RESUMEN
OBJECTIVES: Involvement of the outer membrane protein C (OmpC) of Escherichia coli in neurodegeneration was investigated using a mouse model. RESULTS: OmpC formed protease-resistant fibres that exhibited the diagnostic features of an amyloid. The spectral shift in the Congo Red and the thioflavin T assays produced features similar to neurotoxic peptides. Intramuscular administration of OmpC in mice resulted in spongiform neurodegeneration of the brain through calcium-dependent apoptosis and also showed upregulation of apoptosis related genes. Immunolocalization of OmpC in brain demonstrated the direct involvement of the porin in neurodegeneration and formation of spongiform encephalopathy. CONCLUSION: We have demonstrated the ability of OmpC of E. coli to induce neurodegeneration in mice.
Asunto(s)
Encéfalo/patología , Enfermedades Neurodegenerativas/etiología , Porinas/toxicidad , Animales , Apoptosis , Encéfalo/metabolismo , Calcio/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Inyecciones Intramusculares , Ratones , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Porinas/administración & dosificaciónRESUMEN
Bacteria, as well as higher organisms such as sea anemones or earthworms, have developed sophisticated virulence factors such as the pore-forming toxins (PFTs) to mount their attack against the host. One of the most fascinating aspects of PFTs is that they can adopt a water-soluble form at the beginning of their lifetime and become an integral transmembrane protein in the membrane of the target cells. There is a growing understanding of the sequence of events and the various conformational changes undergone by these toxins in order to bind to the host cell surface, to penetrate the cell membranes and to achieve pore formation. These points will be addressed in this review.
Asunto(s)
Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Animales , Bacterias/patogenicidad , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidad , Membrana Celular/efectos de los fármacos , Colicinas/química , Colicinas/toxicidad , Citotoxinas/química , Citotoxinas/toxicidad , Modelos Moleculares , Estructura Molecular , Oligoquetos/patogenicidad , Proteínas Citotóxicas Formadoras de Poros/fisiología , Porinas/química , Porinas/toxicidad , Conformación Proteica , Anémonas de Mar/patogenicidad , Virulencia/fisiologíaRESUMEN
Campylobacter jejuni is a major food-borne pathogen and a leading cause of diarrhoea. A cytotoxin is most likely involved in the pathogenesis of inflammatory diarrhoea due to C. jejuni. A 45-kDa outer membrane protein encoded by the porA gene was reported to exhibit cytotoxic activity for cultured mammalian cells in vitro. We cloned and expressed the porA gene in Escherichia coli BL21 codon plus RIL strain using the fusion vector pGEX-4T-1. The fusion protein solubilised in urea in denatured form or solubilised in Empigen BB in native form or their thrombin-cleaved products did not exhibit cytotoxic activity for Chinese hamster ovary (CHO) cells. The urea-solubilised fusion protein did not induce fluid accumulation in the rabbit ileal loop assay. All 76 clinical isolates of Campylobacter spp. tested were positive for porA by PCR, but only 13 isolates were positive for cytotoxin on CHO cells. Both cytotoxin-positive as well as cytotoxin-negative strains expressed PorA as determined by immunoblot analysis. These findings show that the porA gene product of C. jejuni is not a cytotoxin mediating inflammatory diarrhoea.
Asunto(s)
Proteínas Bacterianas/toxicidad , Campylobacter jejuni/patogenicidad , Diarrea/microbiología , Porinas/toxicidad , Animales , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Células CHO , Chlorocebus aethiops , Cricetinae , Cricetulus , Citotoxinas/toxicidad , Diarrea/fisiopatología , Expresión Génica , Íleon/fisiopatología , Porinas/metabolismo , Conejos , Proteínas Recombinantes/toxicidad , Células VeroRESUMEN
Alzheimer's and Parkinson's diseases are associated with the formation in the brain of amyloid fibrils from beta-amyloid and alpha-synuclein proteins, respectively. It is likely that oligomeric fibrillization intermediates (protofibrils), rather than the fibrils themselves, are pathogenic, but the mechanism by which they cause neuronal death remains a mystery. We show here that mutant amyloid proteins associated with familial Alzheimer's and Parkinson's diseases form morphologically indistinguishable annular protofibrils that resemble a class of pore-forming bacterial toxins, suggesting that inappropriate membrane permeabilization might be the cause of cell dysfunction and even cell death in amyloid diseases.
Asunto(s)
Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Enfermedad de Parkinson/genética , Porinas/química , Porinas/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cromatografía en Gel , Dicroismo Circular , Humanos , Modelos Biológicos , Peso Molecular , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/toxicidad , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Placa Amiloide/genética , Placa Amiloide/metabolismo , Placa Amiloide/patología , Porinas/metabolismo , Porinas/toxicidad , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Sinucleínas , alfa-SinucleínaRESUMEN
BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is responsible for serious infections in the immunocompromised host. Many skin lesions induced by P. aeruginosa have been described. Few investigations have been performed on the local action of P. aeruginosa components. OBJECTIVES: To shed light on the "in vivo" activity of lipopolysaccharide (LPS) and porins extracted from P. aeruginosa, by verifying their effects after inoculation in mouse skin through the observation of histological changes and immunohistochemical expression of collagen IV. RESULTS: Both substances were able to induce a similar inflammatory process and a characteristic reversible change in collagen IV distribution. Interestingly, a fibroblast increase was observed at 24 h in the skin treated with porins, while it appeared later in the skin treated with LPS. Besides these changes, porins particularly increased collagen edema, together with disgregation of hypodermal structures. Moreover "in vitro", porins were able to stimulate fibloblasts 3T3 to convert 72 kDa type IV collagenase into the activated 62 kDa form and to release the 92 kDa collagenase. CONCLUSION: LPS and porins, released by gram-negative bacteria during cell growth and lysis, interact with the host at target cells, such as keratinocytes, fibroblasts and immunocompetent cells, thus contributing significantly to the pathogenesis of P aeruginosa skin infections.
Asunto(s)
Colágeno Tipo IV/metabolismo , Lipopolisacáridos/toxicidad , Porinas/toxicidad , Piel/efectos de los fármacos , Piel/patología , Células 3T3 , Animales , Colagenasas/química , Colagenasas/metabolismo , Inmunohistoquímica , Ratones , Peso Molecular , Infecciones por Pseudomonas/etiología , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Piel/metabolismo , Enfermedades Cutáneas Bacterianas/etiología , Enfermedades Cutáneas Bacterianas/metabolismo , Enfermedades Cutáneas Bacterianas/patologíaRESUMEN
Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three different regions of the molecule. In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp(45), EqtII Trp(116/117) and EqtII Trp(149), were prepared in an Escherichia coli expression system [here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein]. They all possess a single intrinsic fluorescent centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp(116/117) resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp(45) and EqtII Trp(149), was altered to a certain extent. EqtII Trp(149) was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with (2)H(2)O, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp(45) and EqtII Trp(149) were more exposed to water, and also remained as such in the membrane-bound form.
Asunto(s)
Venenos de Cnidarios/química , Venenos de Cnidarios/metabolismo , Citotoxinas/metabolismo , Mutación/genética , Anémonas de Mar/química , Triptófano/metabolismo , Acrilamida/metabolismo , Secuencia de Aminoácidos , Animales , Bromosuccinimida/metabolismo , Bovinos , Muerte Celular/efectos de los fármacos , Venenos de Cnidarios/genética , Venenos de Cnidarios/toxicidad , Citotoxinas/química , Citotoxinas/genética , Citotoxinas/toxicidad , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Fluorescencia , Hemólisis/efectos de los fármacos , Liposomas/química , Liposomas/metabolismo , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Porinas/química , Porinas/genética , Porinas/metabolismo , Porinas/toxicidad , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Espectroscopía Infrarroja por Transformada de Fourier , Relación Estructura-Actividad , Triptófano/genética , Agua/metabolismoRESUMEN
Bacteria or bacterial products may constitute important inducers of surface molecule expression on endothelial cells and leucocytes. This study was undertaken to determine the effects of the Salmonella typhimurium porins, LPS-S and LPS-R on the transendothelial migration of leucocytes through human umbilical vein endothelial cells (HUVEC). Treatment of the HUVEC with either porins or LPS-S or LPS-R increased the transmigration of different leucocyte populations, in particular that of neutrophils. The maximal increase occurred using LPS-S treatment, whereas porin stimulation fell between LPS-S and LPS-R. The transmigration increase was dose-dependent and reached its maximum at about 100-1000 ng/ml of stimulus. Optimal endothelial activation occurred after 2-4 h and 4-6 h using LPS and porin, respectively. Stimulation of leucocytes with either porins or LPS slightly increased their transmigration through non-activated endothelial cells. Transmigration increased remarkably during the simultaneous stimulation of endothelial cells by IL-1ss together with either porins or LPS. To assess participation of E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and leucocyte adhesion complex (CD11/18) in porin- or LPS-mediated leucocyte migration, blocking MoAbs were used. Each blocking MoAb partially and selectively decreased leucocyte transmigration. The obtained results contribute to clarify some aspects of the inflammatory process at sites of infection.
Asunto(s)
Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Lipopolisacáridos/toxicidad , Porinas/toxicidad , Salmonella typhimurium/patogenicidad , Anticuerpos Monoclonales/farmacología , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Endotelio Vascular/inmunología , Humanos , Técnicas In Vitro , Inflamación/etiología , Interleucina-1/farmacología , Leucocitos/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Linfocitos/fisiología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/fisiologíaRESUMEN
A clinical isolate of Campylobacter jejuni, previously found to produce a toxin active in cell culture assays, was used for identification and characterisation of a cytotoxic porin-lipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by high-performance liquid chromatography of crude concentrated culture supernate and DEAE-anion exchange chromatography. The complex had a toxic activity of 20.1 tissue culture dose50 (TCD50)/microg of protein for HEp-2 cells, 7.49 TCD50/microg of protein for HeLa cells and 1.87 TCD50/microg of protein for Chinese hamster ovary cells. Analysis by SDS-PAGE revealed a single protein band of 45 kDa and a high mol. wt carbohydrate moiety. The complex gave a positive result in the Limulus amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins Galanthus nivalis agglutinin, Maackia amurensis agglutinin and Datura stramonium agglutinin. The cytotoxic activity associated with the complex was heat-labile at 70 degrees C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperiodate and the glycosidases neuraminidase and N-glycosidase F. Sequencing of the N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C. jejuni. The cytotoxic activity of the complex was neutralised by a polyclonal, homologous antiserum, which reacted on Western blot with the 45-kDa protein, but not by polyclonal antisera raised against a number of other bacterial toxins.
Asunto(s)
Campylobacter jejuni/metabolismo , Citotoxinas/química , Lipopolisacáridos/química , Porinas/química , Secuencia de Aminoácidos , Animales , Western Blotting , Células CHO , Campylobacter jejuni/genética , Secuencia de Carbohidratos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cricetinae , Citotoxinas/aislamiento & purificación , Citotoxinas/toxicidad , ADN Bacteriano/análisis , Relación Dosis-Respuesta a Droga , Galanthus , Células HeLa , Humanos , Prueba de Limulus , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/toxicidad , Datos de Secuencia Molecular , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Porinas/aislamiento & purificación , Porinas/toxicidad , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro-apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage-dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis.
Asunto(s)
Apoptosis/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Calcio/metabolismo , Cisteína Endopeptidasas/metabolismo , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/patogenicidad , Porinas/metabolismo , Adenosina Trifosfato/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/toxicidad , Calpaína/metabolismo , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/efectos de los fármacos , Células HeLa , Humanos , Transporte Iónico/efectos de los fármacos , Mutación , Neisseria gonorrhoeae/genética , Porinas/genética , Porinas/toxicidadRESUMEN
Prominent antigens of Treponema denticola have been suggested to be mediators of the cytopathic effects typically seen in periodontal disease. In the present study of the T. denticola major surface protein (Msp) and the surface-expressed chymotrypsinlike protease complex (CTLP), we characterized the ability of these proteins to adhere to and lyse epithelial cells. Msp and CTLP were closely associated in spirochete outer membranes. Purified Msp, both native and recombinant, and CTLP bound to glutaraldehyde-fixed periodontal ligament epithelial cells. Adherence of Msp was partially blocked by specific antibodies. Adherence of CTLP was partially blocked by serine protease inhibitors and was further inhibited by specific antibodies. Both native Msp and CTLP were cytotoxic toward periodontal ligament epithelial cells, and their cytotoxicity was inhibited by the same treatments that inhibited adherence. Msp, but not CTLP, lysed erythrocytes. Msp complex (partially purified outer membranes free of protease activity) was cytotoxic toward a variety of different cell types. Pore-forming activities of recombinant Msp in black lipid model membrane assays and in HeLa cell membranes were similar to those reported for the native protein, supporting the hypothesis that Msp cytotoxicity was due to its pore-forming activity.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/toxicidad , Serina Endopeptidasas/toxicidad , Treponema/patogenicidad , Animales , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Células CHO , Quimasas , Cricetinae , Células HeLa , Hemólisis , Humanos , Peso Molecular , Porinas/toxicidad , Conejos , PorcinosRESUMEN
BACKGROUND/AIMS: Several bacterial components may contribute to the development of renal injury during Gram-negative sepsis. In the present study, we evaluated the proinflammatory effect on cultured human proximal tubular epithelial cells (PTEC) of a 36-kD porin purified from escherichia coli METHODS: PTEC were stimulated with E. coli porin to evaluate 45Ca2+ influx, cytoskeleton changes, generation of reactive oxygen species (ROS) as detected by chemiluminescence and cytochrome c reduction. Production of TNF-alpha, IL-6 and IL-8 was evaluated at the mRNA and protein levels. RESULTS: Stimulation of PTEC with porin was followed by a rapid and sustained 45Ca2+ influx and by an altered distribution of actin fibers and of vinculin streaks. Porin was able to induce generation of ROS and production of proinflammatory cytokines from PTEC. TNF-alpha production peaked at 6 h after exposure to porin and preceded that of IL-6 and IL-8. CONCLUSIONS: E. coli porin - at doses attainable in vivo - appears to stimulate PTEC to produce ROS and cytokines. Porin-induced PTEC activation may contribute to renal injury in the course of Gram-negative infection.
Asunto(s)
Citocinas/biosíntesis , Escherichia coli , Túbulos Renales Proximales/efectos de los fármacos , Porinas/toxicidad , Actinas/análisis , Células Cultivadas , Epitelio/efectos de los fármacos , Epitelio/inmunología , Epitelio/patología , Humanos , Técnicas In Vitro , Inflamación , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Túbulos Renales Proximales/inmunología , Túbulos Renales Proximales/patología , Mediciones Luminiscentes , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Reacción en Cadena de la Polimerasa , Porinas/aislamiento & purificación , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We tested the ability of recombinant outer membrane proteins of Pseudomonas aeruginosa to serve as a protective vaccine against this gram negative pathogen under two main pathophysiological events leading to P. aeruginosa sepsis. i) systemic infection during immunosuppression, and ii) bacterial translocation. A hybrid vaccine was cloned combining protective epitopes of outer membrane protein F (OprF) and outer membrane protein I (OprI). This vaccine proved to be highly protective against an intraperitoneal challenge with P. aeruginosa in immunosuppressed mice. Oral immunization of mice, with recombinant Salmonella dublin expressing OprI induced s-IgA antibodies in the gut mucosa against OprI and provided protection against translocation of P. aeruginosa in an immunosuppressed mouse model. To test whether OprI is safe for use in humans, recombinant OprI was purified and used for immunization of volunteers. Vaccination was well tolerated and no major side effects were observed. The induction of serum antibodies against OprI was found to be dose-dependent and was observed in total in 65% of the volunteers.
Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Lipoproteínas/inmunología , Porinas/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Anticuerpos Monoclonales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/toxicidad , Vacunas Bacterianas/biosíntesis , Vacunas Bacterianas/toxicidad , Secuencia de Bases , Cartilla de ADN , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Femenino , Humanos , Lipoproteínas/biosíntesis , Lipoproteínas/toxicidad , Ratones , Ratones Endogámicos BALB C/inmunología , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Porinas/biosíntesis , Porinas/toxicidad , Infecciones por Pseudomonas/prevención & control , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/toxicidad , Salmonella , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/toxicidadRESUMEN
Porins isolated from Salmonella typhimurium and found to contain less than 0.1% w/w of LPS, were found to be lethal at a dose of 100 ng to both LPS-responder (BALB/cByJ) and non-responder (C3H/HeJ) mice sensitized with D-galactosamine. This lethal action could be prevented by anti-TNF-alpha serum given intravenously 10 min before the porin injection but not by polymyxin-B mixed with the porins in a ratio of approximately 300 moles polymyxin-B per mole of porin. The porin preparation was also pyrogenic to rabbits at a dose of 1 microgram/kg and elicited a local Shwartzman reaction when used as the sensitizing and eliciting agent; these reactions were also present when the porins were mixed with polymyxin-B.