RESUMO
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.
Assuntos
Brônquios/microbiologia , Pseudomonas aeruginosa/fisiologia , Brônquios/citologia , Brônquios/ultraestrutura , Diferenciação Celular , Polaridade Celular , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Imunofluorescência , Humanos , Microscopia EletrônicaRESUMO
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.
Assuntos
Aderência Bacteriana/fisiologia , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA , Endotélio Vascular/microbiologia , Fímbrias Bacterianas/fisiologia , Pseudomonas aeruginosa/fisiologia , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico/efeitos dos fármacos , Divisão Celular , Células Cultivadas , Citocalasina D/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Proteínas de Escherichia coli , Proteínas de Fímbrias , Fímbrias Bacterianas/genética , Genes Bacterianos/genética , Humanos , L-Lactato Desidrogenase/análise , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/ultraestrutura , RNA Polimerase Sigma 54 , Fator sigma/genética , Veias Umbilicais/citologiaRESUMO
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.