RESUMEN

Achromobacter spp. are opportunistic pathogens increasingly recovered from adult patients with cystic fibrosis (CF). We report the characterization of 122 Achromobacter spp. isolates recovered from 39 CF patients by multilocus sequence typing, virulence traits, and susceptibility to antimicrobials. Two species, A. xylosoxidans (77%) and A. ruhlandii (23%) were identified. All isolates showed a similar biofilm formation ability, and a positive swimming phenotype. By contrast, 4·3% and 44·4% of A. xylosoxidans and A. ruhlandii, respectively, exhibited a negative swarming phenotype, making the swimming and swarming abilities of A. xylosoxidans significantly higher than those of A. ruhlandii. A. xylosoxidans isolates from an outbreak clone also exhibited significantly higher motility. Both species were generally susceptible to ceftazidime, ciprofloxacin, imipenem and trimethoprim/sulphamethoxazole and there was no significant difference in susceptibility between isolates from chronic or sporadic infection. However, A. xylosoxidans isolates from chronic and sporadic cases were significantly more resistant to imipenem and ceftazidime than isolates of the outbreak clone.

Asunto(s)

Achromobacter/aislamiento & purificación , Fibrosis Quística/complicaciones , Infecciones por Bacterias Gramnegativas/microbiología , Factores de Virulencia/análisis , Achromobacter/clasificación , Achromobacter/efectos de los fármacos , Achromobacter/fisiología , Antibacterianos/farmacología , Biopelículas/crecimiento & desarrollo , Humanos , Locomoción , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus

RESUMEN

The present study addressed the question whether ExoU, a Pseudomonas aeruginosa toxin with phospholipase A2 (PLA2) activity, may induce airway epithelial cells to overexpress tissue factor (TF) and exhibit a procoagulant phenotype. Cells from the human bronchial epithelial BEAS-2B line were infected with an ExoU-producing P. aeruginosa strain, pre-treated or not with the cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP), or with two ExoU-deficient mutants. Control noninfected and infected cells were assessed for the expression of: 1) TF mRNA by RT-PCR; 2) cell-associated TF by enzyme immunoassay and flow cytometry; 3) procoagulant activity by a colorimetric assay; and 4) microparticle-associated TF by flow cytometry. An enzyme immunoassay was also used to assess cell-associated TF in lung extracts from mice infected intratracheally with ExoU-producing and -deficient bacteria. Cells infected with the wild-type bacteria had higher levels of TF mRNA, cell-associated TF expression, procoagulant activity and released microparticle-associated TF than cells infected with the mutants. Bacterial treatment with MAFP significantly reduced the expression of TF by infected cells. Lung samples from mice infected with the wild-type bacteria exhibited higher levels of cell-associated TF and procoagulant activity. The present results demonstrate that ExoU may contribute to the pathogenesis of lung injury by inducing a tissue factor-dependent procoagulant activity in airway epithelial cells.

Asunto(s)

Proteínas Bacterianas/fisiología , Bronquios/microbiología , Coagulantes/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Proteínas Bacterianas/metabolismo , Bronquios/citología , Citosol/metabolismo , Femenino , Humanos , Ratones , Modelos Biológicos , Mutación , Organofosfonatos/metabolismo , Fosfolipasas A2/metabolismo , Infecciones por Pseudomonas/diagnóstico , Tromboplastina/metabolismo

RESUMEN

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.

Asunto(s)

Eicosanoides/biosíntesis , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiología , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Ácido Araquidónico/antagonistas & inhibidores , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/farmacología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Línea Celular , Dinoprostona/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Epoprostenol/metabolismo , Femenino , Fosfolipasas A2 Grupo IV , Humanos , Ibuprofeno/uso terapéutico , Indometacina/uso terapéutico , Inflamación/patología , Inhibidores de la Lipooxigenasa/uso terapéutico , Masoprocol/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Organofosfonatos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad

RESUMEN

AIM: To evaluate the cytotoxic effects of two brands of mineral trioxide aggregate (MTA) (Pro-Root MTA and MTA Angelus) and Portland cement (PC) on the human ECV 304 endothelial cell line. METHODOLOGY: Endothelial ECV 304 cells were incubated at 37 degrees C in an atmosphere of 95% air, 5% carbon dioxide and 100% humidity for 7 days and grown in F12 medium supplemented with 10% fetal bovine serum with 50 microg mL(-1) of gentamicin sulphate. Effects of the materials on mitochondrial functions were measured by a colorimetric assay. At each experimental time interval (24, 48 and 72 h), a dimethyl-thiazol-diphenyl tetrazolium bromid assay was conducted to measure cell viability. All assays were repeated three times to ensure reproducibility. Results were expressed as average absorbance (A(570/nm)) +/- SD and the data were analysed statistically by one-way analysis of variance and the Bonferroni post-test. A P-value < 0.05 was considered statistically significant. RESULTS: No statistically significant difference was shown between any of the experimental materials (P > 0.05). CONCLUSIONS: The two brands of MTA analysed, as well as the PC, initially showed a similar elevated cytotoxic effect that decreased gradually with time allowing the cell culture to become reestablished.

Asunto(s)

Células Endoteliales/efectos de los fármacos , Materiales de Obturación del Conducto Radicular/toxicidad , Compuestos de Aluminio/toxicidad , Análisis de Varianza , Compuestos de Calcio/toxicidad , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Colorantes/metabolismo , Cementos Dentales/toxicidad , Combinación de Medicamentos , Humanos , Modelos Lineales , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxidos/toxicidad , Silicatos/toxicidad , Sales de Tetrazolio/metabolismo , Tiazoles/metabolismo

RESUMEN

Bacteria of Stenotrophomonas maltophilia have been isolated with increasing frequency from the airways of cystic fibrosis (CF) patients, usually following P. aeruginosa infections, but their adherence to human epithelial respiratory cells has never been investigated. In this study, various S. maltophilia strains were seen to adhere to epithelial respiratory cells in vitro, mainly along intercellular junctions. Bacteria could also enter into host cells, as determined by the gentamicin exclusion assay and transmission electron microscopy. Cells co-incubated with P. aeruginosa and S. maltophilia exhibited a significantly decreased adherence of these latter bacteria. No decrease in S. maltophilia adherence was observed when co-infection was carried out with heat-killed P. aeruginosa or when respiratory cells were first incubated with P. aeruginosa, before incubation with S. maltophilia. Our data suggest that P. aeruginosa infections do not account for the increased prevalence of S. maltophilia in CF patient airways, that thermolabile products from P. aeruginosa can control the adherence of S. maltophilia to respiratory cells and also that these two bacteria do not compete for cell receptors.

Asunto(s)

Adhesión Bacteriana , Mucosa Respiratoria/microbiología , Stenotrophomonas maltophilia/patogenicidad , Bronquios/citología , Bronquios/microbiología , Fibrosis Quística/microbiología , Células Epiteliales/citología , Células Epiteliales/microbiología , Humanos , Uniones Intercelulares/microbiología , Pseudomonas aeruginosa/patogenicidad , Mucosa Respiratoria/citología

RESUMEN

Pseudomonas aeruginosa has been shown to enter into human endothelial cells in vitro. To ascertain the effects of bacterial intracellular (IC) infection, endothelial cells were exposed to PAK and PAO-1 strains for 1 h and treated with gentamicin in culture medium for different periods. P. aeruginosa induced a significant production of superoxide and hydrogen peroxide by endothelial cells. Concentrations of IC bacteria were reduced progressively with time and no viable PAO-1 was detected at 24 h after infection. However, IC infection led to killing of 32.2%+/-2.9 and 51.8%+/-3.5 of the cells infected with PAK and PAO-1, respectively, as determined by the MTT assay. By three criteria (transmission electron microscopy, DNA electrophoresis and reactivity with annexin V) infected cells exhibited features of apoptosis. Treatment of infected cells with anti-oxidants (catalase, tocopherol and N -acetyl-L-cysteine) significantly decreased the percentage of cell death. In contrast, treatment with aminoguanidine, an inhibitor of inducible NO synthase, increased significantly the killing of PAO-1 infected cells. Based on these results we speculate that in response to P. aeruginosa infection, endothelial cells increase the production of reactive oxygen intermediates to eliminate IC pathogens, but cells do not resist the oxidative stress and die by apoptosis.

Asunto(s)

Apoptosis/fisiología , Endotelio/microbiología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Acetilcisteína/farmacología , Antioxidantes/farmacología , Catalasa/farmacología , Línea Celular , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Electroforesis en Gel de Agar , Endotelio/citología , Citometría de Flujo , Colorantes Fluorescentes/química , Formazáns/química , Guanidinas/farmacología , Humanos , Lipopolisacáridos , Microscopía Electrónica , Microscopía Fluorescente , Fenantridinas/química , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Especies Reactivas de Oxígeno/fisiología , Sales de Tetrazolio/química , Vitamina E/farmacología

RESUMEN

Proinflammatory cytokines have been shown to activate endothelial cells. To investigate the effect of cytokines on the interaction of human umbilical vein endothelial cells (HUVEC) with Pseudomonas aeruginosa, cells were treated with interferon-gamma (IFN-gamma) plus tumour necrosis factor-alpha (TNF-alpha) for 24 hr and exposed to P. aeruginosa suspension for 1 hr. Light microscopy showed that activated cells internalized significantly more bacteria than control cells. To ascertain the effect of cytokines on the microbicidal activity of HUVEC, the concentrations of viable intracellular (IC) bacteria in control and activated cells were determined, at 1 and 5 hr postinfection, by the gentamicin exclusion assay. In control cells, no significant decrease in the concentration of bacteria was detected 5 hr postinfection. In contrast, in activated cells the concentration of viable bacteria at 5 hr was significantly lower. Concentrations of superoxide and hydrogen peroxide detected in supernatants of activated cells were significantly higher than in control cell supernatants. HUVEC anti-P. aeruginosa activity was insensitive to the antioxidants superoxide dismutase, dimethylthiourea and allopurinol as well as to the L-arginine analogues aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), but was significantly inhibited by catalase. Our results indicate that HUVEC can be activated by IFN-gamma plus TNF-alpha to kill IC P. aeruginosa and suggest a role for reactive oxygen radicals, notably hydrogen peroxide, in HUVEC antibacterial activity.

Asunto(s)

Endotelio Vascular/inmunología , Interferón gamma/inmunología , Pseudomonas aeruginosa/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Técnicas de Cultivo de Célula , Supervivencia Celular/inmunología , Endocitosis/inmunología , Endotelio Vascular/microbiología , Humanos , Óxido Nítrico/inmunología , Especies Reactivas de Oxígeno/inmunología , Venas Umbilicales/inmunología , Venas Umbilicales/microbiología

RESUMEN

Internalization of Pseudomonas aeruginosa by epithelial respiratory cell lines has been suggested to be dependent on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Because we have observed intracellular (IC) P. aeruginosa only in cells that do not express apical CFTR, we addressed the question of whether bacterial internalization by epithelial cells depends on the degree of cell differentiation and polarity. Internalization of piliated P. aeruginosa PAO-1 and PAK by human epithelial respiratory cells in primary culture and by the 16 human bronchial epithelial 14o- cell line cultured either on thick collagen gels or on thin collagen films was evaluated by the gentamicin exclusion assay. Cells cultured on thick gels were differentiated, polarized, and tight. They exhibited CFTR at their apical membranes, expressed beta1 integrins at their basal membranes, excluded lanthanum nitrate, and uniformly expressed ZO-1 protein. In contrast, in cells cultured on thin films, CFTR was present mainly in the cytoplasm, whereas beta1 integrins were detected at apical membranes. Most cells cultured on thin films did not exclude lanthanum nitrate and rarely expressed ZO-1 protein. Cells grown on thick and thin collagen substrates differed markedly in bacterial internalization: no IC bacteria could be detected in cells cultured on gels, whereas high IC bacterial concentrations were isolated from cells cultured on thin films. Treatment of cells cultured on thin films with ethylenediaminetetraacetic acid, to disrupt intercellular junctions further, significantly enhanced P. aeruginosa internalization. Our results suggest that P. aeruginosa internalization by epithelial respiratory cells does not depend on CFTR protein expression at the epithelial cell surface but rather on cell polarity and junctional complex integrity.

Asunto(s)

Bronquios/microbiología , Pseudomonas aeruginosa/fisiología , Bronquios/citología , Bronquios/ultraestructura , Diferenciación Celular , Polaridad Celular , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Electrónica

RESUMEN

Intravenous administration of lipopolysaccharide (LPS) to rats increased the production of nitric oxide (NO) metabolites (NOx) by blood polymorphonuclear neutrophils (PMN) in vitro. Both dexamethasone and L-NMMA, added in vitro to neutrophil cultures, inhibited the production of NO. On the other hand, the production of NO was not affected by the treatment, in vivo or in vitro, with different inhibitors of cyclooxygenase or 5-lipoxygenase or with a platelet-activating factor (PAF) antagonist. The incubation of blood PMN from normal rats in vitro with neutrophil activators (PAF, leukotriene B4, and interleukin-8) and different cytokines [interleukin-1, tumor necrosis factor alpha, and interferon-gamma (IFN-gamma)] showed that only IFN-gamma was able to induce the production of high amounts of NO. This induction was directly correlated with the expression of iNOS and an increase in in the enzyme activity in blood PMN. The tyrosine kinase inhibitor genistein inhibited NO production induced by IFN-gamma, suggesting that the signal transduction pathway leading to NOS induction in rat PMN involves phosphorylation by tyrosine kinase. We also showed that NO produced by IFN-gamma activated rat blood PMN involved in the killing of Pseudomonas aeruginosa.

Asunto(s)

Actividad Bactericida de la Sangre/inmunología , Neutrófilos/inmunología , Óxido Nítrico Sintasa/inmunología , Proteínas Tirosina Quinasas/biosíntesis , Animales , Actividad Bactericida de la Sangre/efectos de los fármacos , Citocinas/fisiología , Dexametasona/farmacología , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/inmunología , Interferón gamma/farmacología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Masculino , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/sangre , Óxido Nítrico Sintasa de Tipo II , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/inmunología , Ratas , Ratas Wistar , omega-N-Metilarginina/farmacología

RESUMEN

Release of lactate dehydrogenase (LDH) from the cytoplasmic compartment, trypan blue exclusion and methylthiazole tetrazolium (MTT) colorimetric assays were compared with regard to their sensitivity in detecting damage of human cultured epithelial cells induced by sodium fluoride or puromycin. LDH assay did not detect any difference between controls and cells treated with either of the two drugs. Cell monolayers treated with 0.3 sodium fluoride or 10(-2) M puromycin presented higher percentages of cells that took up the trypan blue dye than controls but monolayers treated with lower drug concentrations did not differ from controls. Viability measured by MTT assay was the most sensitive assay, detecting a dose-dependent impairment of cell function after treatment with the two drugs. Moreover, MTT offered major advantages in speed, simplicity and precise quantitation over the other viability assays

Asunto(s)

Humanos , Animales , Estudio Comparativo , Técnicas de Cultivo de Célula/métodos , Hígado/citología , Chlorocebus aethiops , Supervivencia Celular , Colorantes , L-Lactato Deshidrogenasa/metabolismo , Mamíferos , Sales de Tetrazolio , Tiazoles , Azul de Tripano , Células Vero

RESUMEN

Release of lactate dehydrogenase (LDH) from the cytoplasmic compartment, trypan blue exclusion and methylthiazole tetrazolium (MTT) colorimetric assays were compared with regard to their sensitivity in detecting damage of human cultured epithelial cells induced by sodium fluoride or puromycin. LDH assay did not detect any difference between controls and cells treated with either of the two drugs. Cell monolayers treated with 0.3 sodium fluoride or 10(-2) M puromycin presented higher percentages of cells that took up the trypan blue dye than controls but monolayers treated with lower drug concentrations did not differ from controls. Viability measured by MTT assay was the most sensitive assay, detecting a dose-dependent impairment of cell function after treatment with the two drugs. Moreover, MTT offered major advantages in speed, simplicity and precise quantitation over the other viability assays

Asunto(s)

Humanos , Animales , Técnicas de Cultivo de Célula , Hígado/citología , Supervivencia Celular , Chlorocebus aethiops , Colorantes , L-Lactato Deshidrogenasa , Mamíferos , Sales de Tetrazolio , Tiazoles , Azul de Tripano , Células Vero

RESUMEN

Release of lactate dehydrogenase (LDH) from the cytoplasmic compartment, trypan blue exclusion and methylthiazole tetrazolium (MTT) colorimetric assays were compared with regard to their sensitivity in detecting damage of human cultured epithelial cells induced by sodium fluoride or puromycin. LDH assay did not detect any difference between controls and cells treated with either of the two drugs. Cell monolayers treated with 0.3% sodium fluoride or 10(-2) M puromycin presented higher percentages of cells that took up the trypan blue dye than controls but monolayers treated with lower drug concentrations did not differ from controls. Viability measured by MTT assay was the most sensitive assay, detecting a dose-dependent impairment of cell function after treatment with the two drugs. Moreover, MTT offered major advantages in speed, simplicity and precise quantitation over the other viability assays.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Hígado/citología , Animales , Chlorocebus aethiops , Colorantes , Humanos , L-Lactato Deshidrogenasa/metabolismo , Mamíferos , Sales de Tetrazolio , Tiazoles , Azul de Tripano , Células Vero

RESUMEN

Release of lactate dehydrogenase (LDH) from the cytoplasmic compartment, trypan blue exclusion and methylthiazole tetrazolium (MTT) colorimetric assays were compared with regard to their sensitivity in detecting damage of human cultured epithelial cells induced by sodium fluoride or puromycin. LDH assay did not detect any difference between controls and cells treated with either of the two drugs. Cell monolayers treated with 0.3

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sodium fluoride or 10(-2) M puromycin presented higher percentages of cells that took up the trypan blue dye than controls but monolayers treated with lower drug concentrations did not differ from controls. Viability measured by MTT assay was the most sensitive assay, detecting a dose-dependent impairment of cell function after treatment with the two drugs. Moreover, MTT offered major advantages in speed, simplicity and precise quantitation over the other viability assays.

RESUMEN

Human respiratory cells participating in the repair of epithelial wounds have been shown to be highly susceptible to Pseudomonas aeruginosa adherence. To ascertain whether such susceptibility is a common feature of different repairing epithelial cells, Caco-2 cell monolayers were chemically injured, reincubated for 48 h to partially repair and exposed to bacteria. Cells edging the wounds that spread and migrate to re-establish cell confluence were called 'repairing cells' while cells far from the wounds were called 'non-repairing cells'. By light microscopy, bacteria were seen to adhere to and to enter into both repairing and non-repairing cells. The percentage of intracellular bacteria in repairing cells was significantly higher than in non-repairing cells. The higher susceptibility of repairing monolayers to bacterial entry was confirmed by the gentamicin exclusion assay. P. aeruginosa entry into Caco-2 cells was greatly enhanced in non-injured confluent monolayers treated with EDTA to disrupt intercellular junctions. As tight junction disfunctions have been described during the wound repair process, we speculate that exposure of basolateral receptors to bacterial ligands may account for the enhancement of P. aeruginosa internalization by repairing monolayers.

Asunto(s)

Células Epiteliales/microbiología , Pseudomonas aeruginosa/crecimiento & desarrollo , Cicatrización de Heridas , Adhesión Bacteriana , Células CACO-2 , Polaridad Celular , Células Epiteliales/patología , Humanos , Infecciones Oportunistas/etiología , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/patogenicidad , Receptores de Superficie Celular

RESUMEN

P. aeruginosa is selectively internalized by human endothelial cells but is not efficiently killed in the intracellular (IC) compartment. To investigate whether IC survival is associated with failure in bacteria-containing endosome-lysosome (E-L) fusion, endothelial cells were exposed to albumin-colloidal gold complex and to bacterial suspension and submitted to transmission electron microscopy (TEM). Gold granules were detected in P. aeruginosa-containing vacuoles, indicating that E-L fusion had occurred. Bacteria were also seen apparently free in the cell cytoplasm, suggesting disruption of endosome membranes. To ascertain whether phospholipase C (PLC) could account for vacuolar lysis, PLC producing PAO1 and PAK strains were compared with a PLC deficient mutant (PLCN) in their IC survival. All three strains were equally uptaken by the endothelial cells, as determined by the gentamicin exclusion assay. After 3 h of infection, the IC concentration of PAK and PAO1 increased significantly while the concentration of the mutant decreased to 56.8 +/- 18.2% of the viable counts at 1 h of infection. After 5 h, the IC concentration of P. aeruginosa corresponded to 83.1 +/- 34.6%, 109 +/- 22.6% and 26.2 +/- 14.7% of the viable counts detected at 1 h, for PAK, PAO1 and the mutant, respectively. By TEM, while most PAO1-containing vacuoles presented partially lysed membranes, in cells infected with the PLCN mutant bacteria were most often observed in vacuoles with intact membranes. These observations suggest that the IC survival of P. aeruginosa results from a competition between the microbicidal activity of endothelial cells following E-L fusion and the capacity of bacteria to escape from endosomes.

Asunto(s)

Endosomas/ultraestructura , Endotelio Vascular/microbiología , Endotelio Vascular/ultraestructura , Fagosomas/ultraestructura , Pseudomonas aeruginosa/fisiología , Supervivencia Celular , Células Cultivadas , Cloroquina/farmacología , Citoplasma/microbiología , Citoplasma/ultraestructura , Endosomas/fisiología , Endotelio Vascular/fisiología , Humanos , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Fusión de Membrana , Microscopía Electrónica , Fagosomas/fisiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/ultraestructura , Fosfolipasas de Tipo C/metabolismo , Vacuolas/microbiología , Vacuolas/fisiología , Vacuolas/ultraestructura

RESUMEN

Pseudomonas aeruginosa is a major human pathogen known to infect tissues that have been previously damaged in some way. In wounded human respiratory tissues, P. aeruginosa cells were found attached to exposed basement membranes following epithelial denudation, suggesting that the affinity for extracellular matrix proteins may account for the bacterium's opportunistic character. By using microtiter wells coated with different P. aeruginosa strains, we demonstrated that laminin binds to both colonizing bacterial strains, isolated from asymptomatic carriers, and strains isolated from infected patients. Binding of soluble laminin to piliated P. aeruginosa PAK and to the nonpiliated isogenic mutant PAK/p--was shown to be saturable. Binding of laminin to the piliated PAK strain was not different from binding to th nonpiliated PAK/p--strain but was significantly higher than binding to the avirulent, nonpiliated PAK-N1 rpoN mutant. By transmission electron microscopy, we localized the laminin-binding sites on a loose material in the outermost layer of the bacteria. Western immunoblotting results suggested that 57- and 59-kDa nonpilus adhesins from the microbial outer membranes account for the binding of P. aeruginosa to laminin. We speculate that bacterial affinity for laminin may be of biological significance in the pathogenesis of P. aeruginosa infection of injured tissues.

Asunto(s)

Adhesinas Bacterianas/análisis , Adhesión Bacteriana , Laminina/metabolismo , Lectinas , Pseudomonas aeruginosa/fisiología , Adhesinas Bacterianas/metabolismo , Sitios de Unión , Humanos

RESUMEN

The pathogenesis of Pseudomonas aeruginosa disseminated infections depends on bacterial interaction with blood vessels. We have hypothesized that in order to traverse the endothelial barrier, bacteria would have to adhere to and damage endothelial cells. To test this hypothesis, we studied the adherence to human endothelial cells in primary culture of the piliated P. aeruginosa strain PAK and of two isogenic nonpiliated strains: PAK/p-, which carries a mutation in the pilin structural gene, and PAK-N1, a mutant defective in the regulatory rpoN gene. PAK adhered significantly more than did the pilus-lacking strains. P. aeruginosa was also taken up by endothelial cells, as determined by quantitative bacteriologic assays and by transmission electron microscopy. This internalization of P. aeruginosa seems to be a selective process, since the piliated strain was taken up significantly more than the nonpiliated bacteria and the avirulent Escherichia coli DH5 alpha, even following bacterial centrifugation onto the cell monolayers. A significant fraction of the internalized P. aeruginosa PAK was recovered in a viable form after 6 h of residence within endothelial cells. Progressive endothelial cell damage resulted from PAK intracellular harboring, as indicated by the release of lactate dehydrogenase. An increasing concentration of PAK cells was recovered from the extracellular medium with time, suggesting that ingested bacteria were released from endothelial cells and multiplied freely. We speculate that in vivo the ability of some P. aeruginosa strains to resist intracellular residence would afford protection from host defenses and antibiotics and that the release of viable bacteria into bloodstream may represent a central feature of the pathogenesis of bacteremia in compromised patients.

Asunto(s)

Adhesión Bacteriana/fisiología , Proteínas de Unión al ADN , ARN Polimerasas Dirigidas por ADN , Endotelio Vascular/microbiología , Fimbrias Bacterianas/fisiología , Pseudomonas aeruginosa/fisiología , Adhesión Bacteriana/genética , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico/efectos de los fármacos , División Celular , Células Cultivadas , Citocalasina D/farmacología , Endotelio Vascular/citología , Endotelio Vascular/patología , Endotelio Vascular/ultraestructura , Proteínas de Escherichia coli , Proteínas Fimbrias , Fimbrias Bacterianas/genética , Genes Bacterianos/genética , Humanos , L-Lactato Deshidrogenasa/análisis , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/ultraestructura , ARN Polimerasa Sigma 54 , Factor sigma/genética , Venas Umbilicales/citología

RESUMEN

Different bacterial species adhere avidly to respiratory mucus. Such adhesion, when followed by ciliary clearance, represents an important stage of the airway defense system. However, in pathological conditions, the mucociliary clearance may be severely reduced, and mucus-associated bacteria may multiply and infect the underlying epithelium. Only a few bacteria have been shown to adhere to ciliary membranes of functionally active ciliated cells. Therefore, the first way in which most of the respiratory pathogens associate with the airway epithelium is likely to be by their adhesion to mucus. Some bacteria also secrete products that may affect ciliary function and/or cause cell death and epithelial disruption. Respiratory pathogens that do not bind to normal ciliated cells may readily adhere to injured epithelial cells, or to the unmasked extracellular matrix. Furthermore, following injury, epithelial respiratory cells in the process of migration, in order to repair the wounds, may present receptors to which bacteria adhere. The adhesion to all of these epithelial receptors may contribute to the chronicity of many bacterial respiratory infections.

Asunto(s)

Adhesión Bacteriana , Sistema Respiratorio/microbiología , Animales , Adhesión Bacteriana/fisiología , Movimiento Celular , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Matriz Extracelular/microbiología , Humanos , Membrana Mucosa/citología , Membrana Mucosa/microbiología , Pseudomonas aeruginosa/fisiología , Sistema Respiratorio/citología

RESUMEN

Human nasal polyps in outgrowth culture were used to study the adhesion of Pseudomonas aeruginosa to respiratory cells. By transmission electron microscopy, bacteria associated with ciliated cells were identified trapped at the extremities of cilia, usually as aggregates of several bacterial cells. They were never seen at the interciliary spaces or attached along cilia. Bacteria were also seen to adhere avidly to migrating cells of the periphery of the outgrowth culture. Using a model of repair of wounded respiratory epithelial cells in culture, we observed that the adhesion of P. aeruginosa to migrating cells of the edges of the repairing wounds was significantly higher than the adhesion to non-migrating cells and that adherent bacteria were surrounded by a fibronectin-containing fibrillar material. The secretion of extracellular matrix components is involved in the process of epithelium repair following injury. To investigate the molecular basis of P. aeruginosa adhesion to migrating cells, bacteria were treated with a fibronectin solution before their incubation with the respiratory cells. P. aeruginosa treatment by fibronectin significantly increased their adhesion to migrating cells. Accordingly, we hypothesize that during cell migration, fibronectin secreted by epithelial cells may favour P. aeruginosa adhesion by establishing a bridge between the bacteria and the epithelial cell receptors. Such a mechanism may represent a critical step for P. aeruginosa infection of healing injured epithelium.

Asunto(s)

Mucosa Nasal/microbiología , Pseudomonas aeruginosa/fisiología , Adhesión Bacteriana , Movimiento Celular , Cilios/microbiología , Epitelio/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Glicoconjugados/metabolismo , Humanos , Mucosa Nasal/lesiones , Pólipos Nasales/patología , Técnicas de Cultivo de Órganos , Polímeros , Solubilidad , Cicatrización de Heridas

RESUMEN

Human nasal polyps outgrowth culture were used to study the adhesion of Pseudomonas aeruginosa to respiratory cells. By transmission electron microscopy, bacteria associated with ciliated cells were identified trapped at the extremities of cilia, usually as aggregates of several bacterial cells. They were never seen at the interciliary spaces or attached along cilia. Bacteria were also seen to adhere to migrating cells of the periphery of the outgrowth culture. Using a model of repair of wounded respiratory epithelial cells in culture, we observed that the adhesion of P. aeruginosa to migrating cells of the edges of the repairing wounds was significantly higher than the adhesion to non-migrating cells and that adherent bacteria were surrounded by a fibrocnectin-containing fibrillar material The secretion of extracellular matrix components is involved in the process of epithelium repair following injury. To investigate the molecular basis of P. aeruginosa adhesion to migrating cells, bacteria were treated with a fibronectin solution before their incubation with the respiratory cells. P. aeruginosa treatment by fibronectin significantly increased their adhesion to migrating cells. Accordingly, we hypothesize that during cell migration, fibronectin secreted by epithelial cells may favour P. aeruginosa adhesion by establishing a bridge between the bacteria and the epithelial cell receptors. Such a mechanism may represent a critical step for P. aeruginosa infection of healing injured epithelium

Asunto(s)

Adhesión Bacteriana , Cilios/parasitología , Fibronectinas , Pseudomonas aeruginosa/patogenicidad , Sistema Respiratorio/parasitología