RESUMO
The constitutive reverter of eIF2α phosphorylation (CReP)/PPP1r15B targets the catalytic subunit of protein phosphatase 1 (PP1c) to phosphorylated eIF2α (p-eIF2α) to promote its dephosphorylation and translation initiation. Here, we report a novel role and mode of action of CReP. We found that CReP regulates uptake of the pore-forming Staphylococcus aureus α-toxin by epithelial cells. This function was independent of PP1c and translation, although p-eIF2α was involved. The latter accumulated at sites of toxin attack and appeared conjointly with α-toxin in early endosomes. CReP localized to membranes, interacted with phosphomimetic eIF2α, and, upon overexpression, induced and decorated a population of intracellular vesicles, characterized by accumulation of N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a lipid marker of exosomes and intralumenal vesicles of multivesicular bodies. By truncation analysis, we delineated the CReP vesicle induction/association region, which comprises an amphipathic α-helix and is distinct from the PP1c interaction domain. CReP was also required for exocytosis from erythroleukemia cells and thus appears to play a broader role in membrane traffic. In summary, the mammalian traffic machinery co-opts p-eIF2α and CReP, regulators of translation initiation.
Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Células Epiteliais/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Iniciação Traducional da Cadeia Peptídica/fisiologia , Proteína Fosfatase 1/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Membrana Celular/genética , Endossomos/genética , Células Epiteliais/citologia , Fator de Iniciação 2 em Eucariotos/genética , Humanos , Células K562 , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Coelhos , Staphylococcus aureus/metabolismoRESUMO
Autophagy is a catabolic process of paramount importance for cellular homeostasis during starvation. Generally, autophagy and translation are inversely regulated. Many kinds of stress lead to attenuation of translation via phosphorylation of eukaryotic translation initiation factor alpha (eIF2α). This response is conserved from yeast to man and can be either protective or detrimental depending on strength and duration of stress, and additional factors. During starvation or viral infection, phosphorylation of eIF2α is required for induction of autophagy. As exemplified here by α-hemolysin, a small pore-forming toxin (PFT) of Staphylococcus aureus and (S)-3-oxo-C12-homoserine lactone [(S)-3-oxo-C12-HSL], a quorum-sensing hormone of Pseudomonas aeruginosa, bacterial exoproducts may also impact translation and autophagy. Thereby, PFT and (S)-3-oxo-C12-HSL act differentially. Damage of the plasma membrane by PFT causes efflux of potassium, which leads to amino acid starvation and energy loss. This triggers amino acid-sensitive eIF2α-kinase GCN2, as well as energy sensor AMPK, and deactivates mTORC1. The output of this response, that is, transient metabolic reprogramming is an essential part of a defense program which enables cells to survive attack by a pore-forming agent. Thus, nutrient/energy sensors serve as sentinels of plasma membrane integrity. In contrast to PFT, (S)-3-oxo-C12-HSL does not cause acute loss of ATP or activation of GCN2, but also triggers phosphorylation of eIF2α and inhibits translation. This response appears not to depend on efflux of potassium and requires eIF2α-kinase PKR. Like α-toxin, (S)-3-oxo-C12-HSL increases lipidation of LC3 and accumulation of autophagosomes in cells. Apart from directly affecting host-cell viability, bacterial exoproducts might galvanize bystander cells to prepare for close combat with microbial offenders or inadvertently accommodate some of them.
Assuntos
Autofagia , Fator de Iniciação 2 em Eucariotos/metabolismo , Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/patogenicidade , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Homosserina/análogos & derivados , Homosserina/metabolismo , Humanos , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Mitogen activated protein kinase (MAPK) p38 has emerged as a survival protein in cells that are attacked by bacterial toxins forming small membrane pores. Activation of p38 by pore forming toxins (PFT) has been attributed to osmotic stress, but here we show that loss of K+ is likely to be the critical parameter. Several lines of evidence support this conclusion: first, osmoprotection did not prevent p38-phosphorylation in alpha-toxin-loaded cells. Second, treatment of cells with a K+ ionophore, or simple incubation in K+-free medium sufficed to cause robust p38-phosphorylation. Third, media containing high [K+] prevented p38-activation by Staphylococcus aureus alpha-toxin, Vibrio cholerae cytolysin (VCC), Streptolysin O (SLO), or Escherichia coli hemolysin (HlyA), but did not impair activation by H2O2. Fourth, potential roles of LPS, TLR4, or calcium-influx were ruled out. Therefore, we propose that PFT trigger the p38 MAPK-pathway by causing loss of cellular K+.
Assuntos
Toxinas Bacterianas/toxicidade , Membrana Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Potássio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Ativação Enzimática , Células Epiteliais/enzimologia , Proteínas de Escherichia coli/toxicidade , Proteínas Hemolisinas/toxicidade , Humanos , FosforilaçãoRESUMO
Vibrio cholerae cytolysin (VCC) is a pore-forming toxin that is secreted in precursor form (pro-VCC) and requires proteolytic cleavage in order to attain membrane-permeabilizing properties. Pro-VCC can be activated both in solution and membrane-bound state. Processing of membrane-bound pro-VCC can in turn be achieved through the action of both cell-associated and soluble proteases. The current investigation describes the interaction of VCC with human neutrophil granulocytes. It is shown that pro-VCC binds to these cells and is cleaved by cell-bound serine proteases. Membrane permeabilization leads to granulocyte activation, as witnessed by the generation of reactive oxygen metabolites and liberation of granule constituents. A mutant toxin with unaltered binding properties but devoid of pore-forming activity did not elicit these effects. The secreted proteases cleave and activate further bound- and non-bound pro-VCC. A positive feedback loop is thus created that results in enhanced cytotoxicity towards both the targeted granulocytes and towards bystander cells that are not primarily killed by the protoxin. Thus, activation of neutrophil granulocytes by VCC fuels a positive feedback cycle that will cripple immune defence, augment inflammation, and enhance the cytotoxic action of the toxin on neighbouring tissue cells.
Assuntos
Degranulação Celular , Inflamação/metabolismo , Neutrófilos/metabolismo , Perforina/metabolismo , Vibrio cholerae/metabolismo , Toxinas Bacterianas/metabolismo , Permeabilidade da Membrana Celular , Cólera/metabolismo , Retroalimentação Fisiológica , Humanos , Inflamação/microbiologia , Neutrófilos/microbiologia , Neutrófilos/fisiologia , Explosão Respiratória , Serina Endopeptidases/metabolismoRESUMO
Abstract Escherichia coli hemolysin is a pore-forming protein belonging to the RTX toxin family. Cysteine scanning mutagenesis was performed to characterize the putative pore-forming domain of the molecule. A single cysteine residue was introduced at 48 positions within the sequence spanning residues 170-400 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that several amino acids in this domain are inserted into the lipid bilayer during pore formation. An amphipathic alpha-helix spanning residues 272-298 was identified that may line the aqueous pore. The importance of this sequence was highlighted by the introduction of two prolines at positions 284 and 287. Disruption of the helix structure did not affect binding properties, but totally abolished the hemolytic activity of the molecule.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Porinas/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Membrana Eritrocítica , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , CoelhosRESUMO
Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with tritiated N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a nonsaturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulocytes. An oxidative burst coincided with elevation of intracellular Ca(2+), which was likely because of passive influx of Ca(2+) through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a nonlytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the beta(2) integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.
Assuntos
Eritrócitos/metabolismo , Escherichia coli/metabolismo , Granulócitos/metabolismo , Proteínas Hemolisinas/metabolismo , Superóxidos/metabolismo , Acilação , Substituição de Aminoácidos , Toxinas Bacterianas , Sítios de Ligação , Cálcio/metabolismo , Eritrócitos/citologia , Granulócitos/citologia , Proteínas Hemolisinas/genética , Humanos , Células K562 , Antígeno-1 Associado à Função Linfocitária/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Explosão Respiratória , Deleção de SequênciaRESUMO
Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a beta-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel beta-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the beta-barrel-forming sequence of staphylococcal cytolysins and identify the beta-barrel as a membrane-perforating structure that is highly conserved in evolution.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Sequência de Aminoácidos , Animais , Sequência Conservada , Cisteína/química , Dissulfetos/química , Corantes Fluorescentes/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Perforina , Mutação Puntual , Proteínas Citotóxicas Formadoras de Poros , Conformação Proteica , Estrutura Terciária de Proteína , Coelhos , Compostos de Sulfidrila/química , Vibrio choleraeRESUMO
Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores in animal cells. The molecule is secreted as a procytolysin (pro-VCC) of 79 kDa that must be cleaved at the N terminus to generate the active 65-kDa toxin. Processing can occur in solution, and previous studies have described the action of mature VCC thus generated. However, little is known about the properties of pro-VCC itself. In this study, it is shown that pro-VCC exist as a monomer in solution and binds as a monomer to eukaryotic cells. Bound pro-VCC can then be activated either by exogenous, extracellular, or by endogenous, cell-bound proteases. In both cases, cleavage generates the 65-kDa VCC that oligomerizes to form transmembrane pores. A wide variety of exogenous proteinases can mediate activation. In contrast, the activating cellular protease is selectively inhibited by the hydroxamate inhibitor TAPI, and thus probable candidates are members of the ADAM-metalloproteinase family. Furin, MMP-2, MMP-9, and serine proteinases were excluded. Cells over-expressing ADAM-17, also known as tumor necrosis factor alpha converting enzyme, displayed increased activation of VCC, and knockout cells lacking ADAM-17 had a markedly decreased capacity to cleave the protoxin. The possibility is raised that pro-VCC is targeted to membrane sites that selectively contain or are accessible to cellular ADAM-metalloproteinases. Although many microbial toxins are activated by furin, this is the first evidence for processing by a cellular metalloproteinase. We identified ADAM-17 as a potent activator of pro-VCC.