RESUMEN
Ruffles are specialized plasma membrane ultrastructures of mammalian cells though to be integral to growth, development and locomotion. Induced by growth factors, mitogens or oncogene expression, ruffles are sites of filamentous actin rearrangement and are temporally associated with enhanced pinocytosis. But the function of ruffles, their mechanism of induction and their role in pinocytosis are not understood. We have observed formation of structures resembling ruffles associated with the site of entry of invasive Salmonella typhimurium. Here we report that ruffles elicited by invasive Salmonella directly mediate internalization of non-invasive bacteria in a macropinocytotic fashion, a phenomenon we term 'passive entry'. Furthermore, ruffles induced in the absence of Salmonella also facilitate passive entry. We present evidence that ruffles, common to many signalling events, comprise the macropinocytotic machinery mediating pinocytosis and are subverted by Salmonella so as to enter mammalian cells.
Asunto(s)
Membrana Celular/ultraestructura , Pinocitosis/fisiología , Salmonella typhimurium/fisiología , Animales , Adhesión Bacteriana/fisiología , Membrana Celular/microbiología , Membrana Celular/fisiología , Receptores ErbB/fisiología , Escherichia coli/fisiología , Humanos , Ratas , Salmonella typhimurium/patogenicidad , Células Tumorales Cultivadas , Yersinia pseudotuberculosis/fisiologíaRESUMEN
Salmonella typhimurium grown under oxygen-limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp-2 cells within 15 min after infection. Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence. Polarized Caco-2 cell morphology was affected 40 s after infection with low-oxygen-grown S. typhimurium. Stationary-phase S. typhimurium did not elicit these phenomena within this time-period even when adherence was enhanced with the afimbial adhesin, AFA-I. Thus, environmental cues regulate S. typhimurium invasion factors, allowing for immediate entry into host cells. Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.
Asunto(s)
Actinas/metabolismo , Adhesión Bacteriana/fisiología , Epitelio/microbiología , Oxígeno/fisiología , Salmonella typhimurium/patogenicidad , Adhesinas de Escherichia coli , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Línea Celular , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Perros , Células Epiteliales , Epitelio/ultraestructura , Humanos , Microscopía , Salmonella typhimurium/crecimiento & desarrollo , Células Tumorales CultivadasRESUMEN
Previous studies have identified two bacterial factors involved in enteropathogenic Escherichia coli (EPEC) infection. A plasmid-mediated EPEC adherence factor (EAF) is responsible for initial and localized adherence. A chromosomally encoded E. coli attachment and effacement factor (eae) is involved in effacement of the eukaryotic cell surface and characteristic "pedestal" formation. By using isogenic strains deficient in either EAF, eae, or both, the process of EPEC adherence and entry in vitro was examined. While EAF proved necessary and sufficient for efficient bacterial association with HEp-2 cells, both EAF and eae were required for efficient effacement of and entry into these cells and other cultured cell lines. Invasion mediated by eae was markedly inhibited by cytochalasin D and colchicine. Afimbrial adhesion or type I pili from uropathogenic strains of E. coli substituted for EAF in EAF-Eae+ strains to provide initial adherence to HEp-2 cells and to facilitate actin condensation.
Asunto(s)
Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Adhesinas de Escherichia coli , Adhesión Bacteriana/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/genética , Carcinoma de Células Escamosas , Línea Celular , Supervivencia Celular , Colchicina/farmacología , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Escherichia coli/genética , Humanos , Neoplasias Laríngeas , Microscopía Electrónica , Microvellosidades/ultraestructura , Plásmidos , Células Tumorales CultivadasRESUMEN
In Escherichia coli, the outer membrane protein FepA is a receptor for the siderophore complex ferric enterobactin and for colicins B and D. To identify protein domains important for FepA activity, the effects of deletion and linker insertion mutations on receptor structure and function were examined. In-frame internal deletion mutations removing sequences encoding up to 304 amino acid residues resulted in functionally defective FepA polypeptides, although most were translocated efficiently to the outer membrane. One exception, a derivative lacking 87 internal amino acid residues near the N terminus, showed an inability to transport ferric enterobactin but retained limited colicin receptor function. Analysis of cells carrying 3'-terminal fepA deletion mutations suggested that residues within the C terminus of FepA may be involved in secretion and proper translocation of the protein to the outer membrane. Introduction of the peptide Leu-Glu after FepA residues 55, 142, or 324 severely impaired receptor function for all three ligands, while the same insertion after residues 339 or 359 had virtually no detrimental effect on FepA function. Foreign peptides inserted after residues 204 or 635 restricted colicin B and D function only, leaving ferric enterobactin transport ability at near wild-type levels. The results presented in this study have identified key regions of FepA potentially involved in receptor function and demonstrate the presence of both shared and unique ligand-responsive domains.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/genética , Escherichia coli/genética , Receptores de Superficie Celular , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Secuencia de Bases , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Deleción Cromosómica , Mapeo Cromosómico , Colicinas/farmacología , Electroforesis en Gel de Poliacrilamida , Escherichia coli/efectos de los fármacos , Genes Bacterianos , Hierro/metabolismo , Cinética , Datos de Secuencia Molecular , Mutación , Sondas de Oligonucleótidos , Plásmidos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.