. subtilis monitors its metabolic potential before committing itself to spore formation.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/biossíntese , Ciclo do Ácido Cítrico , Expressão Gênica , Fatores de Transcrição/biossíntese , Bacillus subtilis/crescimento & desenvolvimento , Deleção de Genes , Genótipo , Glucose/metabolismo , Cinética , Mutagênese Insercional , Fosforilação , Regiões Promotoras Genéticas , Esporos Bacterianos/fisiologiaRESUMO
Multiple physiological and environmental signals are needed to initiate endospore formation in Bacillus subtilis. One key event controlling sporulation is activation of the Spo0A transcription factor. Spo0A is a member of a large family of conserved regulatory proteins whose activity is controlled by phosphorylation. We have isolated deletion mutations that remove part of the conserved amino terminus of Spo0A and make the transcription factor constitutively active, indicating that the amino terminus normally functions to keep the protein in an inactive state. Expression of an activated gene product is sufficient to activate expression of several sporulation genes in the absence of signals normally needed for initiation of sporulation. Our results indicate that nutritional, cell density, and cell-cycle signals are integrated through the phosphorylation pathway that controls activation of Spo0A.