Adherence of Pseudomonas aeruginosa to human buccal epithelial cells

The aim of this study was to evaluate adherence of 83 strains of Pseudomonas aeruginosa isolated from humans and different animals to trypsin-treated buccal cells. We have demonstrated that Pseudomonas aeruginosa attached to trypsin-treated buccal cells in far greater numbers than to cells from controls (normal buccal epithelial cells). The mean number of bacteria adhering to trypsin-treated cells amounted 107.05 +/- 102.16 and to normal cells - 6.97 +/- 3.53. We conclude that exposure of cells to proteolytic enzymes increases Pseudomonas aeruginosa binding to buccal cells.     

The binding of Pseudomonas aeruginosa outer membrane ghosts to human buccal epithelial cells

The binding of outer membrane (OM) ghosts derived from Pseudomonas aeruginosa strain 492c to human buccal epithelial cells (BECs) was examined. Electron microscopic examination of the binding of OM ghosts to BECs revealed direct OM ghost-BEC interaction. Equilibrium analysis of the binding of OM ghosts to trypsinized BECs employing the Langmuir adsorption isotherm indicated the number of binding sites (N) to be 1.3 X 10(-4) micrograms protein per BEC with an apparent association constant (Ka) of 3.4 X 10(-2) mL/microgram protein. The Langmuir analysis of binding of OM ghosts to untrypsinized BECs was complex, suggesting two possible classes of receptors, a high affinity-low copy number class (Ka, 7.8 X 10(-2)mL/microgram protein; N, 8.6 X 10(-5) microgram protein per BEC) and a low affinity-high copy number class (Ka, 3.7 X 10(-3)mL/microgram protein; N, 9.2 X 10(-4)microgram protein per BEC). Sugar inhibition studies incorporating D-galactose enhanced binding to each BEC type. N-Acetylneuraminic acid and N-acetylglucosamine both enhanced binding of OM ghosts to untrypsinized BECs, while inhibiting binding to trypsinized BECs. D-Arabinose inhibited binding to both BEC types. Binding of OM ghosts to both BEC types was greatly inhibited by D-fucose, while L-fucose only greatly inhibited binding to untrypsinized BECs. These sugar inhibition data demonstrated a difference in the binding of OM ghosts to trypsinized and untrypsinized BECs and possibly reveal the nature of the receptor(s), free of possible bacterial metabolic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adhesion of Pseudomonas aeruginosa to human buccal epithelial cells: evidence for two classes of receptors

The adhesion of Pseudomonas aeruginosa strain 492c to trypsinized and untrypsinized buccal epithelial cells (BECs) was studied. Kinetic analysis of the adhesion data, employing a Langmuir absorption isotherm, indicated the presence of two classes of binding sites on untrypsinized BECs: a high affinity - low copy number site (apparent association constant (Ka) approximately equal to 1.57 X 10(-8) mL/cell with ca. 29 binding sites/cell) and a low affinity - high copy number class of binding sites (Ka approximately equal to 4.78 X 10(-10) mL/cell with ca. 264 binding sites/cell). The low affinity - high copy number class of sites was found to be trypsin sensitive. A single class of binding sites was found on trypsinized BECs exhibiting a high affinity - low copy number (Ka approximately equal to 3.70 X 10(-7) mL/cell with ca. 31 binding sites/cell). Positive cooperativity in binding of P. aeruginosa strain 492c to the low affinity - high copy number class site on untrypsinized BECs was demonstrated by analysis of Hill plots of the adhesion data. Sugar inhibition data using a preincubation methodology showed an inhibition of adhesion to trypsinized BECs in the presence of N-acetylneuraminic acid and D-arabinose, while these same two sugars enhanced adhesion to untrypsinized BECs. D-Galactose and N-acetylglucosamine enhanced adhesion to both types of BECs though the latter did to different extents. D-Fucose only inhibited adhesion to untrypsinized BECs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Candida adhesion to buccal epithelial cells by an aqueous extract of Allium sativum (garlic)

The effect of pre-incubation of either Candida or buccal epithelial cells (BEC) with different concentrations of aqueous garlic extract (AGE) was investigated, as well as the effect of mouth rinse with AGE on the adhesion of yeast to BEC. Adhesion of Candida spp. to BEC was significantly reduced after both short and long time exposure of yeast to AGE. A similar inhibition of adherence was observed upon pre-incubation of BEC with AGE. The adherence-inhibition activity of AGE treatment was antagonized by thiols such as l-cysteine, glutathione and 2-mercaptoethanol. In addition, germ-tube formation was suppressed when C. albicans cells were pretreated with AGE. There was a significant reduction in the adherence of yeasts to BEC collected immmediately or 15 min after an oral rinse with AGE. No statistical significance in the adhesion of BEC collected 30 min after oral rinse with AGE and control BEC was observed. The diminished adherence of C. albicans to BEC after exposure to various concentrations of garlic may have clinical relevance.     

Inhibition of Candida adhesion to buccal epithelial cells by an aqueous extract of Allium sativum (garlic)

The effect of pre-incubation of either Candida or buccal epithelial cells (BEC) with different concentrations of aqueous garlic extract (AGE) was investigated, as well as the effect of mouth rinse with AGE on the adhesion of yeast to BEC. Adhesion of Candida spp. to BEC was significantly reduced after both short and long time exposure of yeast to AGE. A similar inhibition of adherence was observed upon preincubation of BEC with AGE. The adherence-inhibition activity of AGE treatment was antagonized by thiols such as L-cysteine, glutathione and 2-mercaptoethanol. In addition, germ-tube formation was suppressed when C. albicans cells were pretreated with AGE. There was a significant reduction in the adherence of yeasts to BEC collected immediately or 15 min after an oral rinse with AGE. No statistical significance in the adhesion of BEC collected 30 min after oral rinse with AGE and control BEC was observed. The diminished adherence of C. albicans to BEC after exposure to various concentrations of garlic may have clinical relevance.     

口腔细菌对铜绿假单胞菌黏附及侵入肺上皮细胞能力的影响

目的 通过比较铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入细胞的能力,探讨细菌间相互作用在呼吸道感染的最初阶段的作用机制.方法 应用培养法和抗生素保护法检测铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入肺上皮细胞的能力.结果 铜绿假单胞菌与口腔细菌共同作用于肺上皮细胞,牙龈卟啉单胞菌和伴放线放线杆菌降低了铜绿假单胞菌的黏附能力,却增强了其侵入能力;而铜绿假单胞菌能够影响口腔细菌对肺上皮细胞的黏附,同时增强口腔细菌侵入肺上皮细胞的能力.结论 口腔细菌,尤其是牙周可疑致病菌主要通过增强铜绿假单胞菌对肺上皮细胞的侵入而影响呼吸道感染过程.     

Development of a high throughput Pseudomonas aeruginosa epithelial cell adhesion assay

Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis and mechanically ventilated patients by binding to specific carbohydrate residues on the surface of lung epithelial cells. Studies have shown that blocking this interaction may have therapeutic effects in vivo. To test compounds that may have an effect on the binding of P. aeruginosa to epithelial cells, we have developed a pseudomonal adhesion assay that is compatible with high throughput technology. This assay utilizes a 96-well culture plate assay and P. aeruginosa strains that have been modified to bioluminesce. This method has proven to be a rapid, sensitive and reproducible system for screening agents that inhibit bacterial adhesion.     

Sulfatide mediates attachment of Pseudomonas aeruginosa to human pharyngeal epithelial cells

Pseudomonas aeruginosa infections are particularly common in people with cystic fibrosis and despite regular treatment with antibiotics, lung damage due to chronic infection with P. aeruginosa remains the major cause of death in those patients. In order to initiate an infection, P. aeruginosa needs contact with the respiratory epithelial surface and by means of its adhesins i.e., fimbria, hemagglutinins,etc., it recognizes and adheres to the corresponding epithelial receptors. We treated P. aeruginosa strains isolated from sputum of cystic fibrosis patients with several glycolipids such as sulfatide, sulfated ganglioside mixture (GM1a, GD1b, GT1b), asialo-GM1 and galactocerebrosides to determine their effect on attachment with pharyngeal epithelial cells. Sulfated ganglioside mixture and sulfatide inhibited the attachment of P. aeruginosa significantly, whereas asialo-GM1, Gal-Cer and sodium sulfite had no effect on attachment inhibition. This finding suggests that sulfated glycoconjugates found in the extracellular matrix, in mucus and on the surface of epithelial cells of human trachea and lung mediates attachment of P. aeruginosa.     

Correlation between cell-surface hydrophobicity of Candida albicans and adhesion to buccal epithelial cells

Adhesion of four isolates of Candida albicans to buccal epithelial cells was determined after growth of the yeasts in defined medium containing 50 mM glucose or 500 mM galactose as the carbon source. With each isolate, adhesion of galactose-grown yeasts was significantly higher than that of glucose-grown organisms. Yeast cell-surface hydrophobicity was assessed by two methods, a modified hydrocarbon adhesion assay and a more sensitive polystyrene microsphere assay. All four isolates were significantly more hydrophobic after growth on 500 mM galactose than after growth on 50 mM glucose. Overall, a strong positive correlation between adhesion and surface hydrophobicity was observed (r = 0.965). These results are discussed in relation to the role of yeast-surface hydrophobicity in pathogenesis.     

Inhibition of Pseudomonas aeruginosa adhesion to fibronectin by PA-IL and monosaccharides: involvement of a lectin-like process

Pseudomonas aeruginosa adherence to fibronectin has been shown to be important to bacterial colonization and infection. To better understand the mechanisms involved in this interaction, the role of the carbohydrate moiety of the fibronectin molecule in P. aeruginosa adhesion was studied. Strain NK 125 502 adhered to immobilized fibronectin with an adherence index of 4.8 x 10(5) CFU/ micro g. Periodic oxidation of fibronectin markedly reduced the adhesion of P. aeruginosa, while a neuraminidase treatment increased bacteria adhesion. N-Acetylgalactosamine, N-acetylglucosamine, sialic acid, and also lectin PA-IL worked as efficient inhibitors in adhesion assays: 59%, 70.7%, 100%, and 60% of inhibition, respectively. We have demonstrated here the involvement of a lectin-like process in the interaction of P. aeruginosa NK 125 502 with immobilized fibronectin.     

Prevention of Pseudomonas aeruginosa adhesion by electric currents

The process of controlling bacterial adhesion using an electric current deserves attention because of its ease of automation and environmentally friendly nature. This study investigated the role of electric currents (negative, positive, alternating) for preventing adhesion of Pseudomonas aeruginosa and achieving bacterial inactivation. Indium tin oxide (ITO) film was used as a working electrode to observe adhesion and inactivation under electric polarization. Electric current types were classified into negative, positive, and alternating current. The working electrode acted as a cathode or anode by applying a negative or positive current, and an alternating current indicates that the negative current was combined sequentially with the positive current. The numbers of adhered cells were compared under a flow condition, and the in situ behavior of the bacterial cells and the extent of their inactivation were also investigated using time-lapse recording and live/dead staining, respectively. The application of a negative current prevented bacterial adhesion significantly (～81% at 15.0 μA cm(-2)). The positive current did not significantly inhibit adhesion (<20% at 15.0 μA cm(-2)), compared to the nonpolarized case. The alternating current had a similar effect as the negative current on preventing bacterial adhesion, but it also exhibited bactericidal effects, making it the most suitable method for bacterial adhesion control.     

Prevention of Pseudomonas aeruginosa adhesion by electric currents

The process of controlling bacterial adhesion using an electric current deserves attention because of its ease of automation and environmentally friendly nature. This study investigated the role of electric currents (negative, positive, alternating) for preventing adhesion of Pseudomonas aeruginosa and achieving bacterial inactivation. Indium tin oxide (ITO) film was used as a working electrode to observe adhesion and inactivation under electric polarization. Electric current types were classified into negative, positive, and alternating current. The working electrode acted as a cathode or anode by applying a negative or positive current, and an alternating current indicates that the negative current was combined sequentially with the positive current. The numbers of adhered cells were compared under a flow condition, and the in situ behavior of the bacterial cells and the extent of their inactivation were also investigated using time-lapse recording and live/dead staining, respectively. The application of a negative current prevented bacterial adhesion significantly (～81% at 15.0 μA cm^?2). The positive current did not significantly inhibit adhesion (<20% at 15.0 μA cm^?2), compared to the nonpolarized case. The alternating current had a similar effect as the negative current on preventing bacterial adhesion, but it also exhibited bactericidal effects, making it the most suitable method for bacterial adhesion control.     

Regulation of matrilysin expression in airway epithelial cells by Pseudomonas aeruginosa flagellin

Matrilysin (matrix metalloproteinase-7) is expressed by mucosal epithelia throughout the body and functions in host defense by activating murine intestinal alpha-defensins. In normal adult human lung, matrilysin is expressed at low levels in the airway epithelium, but is markedly up-regulated in cystic fibrosis (CF). Because CF lungs support a heavy bacterial load, we assessed if relevant CF pathogens regulate matrilysin expression in human lung epithelial cells. Indeed, acute infection with Pseudomonas aeruginosa (but not Staphylococcus aureus, Haemophilus influenzae, or Klebsiella pneumoniae) induced the expression of matrilysin in Calu-3 lung epithelial cells. Increased matrilysin mRNA levels were detectable at 3 h post-infection and peaked at a 25-fold induction between 6 and 8 h. Both P. aeruginosa CF isolates and laboratory strains induced matrilysin expression to similar levels. Flagellin, the monomeric precursor of bacterial flagella, was identified as the inductive factor released by P. aeruginosa that regulated matrilysin expression. In addition, flagellin-null mutants failed to stimulate matrilysin expression in cultured cells or in lungs infected in vivo. These data show that P. aeruginosa (and specifically flagellin) potently stimulates matrilysin expression in lung epithelial cells and may mediate the overexpression of this proteinase in CF lungs.     

Modulation of lung epithelial functions by Pseudomonas aeruginosa

Microorganisms gain access to the airways and respiratory epithelial surface during normal breathing. Most inhaled microbes are trapped on the mucous layer coating the nasal epithelium and upper respiratory tract, and are cleared by ciliary motion. Microorganisms reaching the alveolar spaces are deposited on the pulmonary epithelium. This contact initiates complex offensive and defensive strategies by both parties. Here, we briefly outline how the pulmonary pathogen Pseudomonas aeruginosa uses multi-pronged strategies that include cell surface appendages, and secreted and injected virulence determinants to switch from an unobtrusive soil bacterium to a pathogen for lung epithelium colonization. Understanding the complex interactions between the lung epithelium and P. aeruginosa might enable more effective therapeutic strategies against infection in cystic fibrosis and immuno-compromised individuals.     

Mapping the surface regions of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells

The adherence of non-mucoid Pseudomonas aeruginosa strains is believed to be mediated by the pilus, which consists of a single protein subunit of 15,000 Daltons called pilin. Ten antipeptide antisera were raised to map the surface regions of pilin from P. aeruginosa strain K (PAK). Only one of the antipeptide antisera to the eight predicted surface regions failed to react with PAK pili in direct ELISA. Five out of eight synthetic peptides representing the eight predicted surface regions reacted with anti-PAK pilus antiserum, indicating their surface exposure. Combining the antipeptide and antipilus antisera results, all eight predicted surface regions were demonstrated to be surface-exposed. The PAK 128-144-OH peptide produced the best binding antiserum to PAK pili. Only antipeptide Fab fragments directed against the disulphide bridged C-terminal region of PAK pilin blocked the adherence of pili to human buccal epithelial cells, which suggests that this region contains the receptor-binding domain of the PAK pilus.     

A beta-linked mannan inhibits adherence of Pseudomonas aeruginosa to human lung epithelial cells

Adherence through carbohydrate-binding adhesins is an earlystep in colonization of the lung by gram-negative organisms,and because published data indicate that binding involves mannosegroups, we tested the ability of a ß-linked acetylmannan(acemannan) to inhibit adherence of Pseudomonus aeruginosa tocultures of human lung epithelial cells. Adherence of radiolabelledP.aeruginosa to A549 cells (a type II-like pneurnocyte line)increased linearly with the duration of the incubation. Acemannaninhibited adherence of bacteria, and the extent of inhibitionwas related to the concentration of the mannan. Inhibition requiredcontinued contact between acemannan and the target epithelialcells; cells washed free of acemannan no longer discouragedbacterial binding. Comparison of binding between seven strainsof P.aeruginosa indicated that fewer mucoid than non-mucoidbacteria adhered, but binding of either phenotype was inhibitedby acemannan. Mannose methyl -D-mannopyranoside, methyl ß-D-mannopyrannosideand dextran did not affect adherence of any of the non-mucoidstrains. Mannose inhibited adherence by one mucoid strain, butnot the other, indicating differences between strains of thesame phenotype. Since prior treatment of epithelial cells withconcanavalin A did not affect acemannan-induced inhibition ofbacterial adherence, we concluded that the inhibitory effectof acemannan probably does not involve mannose-containing receptors. bacterial-host interactions lung epithelium mucoid strains non-mucoid strains     

Human buccal epithelial cell receptors of Pseudomonas aeruginosa: identification of glycoproteins with pilus binding activity

Adherence of Pseudomonas aeruginosa to a patient's epithelial surface is thought to be an important first step in the infection process. Pseudomonas aeruginosa is capable of attaching to epithelial cells via its pili, yet little is known about the epithelial receptors of this adhesin. Using nitrocellulose replicas of polyacrylamide gels of solubilized human buccal epithelial cells (BECs), glycoproteins (Mz: 82,000, and four bands between 40,000 and 50,000) that bound purified pili from P. aeruginosa strain K (PAK) were identified by immunoblotting with a pilus-specific monoclonal antibody that does not affect pilus binding to BECs (PK3B). All pilus-binding glycoproteins were surface localized, as determined by surface radioiodination of intact BECs. Binding of pili to all of the glycoproteins was inhibited by Fab fragments of monoclonal antibody PK99H, which inhibits PAK pili binding to BECs by binding to or near the binding domain of the pilus, but not by Fab fragments of monoclonal antibody PK41C, which binds to PAK pilin but does not inhibit pili binding to BECs, demonstrating that pilus binding to these glycoproteins is likely via the same region of the pilus that binds to intact BECs. Periodate oxidation of the blot eliminated pili binding to all glycoproteins, indicating that a carbohydrate moiety is an important determinant for pilus-binding activity. However, not all of the glycoproteins exhibited the same degree of sensitivity to periodate oxidation. Furthermore, monosaccharide inhibition of pilus binding to BECs implicated L-fucose and N-acetylneuraminic acid as receptor moieties.     

Pseudomonas aeruginosa internalization by corneal epithelial cells involves MEK and ERK signal transduction proteins

Invasion of epithelial cells represents a potential pathogenic mechanism for Pseudomonas aeruginosa. We explored the role of mitogen-activated protein kinase kinases (MEK 1/2) and the extracellular signal-regulated kinases (ERK 1/2) in P. aeruginosa invasion. Treatment of corneal epithelial cells with MEK inhibitors, PD98059 (20 microM) or UO126 (100 microM), reduced P. aeruginosa invasion by approximately 60% without affecting bacterial association with the cells (P=0.0001). UO124, a negative control for UO126, had no effect on bacterial internalization. Infection of cells with an internalization-defective flhA mutant of P. aeruginosa was associated with less ERK 1/2 tyrosine phosphorylation than infection with wild-type invasive P. aeruginosa. An ERK-2 inhibitor, 5-iodotubercidin (20 microM), reduced P. aeruginosa invasion by approximately 40% (P=0.035). Together, these data suggest that P. aeruginosa internalization by epithelial cells involves a pathway(s) that includes MEK and ERK signaling proteins.