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

1986 ◽  
Vol 32 (2) ◽  
pp. 160-166 ◽  
Author(s):  
P. Doig ◽  
A. L. Franklin ◽  
R. T. Irvin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Copy Number ◽  
Electron Microscopic Examination ◽  
Equilibrium Analysis ◽  
Metabolic Effects ◽  
Electron Microscopic ◽  
Buccal Epithelial Cells ◽  
Number Class

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 (iV) to be 1.3 × 10−1 μg protein per BEC with an apparent association constant (Ka) of 3.4 × 10−2 mL/μg 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, 1.8 × 10−2 mL/μg protein; N, 8.6 × 10−5 μg protein per BEC) and a low affinity – high copy number class(Afa, 3.7 × 10−3 mL/μg protein; N, 9.2 × 10−4 μg protein per BEC). Sugar inhibition studies incorporating D-galactose enhanced binding to each BEC type. N-Acetylneuraminic acid and N-acetyl-glucosamine 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. These data indicated that OM ghosts from 492c appear to bind to BECs in a similar manner to the intact bacteria and represent a simple model system to study the adhesion of P. aeruginosa to BECs.

Equilibrium analysis of binding of Candida albicans to human buccal epithelial cells

1990 ◽  
Vol 36 (5) ◽  
pp. 336-340 ◽  
Author(s):  
William Staddon ◽  
Tom Todd ◽  
Randall T. Irvin
Keyword(s):  
Candida Albicans ◽  
Epithelial Cells ◽  
Copy Number ◽  
Incubation Temperature ◽  
Temperature Shift ◽  
Equilibrium Analysis ◽  
High Copy Number ◽  
Buccal Epithelial Cells ◽  
Low Copy Number ◽  
Receptor Interactions

The effect of growth temperature on the binding of Candida albicans to human buccal epithelial cells (BECs) was examined using an equilibrium of binding analysis. Candida albicans was cultured in M9 medium either for 12 h at 25 °C or for 9 h at 25 °C and then shifted to 37 °C for 3 h. The temperature shift did not result in germ tube formation; however, the adherence of C. albicans to BECs was altered. Shifting temperature increased the yeast's ability to bind to BECs. A Langmuir adsorption isotherm was used to calculate the maximum number of available binding sites (N) and the apparent association constants of binding (Ka) for all resolvable adhesin–receptor interactions. Three classes of adhesin–receptor interactions were resolved when the yeast was cultured at 25 °C and included a low copy number site (N = 3.0 cfu/BEC; Ka = 2.11 × 10−6 mL/cfu), a medium copy number site (N = 23.6 cfu/BEC, Ka = 8.21 × 10−7 mL/cfu), and a high copy number site (N = 91.7 cfu/BEC, Ka = 3.35 × 10−8 mL/cfu). Two classes of adhesin–receptor interactions were resolved when the incubation temperature was shifted to 37 °C: a low copy number site (N = 4.5 cfu/BEC, Ka = 3.98 × 10−6 mL/cfu) and a high copy number site (N = 150.5 cfu/BEC, Ka = 8.47 × 10−8 mL/cfu). Augmented C. albicans adherence to BECs due to the elevated growth temperatures appears to result from a temperature-regulated alteration in the C. albicans adhesin that recognizes a high copy number receptor site with relatively low affinity.

Molecular Structure of the Outermost Cell Wall Component of Spirillum Serpens

1977 ◽  
Vol 35 ◽  
pp. 342-343
Author(s):  
Wah Chiu ◽  
David Grano
Keyword(s):  
Molecular Structure ◽  
Cell Wall ◽  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Cell Wall Component ◽  
Protein Components ◽  
Exposure Technique ◽  
Structural Aspects

The periodic structure external to the outer membrane of Spirillum serpens VHA has been isolated by similar procedures to those used by Buckmire and Murray (1). From SDS gel electrophoresis, we have found that the isolated fragments contain several protein components, and that the crystalline structure is composed of a glycoprotein component with a molecular weight of ∽ 140,000 daltons (2). Under an electron microscopic examination, we have visualized the hexagonally-packed glycoprotein subunits, as well as the bilayer profile of the outer membrane. In this paper, we will discuss some structural aspects of the crystalline glycoproteins, based on computer-reconstructed images of the external cell wall fragments.The specimens were prepared for electron microscopy in two ways: negatively stained with 1% PTA, and maintained in a frozen-hydrated state (3). The micrographs were taken with a JEM-100B electron microscope with a field emission gun. The minimum exposure technique was essential for imaging the frozen- hydrated specimens.

Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells

1980 ◽  
Vol 29 (3) ◽  
pp. 1146-1151 ◽  
Author(s):  
D E Woods ◽  
D C Straus ◽  
W G Johanson ◽  
V K Berry ◽  
J A Bass
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Opportunistic Pathogen ◽  
In Vitro System ◽  
Heterologous Antiserum ◽  
Buccal Epithelial Cells ◽  
Serological Studies ◽  
Seriously Ill Patients

Adherence of Pseudomonas aeruginosa organisms to the upper respiratory epithelium of seriously ill patients in vitro is correlated with subsequent colonization of the respiratory tract by this opportunistic pathogen. The role of pili in the attachment to epithelial cells of P. aeruginosa was studied in an in vitro system employing human buccal epithelial cells and P. aeruginosa pretreated by various means. Pretreatment of the bacteria with proteases, heat, or Formalin caused a significant decrease in adherence. A decrease when compared with controls was also noted in the adherence of P. aeruginosa organisms to buccal epithelial cells preincubated with purified pili prepared from the strain used for adherence testing; however, pili prepared from a heterologous strain failed to block adherence. Similar results were obtained in serological studies when antisera to purified pili prepared from the strain used for adherence testing decreased adherence, whereas heterologous antiserum to pili did not decrease adherence. From these results it appears that pili mediate the adherence of P. aeruginosa organisms to human buccal epithelial cells.

Factors Influencing the Adherence of Pseudomonas aeruginosa to Mammalian Buccal Epithelial Cells

1983 ◽  
Vol 5 (Supplement_5) ◽  
pp. S846-S851 ◽  
Author(s):  
Donald E. Woods ◽  
David C. Straus ◽  
W. G. Johanson ◽  
Joe A. Bass
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Factors Influencing ◽  
Buccal Epithelial Cells

scholarly journals Cyclodextrins reduce the ability of Pseudomonas aeruginosa outer-membrane vesicles to reduce CFTR Cl− secretion

2019 ◽  
Vol 316 (1) ◽  
pp. L206-L215 ◽  
Author(s):  
Roxanna Barnaby ◽  
Katja Koeppen ◽  
Bruce A. Stanton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Airway Epithelial Cells ◽  
Outer Membrane Vesicles ◽  
Airway Epithelial ◽  
Ussing Chambers ◽  
Planktonic Growth ◽  
Apical Side

Pseudomonas aeruginosa secretes outer-membrane vesicles (OMVs) that fuse with cholesterol-rich lipid rafts in the apical membrane of airway epithelial cells and decrease wt-CFTR Cl− secretion. Herein, we tested the hypothesis that a reduction of the cholesterol content of CF human airway epithelial cells by cyclodextrins reduces the inhibitory effect of OMVs on VX-809 (lumacaftor)-stimulated Phe508del CFTR Cl− secretion. Primary CF bronchial epithelial cells and CFBE cells were treated with vehicle, hydroxypropyl-β-cyclodextrin (HPβCD), or methyl-β-cyclodextrin (MβCD), and the effects of OMVs secreted by P. aeruginosa on VX-809 stimulated Phe508del CFTR Cl− secretion were measured in Ussing chambers. Neither HPβCD nor MβCD were cytotoxic, and neither altered Phe508del CFTR Cl− secretion. Both cyclodextrins reduced OMV inhibition of VX-809-stimulated Phe508del-CFTR Cl− secretion when added to the apical side of CF monolayers. Both cyclodextrins also reduced the ability of P. aeruginosa to form biofilms and suppressed planktonic growth of P. aeruginosa. Our data suggest that HPβCD, which is in clinical trials for Niemann-Pick Type C disease, and MβCD, which has been approved by the U.S. Food and Drug Administration for use in solubilizing lipophilic drugs, may enhance the clinical efficacy of VX-809 in CF patients when added to the apical side of airway epithelial cells, and reduce planktonic growth and biofilm formation by P. aeruginosa. Both effects would be beneficial to CF patients.

Analysis and modelling of Pseudomonas aeruginosa adherence to human buccal epithelial cells

1997 ◽  
Vol 8 (4-5) ◽  
pp. 267-277
Author(s):  
Anne M.C. Cerf ◽  
Jean-Paul Dehaye ◽  
Michel J. Devleeschouwer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Buccal Epithelial Cells

Alterations in the formation of lipopolysaccharide and membrane vesicles on the surface of Pseudomonas aeruginosa PAO1 under oxygen stress conditions

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2789-2795 ◽  
Author(s):  
W. Sabra ◽  
H. Lünsdorf ◽  
A.-P. Zeng
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Vesicles ◽  
Electron Microscopic Examination ◽  
Surface Characteristics ◽  
Opportunistic Pathogen ◽  
Stress Conditions ◽  
Electron Microscopic ◽  
Oxygen Stress ◽  
Relative Expression ◽  
Hydrophilic Surfaces

It has been postulated that phenotypic variation in the relative expression of two chemically distinct types of lipopolysaccharide (LPS), a serotype-specific LPS (B-band) and a common antigen LPS (A-band) in Pseudomonas aeruginosa is an important mechanism enabling this opportunistic pathogen to alter its surface characteristics to mediate adhesion and to survive under extreme conditions. To further investigate this, the relative expression levels of the two distinct types of LPS in P. aeruginosa PAO1 were investigated with cells grown in a chemostat at different dissolved oxygen tensions (pO2). The A-band LPS was constitutively expressed as pO2 was increased from nearly zero to 350 % of air saturation. In contrast, the B-band LPS showed a remarkable increase with increased pO2. Almost no B-band LPS was found in cells grown at a pO2 of less than 3 % of air saturation. Electron microscopic examination of cells revealed increased formation of membrane vesicles (MVs) on the surface of P. aeruginosa PAO1 under oxygen stress conditions. The toxicity of the supernatant of P. aeruginosa cultures to the growth of a hybridoma cell line significantly increased in samples taken from oxygen-stressed steady-state cultures. Furthermore, studies of adhesion in a continuous-flow biofilm culture revealed an increased adhesiveness for hydrophilic surfaces in P. aeruginosa PAO1 grown at a higher pO2. The oxygen-dependent alterations of cell-surface components and properties observed in this work provide a possible explanation for the emergence of P. aeruginosa lacking the B-band LPS in chronically infected cystic fibrosis patients. The results are also useful for understanding the processes involved in the formation of MVs in P. aeruginosa.

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

1985 ◽  
Vol 31 (6) ◽  
pp. 563-569 ◽  
Author(s):  
David W. McEachran ◽  
Randall T. Irvin
Keyword(s):  
Binding Sites ◽  
Copy Number ◽  
Alginic Acid ◽  
High Copy Number ◽  
Acetylneuraminic Acid ◽  
High Affinity ◽  
Buccal Epithelial Cells ◽  
Low Copy Number ◽  
Inhibition Data ◽  
Number Class

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 ≈ 1.57 × 10−8 mL/cell with ca. 29 binding sites/cell) and a low affinity – high copy number class of binding sites (Ka ≈ 4.78 × 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 ≈ 3.70 × 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. Without preincubation the sugar inhibition data indicated that N-acetylglucosamine had no effect on adhesion while N-acetylneuraminic acid and D-fucose both enhanced adhesion to both types of BECs. D-Arabinose had a slight inhibitory effect on adhesion, while D-galactose had a slight enhancing effect to both cell types, but to different levels. This sugar data suggests a difference in the receptors on the two types of BECs, but the possibility of metabolism of these sugars by P. aeruginosa requires one to interpret this data with caution. Flagella were shown not to be involved in adhesion, while the alginic acid of the capsule was implicated. The low copy number binding site is speculated to be a pili-binding site, while the high copy number class of binding sites is proposed to involve a lectin which binds alginic acid.

ParA proteins of secondary genome elements cross-talk and regulate radioresistance through genome copy number reduction in Deinococcus radiodurans

2019 ◽  
Vol 476 (5) ◽  
pp. 909-930 ◽  
Author(s):  
Ganesh Kumar Maurya ◽  
Swathi Kota ◽  
N. Naveen Kumar ◽  
Raghvendra Tewari ◽  
Hari S. Misra
Keyword(s):  
Copy Number ◽  
Deinococcus Radiodurans ◽  
Bacterial Genome ◽  
Electron Microscopic Examination ◽  
Interaction Patterns ◽  
Electron Microscopic ◽  
Heterotypic Interactions ◽  
Chromosome Ii ◽  
Chromosome I

Abstract Deinococcus radiodurans, an extremely radioresistant bacterium has a multipartite genome system and ploidy. Mechanisms underlying such types of bacterial genome maintenance and its role in extraordinary radioresistance are not known in this bacterium. Chromosome I (Chr I), chromosome II (Chr II) and megaplasmid (Mp) encode its own set of genome partitioning proteins. Here, we have characterized P-loop ATPases of Chr II (ParA2) and Mp (ParA3) and their roles in the maintenance of genome copies and extraordinary radioresistance. Purified ParA2 and ParA3 showed nearly similar polymerization kinetics and interaction patterns with DNA. Electron microscopic examination of purified proteins incubated with DNA showed polymerization on nicked circular dsDNA. ParA2 and ParA3 showed both homotypic and heterotypic interactions to each other, but not with ParA1 (ParA of Chr I). Similarly, ParA2 and ParA3 interacted with ParB2 and ParB3 but not with ParB1 in vivo. ParB2 and ParB3 interaction with cis-elements located upstream to the corresponding parAB operon was found to be sequence-specific. Unlike single mutant of parA2 and parA3, their double mutant (ΔparA2ΔParA3) affected copy number of cognate genome elements and resistance to γ-radiation as well as hydrogen peroxide in this bacterium. These results suggested that ParA2 and ParA3 are DNA-binding ATPases producing higher order polymers on DNA and are functionally redundant in the maintenance of secondary genome elements in D. radiodurans. The findings also suggest the involvement of secondary genome elements such as Chr II and Mp in the extraordinary radioresistance of D. radiodurans.

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