Lectin and E. coli Binding to Carbohydrate-Functionalized Oligo(ethylene glycol)-Based Microgels: Effect of Elastic Modulus, Crosslinker and Carbohydrate Density

The synthesis of carbohydrate-functionalized biocompatible poly(oligo(ethylene glycol) methacrylate microgels and the analysis of the specific binding to concanavalin A (ConA) and Escherichia coli (E. coli) is shown. By using different crosslinkers, the microgels’ size, density and elastic modulus were varied. Given similar mannose (Man) functionalization degrees, the softer microgels show increased ConA uptake, possibly due to increased ConA diffusion in the less dense microgel network. Furthermore, although the microgels did not form clusters with E. coli in solution, surfaces coated with mannose-functionalized microgels are shown to bind the bacteria whereas galactose (Gal) and unfunctionalized microgels show no binding. While ConA binding depends on the overall microgels’ density and Man functionalization degree, E. coli binding to microgels’ surfaces appears to be largely unresponsive to changes of these parameters, indicating a rather promiscuous surface recognition and sufficiently strong anchoring to few surface-exposed Man units. Overall, these results indicate that carbohydrate-functionalized biocompatible oligo(ethylene glycol)-based microgels are able to immobilize carbohydrate binding pathogens specifically and that the binding of free lectins can be controlled by the network density.

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Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb

Biochemical Journal ◽

10.1042/bcj20160575 ◽

2016 ◽

Vol 473 (21) ◽

pp. 3923-3936 ◽

Cited By ~ 3

Author(s):

Dani Zalem ◽

João P. Ribeiro ◽

Annabelle Varrot ◽

Michael Lebens ◽

Anne Imberty ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Cholera Toxin ◽

Carbohydrate Binding ◽

Type I ◽

Type Ii ◽

E Coli ◽

B Subunit ◽

Heat Labile ◽

B Subunits

The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galβ4GlcNAcβ3Galβ4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.

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Anti-Escherichia coli Functionalized Silver-Doped Carbon Nanofibers for Capture of E. coli in Microfluidic Systems

Polymers ◽

10.3390/polym12051117 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1117 ◽

Cited By ~ 3

Author(s):

Soshana Smith ◽

Michael Delaney ◽

Margaret Frey

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Carbon Nanofibers ◽

Specific Binding ◽

Base Material ◽

Fiber Surface ◽

Electrospinning Process ◽

Silver Particles ◽

Microfluidic Systems ◽

E Coli

Silver-doped carbon nanofibers (SDCNF) are used as the base material for the selective capture of Escherichia coli in microfluidic systems. Fibers were spun in a glovebox with dry atmosphere maintained by forced dry air pumped through the closed environment. This affected the evaporation rate of the solvent during the electrospinning process and the distribution of silver particles within the fiber. Antibodies are immobilized on the surface of the silver-doped polyacrylonitrile (PAN) based carbon nanofibers via a three-step process. The negatively charged silver particles present on the surface of the nanofibers provide suitable sites for positively charged biotinylated poly-(L)-lysine-graft-poly-ethylene-glycol (PLL-g-PEG biotin) conjugate attachment. Streptavidin and a biotinylated anti-E. coli antibody were then added to create anti-E. coli surface functionalized (AESF) nanofibers. Functionalized fibers were able to immobilize up to 130 times the amount of E. coli on the fiber surface compared to neat silver doped fibers. Confocal images show E. coli remains immobilized on fiber mat surface after extensive rinsing showing the bacteria is not simply a result of non-specific binding. To demonstrate selectivity and functionalization with both gram negative and gram-positive antibodies, anti-Staphylococcus aureus surface functionalized (ASSF) nanofibers were also prepared. Experiments with AESF performed with Staphylococcus aureus (S. aureus) and ASSF with E. coli show negligible binding to the fiber surface showing the selectivity of the functionalized membranes. This surface functionalization can be done with a variety of antibodies for tunable selective pathogen capture.

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Molecular Characterization of the EhaG and UpaG Trimeric Autotransporter Proteins from Pathogenic Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.06680-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2179-2189 ◽

Cited By ~ 47

Author(s):

Makrina Totsika ◽

Timothy J. Wells ◽

Christophe Beloin ◽

Jaione Valle ◽

Luke P. Allsopp ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Cell Surface ◽

Specific Binding ◽

Negative Regulator ◽

Passenger Domain ◽

Content Type ◽

E Coli ◽

Ecm Proteins

ABSTRACTTrimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagicEscherichia coli(EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenicE. coli(UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from severalE. coligenomes revealed grouping of the proteins in clades almost exclusively represented by distinctE. colipathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in anhnsisogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

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Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.989 ◽

1995 ◽

Vol 15 (2) ◽

pp. 989-996 ◽

Cited By ~ 131

Author(s):

J P McGoldrick ◽

Y C Yeh ◽

M Solomon ◽

J M Essigmann ◽

A L Lu

Keyword(s):

Escherichia Coli ◽

Specific Binding ◽

Polyclonal Antibodies ◽

Gradient Centrifugation ◽

Immunoblot Analysis ◽

Partial Purification ◽

Phosphodiester Bond ◽

E Coli ◽

Nuclear Extracts ◽

Calf Thymus

A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells. Western blot (immunoblot) analysis using polyclonal antibodies to the E. coli MutY protein detected a protein of 65 kDa in both extracts. Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies. Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes). A nicking activity that is either associated with or copurified with MYH was also detected. Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity. The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches. No nicking activity was detected on substrates containing other selected mismatches or homoduplexes. Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters. Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates. Binding is sevenfold greater on A/GO substrates than on A/G substrates. The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E. coli MutY protein.

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Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli

Biochemical Journal ◽

10.1042/bj20041114 ◽

2004 ◽

Vol 384 (2) ◽

pp. 385-390 ◽

Cited By ~ 51

Author(s):

Vesa P. HYTÖNEN ◽

Olli H. LAITINEN ◽

Tomi T. AIRENNE ◽

Heidi KIDRON ◽

Niko J. MELTOLA ◽

...

Keyword(s):

Escherichia Coli ◽

Signal Sequence ◽

Specific Binding ◽

Affinity Purification ◽

Active Form ◽

Efficient Production ◽

E Coli ◽

Secretion Signal ◽

Technology Applications ◽

Biotin Binding

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin–agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380–1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin–biotin technology applications.

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PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells

Glycobiology ◽

10.1093/glycob/cwz059 ◽

2019 ◽

Vol 29 (11) ◽

pp. 789-802 ◽

Cited By ~ 1

Author(s):

Nadine Legros ◽

Stefanie Ptascheck ◽

Gottfried Pohlentz ◽

Helge Karch ◽

Ulrich Dobrindt ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Specific Binding ◽

Human Kidney ◽

Adhesion Assay ◽

Binding Studies ◽

Binding Characteristics ◽

E Coli ◽

Tract Infections ◽

Chain Lengths

Abstract Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1–4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1–4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1–4Galβ1–4Glcβ1–1Cer), globotetraosylceramide (Gb4Cer, GalNAcβ1–3Galα1–4Galβ1–4Glcβ1–1Cer) and Forssman GSL (GalNAcα1–3GalNAcβ1–3Galα1–4Galβ1–4Glcβ1–1Cer) revealed balanced adhesion toward the three GSLs for PapG I–mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III–harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface–exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.

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Specific Immobilization of Escherichia coli Expressing Recombinant Glycerol Dehydrogenase on Mannose-Functionalized Magnetic Nanoparticles

Catalysts ◽

10.3390/catal9010007 ◽

2018 ◽

Vol 9 (1) ◽

pp. 7 ◽

Cited By ~ 7

Author(s):

Fei-Long Li ◽

Meng-Yao Zhuang ◽

Jia-Jia Shen ◽

Xiao-Man Fan ◽

Hyunsoo Choi ◽

...

Keyword(s):

Escherichia Coli ◽

Magnetic Nanoparticles ◽

Hydrophobic Interactions ◽

Immobilized Cells ◽

Specific Binding ◽

Glycerol Dehydrogenase ◽

Buffer Concentration ◽

E Coli ◽

Functionalized Magnetic Nanoparticles ◽

Free Cells

Mannose-functionalized magnetic nanoparticles were prepared for the immobilization of Escherichia coli cells harboring the recombinant glycerol dehydrogenase gene. Immobilization of whole E. coli cells on the carrier was carried out through specific binding between mannose on the nanoparticles and the FimH lectin on the E. coli cell surface via hydrogen bonds and hydrophobic interactions. The effects of various factors including cell concentration, pH, temperature, and buffer concentration were investigated. High degrees of immobilization (84%) and recovery of activity (82%) were obtained under the following conditions: cell/support 1.3 mg/mL, immobilization time 2 h, pH 8.0, temperature 4°C, and buffer concentration 50 mM. Compared with the free cells, the thermostability of the immobilized cells was improved 2.56-fold at 37 °C. More than 50% of the initial activity of the immobilized cells remained after 10 cycles. The immobilized cells were evaluated functionally by monitoring the catalytic conversion of glycerol to 1,3-dihydroxyacetone (DHA). After a 12 h reaction, the DHA produced by the immobilized cells was two-fold higher than that produced by the free cells. These results indicate that mannose-functionalized magnetic nanoparticles can be used for the specific recognition of gram-negative bacteria, which gives them great potential in applications such as the preparation of biocatalysts and biosensors and clinical diagnosis.

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Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins

Microbiology ◽

10.1099/mic.0.28981-0 ◽

2006 ◽

Vol 152 (9) ◽

pp. 2651-2659 ◽

Cited By ~ 19

Author(s):

Robert Manasherob ◽

Mark Itsko ◽

Nadine Sela-Baranes ◽

Eitan Ben-Dov ◽

Colin Berry ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Thuringiensis ◽

Biological Activities ◽

Nitrilotriacetic Acid ◽

Binding Motif ◽

Carbohydrate Binding ◽

High Level Expression ◽

E Coli ◽

C Terminus ◽

High Level

The larvicidal activity of Bacillus thuringiensis subsp. israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a β-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.

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A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-021-01725-w ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Hyunjun Ko ◽

Minsik Kang ◽

Mi-Jin Kim ◽

Jiyeon Yi ◽

Jin Kang ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Expression ◽

Inclusion Bodies ◽

Heterologous Protein ◽

Soluble Expression ◽

Carbohydrate Binding ◽

Heterologous Protein Expression ◽

Carbohydrate Binding Module ◽

Fusion Tag ◽

E Coli

Abstract Background Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. Results A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. Conclusions The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

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I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins

Nucleic Acids Research ◽

10.1093/nar/gkaa014 ◽

2020 ◽

Vol 48 (5) ◽

pp. e28-e28

Author(s):

Sarolta Szentes ◽

Nikolett Zsibrita ◽

Mihály Koncz ◽

Eszter Zsigmond ◽

Pál Salamon ◽

...

Keyword(s):

Escherichia Coli ◽

Specific Binding ◽

Lac Repressor ◽

Host Bacterium ◽

Lambda Phage ◽

Simple Method ◽

E Coli ◽

Laci Gene ◽

Compatible Plasmid ◽

Nhei Site

Abstract We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

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