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.

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.

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.

scholarly journals The structure of siglec-7 in complex with sialosides: leads for rational structure-based inhibitor design

2006 ◽  
Vol 397 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Helen Attrill ◽  
Hirokazu Takazawa ◽  
Simone Witt ◽  
Soerge Kelm ◽  
Rainer Isecke ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Binding Sites ◽  
Cellular Interactions ◽  
Inhibitor Design ◽  
Binding Assays ◽  
Rational Structure ◽  
Transmembrane Receptors ◽  
Acetylneuraminic Acid ◽  
Sialic Acid Binding ◽  
Inhibition Data

Siglecs (sialic acid binding Ig-like lectins) are transmembrane receptors for sialylated glycoconjugates that modulate cellular interactions and signalling events in the haematopoietic, immune and nervous systems. Siglec-7 is a structural prototype for the recently described family of immune inhibitory CD33-related siglecs and is predominantly expressed on natural killer cells and monocytes, as well as subsets of CD8 T-cells. Siglec-specific inhibitors are desired for the detection of masked and unmasked forms of siglecs, to aid in dissection of signalling pathways and as tools to investigate siglecs as potential therapeutic targets. As a first step towards this end, we present the crystal structure of siglec-7 in complex with a sialylated ligand, the ganglioside analogue DSLc4 [α(2,3)/α(2,6) disialyl lactotetraosyl 2-(trimethylsilyl)ethyl], which allows for a detailed description of the binding site, required for structure-guided inhibitor design. Mutagenesis and binding assays were used to demonstrate a key structural role for Lys131, a residue that changes conformation upon sialic acid binding. Differences between the binding sites of siglec family members were then exploited using α-methyl Neu5Ac (N-acetylneuraminic acid) as a basic scaffold. A co-crystal of siglec-7 in complex with the sialoside inhibitor, oxamido-Neu5Ac [methyl α-9-(amino-oxalyl-amino)-9-deoxy-Neu5Ac] and inhibition data for the sialosides gives clear leads for future inhibitor design.

Expression of a Bacillus thuringiensis δ-endotoxin gene by Bacillus pumilus

1998 ◽  
Vol 44 (3) ◽  
pp. 259-269 ◽  
Author(s):  
L B Selinger ◽  
G G Khachatourians ◽  
J R Byers ◽  
M F Hynes
Keyword(s):  
Bacillus Thuringiensis ◽  
Copy Number ◽  
Bacillus Pumilus ◽  
High Copy Number ◽  
Cry Genes ◽  
Bacillus Thuringiensis Subsp ◽  
High Copy Number Plasmid ◽  
Low Copy Number ◽  
High Level ◽  
Lepidoptera Noctuidae

The δ -endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a δ -endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and δ -endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that <0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal inclusions. On low copy number vectors, the cry genes did not inhibit sporulation; however, production of δ -endotoxins was undetectable. Using a heat shock regime for enrichment of sporogenous crystalliferous variants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid, was obtained in which high level δ -endotoxin production occurred concomitant with sporulation. Synthesis of functional δ -endotoxin by this strain was confirmed by Western blot analysis and bioassay with pale western cutworm larvae. These results show that rhizosphere-inhabiting bacilli are indeed a potential route for introduction of δ -endotoxins to the root environment for biocontrol purposes.Key words: Bacillus thuringiensis, δ -endotoxin, conjugation, sporulation, expression.

High-copy-number and low-copy-number plasmid vectors for lacZα-complementation and chloramphenicol- or kanamycin-resistance selection

Gene ◽  
1987 ◽  
Vol 61 (1) ◽  
pp. 63-74 ◽  
Author(s):  
Sunao Takeshita ◽  
Masahiro Sato ◽  
Mari Toba ◽  
Wakako Masahashi ◽  
Tamotsu Hashimoto-Gotoh
Keyword(s):  
Copy Number ◽  
Kanamycin Resistance ◽  
High Copy Number ◽  
Plasmid Vectors ◽  
Resistance Selection ◽  
Copy Number Plasmid ◽  
Low Copy Number

scholarly journals Hyperproduction of Tryptophan byCorynebacterium glutamicum with the Modified Pentose Phosphate Pathway

1999 ◽  
Vol 65 (6) ◽  
pp. 2497-2502 ◽  
Author(s):  
Masato Ikeda ◽  
Ryoichi Katsumata
Keyword(s):  
Late Stage ◽  
Copy Number ◽  
Batch Cultivation ◽  
High Copy Number ◽  
Fed Batch ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Aromatic Biosynthesis ◽  
Fed Batch Cultivation ◽  
Engineered Strain

ABSTRACT A classically derived tryptophan-producing Corynebacterium glutamicum strain was recently significantly improved both by plasmid-mediated amplification of the genes for the rate-limiting enzymes in the terminal pathways and by construction of a plasmid stabilization system so that it produced more tryptophan. This engineered strain, KY9218 carrying pKW9901, produced 50 g of tryptophan per liter from sucrose after 80 h in fed-batch cultivation without antibiotic pressure. Analysis of carbon balances showed that at the late stage of the fermentation, tryptophan yield decreased with a concomitant increase in CO2 yield, suggesting a transition in the distribution of carbon flow from aromatic biosynthesis toward the tricarboxylic acid cycle via glycolysis. To circumvent this transition by increasing the supply of erythrose 4-phosphate, a direct precursor of aromatic biosynthesis, the transketolase gene of C. glutamicum was coamplified in the engineered strain by using low- and high-copy-number plasmids which were compatible with the resident plasmid pKW9901. The presence of the gene in low copy numbers contributed to improvement of tryptophan yield, especially at the late stage, and led to accumulation of more tryptophan (57 g/liter) than did its absence, while high-copy-number amplification of the gene resulted in a tryptophan production level even lower than that resulting from the absence of the gene due to reduced growth and sugar consumption. In order to assemble all the cloned genes onto a low-copy-number plasmid, the high-copy-number origin of pKW9901 was replaced with the low-copy-number one, generating low-copy-number plasmid pSW9911, and the transketolase gene was inserted to yield pIK9960. The pSW9911-carrying producer showed almost the same fermentation profiles as the pKW9901 carrier in fed-batch cultivation without antibiotic pressure. Under the same culture conditions, however, the pIK9960 carrier achieved a final tryptophan titer of 58 g/liter, which represented a 15% enhancement over the titers achieved by the pKW9901 and pSW9911 carriers.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

Sign in / Sign up

Export Citation Format

Share Document