Binding of Selected Extracellular Matrix Proteins to Enterococci and Streptococcus bovis of Animal Origin

Thirty-three enterococcal strains and 10 Streptococcus bovis strains were investigated for their protein-binding cell surface components. Seven extracellular matrix (ECM) proteins were immobilized on Difco latex beads to detect these components on the surface of all enterococcal strains and eight non-autoaggregating S. bovis strains by a particle agglutination assay (PAA). Twenty-three selected strains were also examined in microtiter plate assays. According to the absorbance readings (A_570nm), 11 strains were classified as nonadherent (A_570nm < 0.1), 10 strains as weakly adherent (0.1 < A_570nm > 0.3), and 2 strains as strongly adherent (A_570nm > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A_570nm readings of microtiter plate assays. Binding of ¹²⁵I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%–30% and of ¹²⁵I-labeled human vitronectin in the range of 9%–33% to streptococci. The binding of ¹²⁵I-labeled ECM proteins to selected strains was much more effectively inhibited by sulfated carbohydrates than by non-sulfated hyaluronic acid, indicating the importance of the sulfate groups of these inhibitors. An inhibition effect of heparin on bLf binding to four selected strains was higher in comparison with fucoidan in the microtiter plates. Thirty-five out of 44 strains had agglutinated rabbit erythrocytes. However, these strains showed no ability to agglutinate bovine or sheep erythrocytes. Received: 28 April 1999 / Accepted: 26 July 1999     

Binding of extracellular matrix proteins by lactobacilli

Ten gut and ten vaginalLactobacillus strains were investigated for their ability to bind type I collagen (Cn-I) and four selected gut lactobacilli were investigated for their binding to other extracellular matrix (ECM) molecules. Immobilized Cn-I (100 mg/L) in wells of microtitre plates was bound by all 10 autoaggregating vaginal strains and by 3 strains of gut lactobacilli from piglets in the range ofA ₅₇₀ readings 0.114–1.806.L. acidophilus strain SV31 was much more adherent than the rest of strains. All four gut lactobacilli tested for binding to other ECM molecules displayed good binding to porcine fibronectin and heparin and some of them bound weakly to fetuin and porcine mucin. No binding of these strains was observed to bovine mucin, bovine fibrinogen and bovine lactoferrin.     

Binding of extracellular matrix molecules by probiotic bacteria

AIMS: The aim of this study was to investigate extracellular matrix (ECM) and mucin binding of selected bacterial isolates with probiotic features in comparison with commercially used probiotic bacteria. METHODS AND RESULTS: ECM molecules were immobilized in microtitre plates (mucin and fetuin) or on the surface of latex beads. Porcine mucin was bound by all 13 probiotic strains tested with important inter-strain differences; however, fetuin binding was similar (weak) for all 14 strains tested. Strongly positive (three) binding of bovine fibrinogen was expressed by strains from fermented food (Lactobacillus rhamnosus GG, L. casei Shirota and L. johnsonii La1) as well as by L. casei L.c., Lactobacillus sp. 2I3 and by L. plantarum LP. The other strains expressed moderate (2) or weakly positive (1) binding of bovine fibrinogen. Strongly positive (3) binding of porcine fibronectin was observed only with two strains; however, all other strains also bound this molecule. Bovine lactoferrin was bound to a higher extent than transferrins. SIGNIFICANCE AND IMPACT OF THE STUDY: Some animal strains (at least L. casei L.c. and Lactobacillus sp. 2I3) are comparable with the commercially used strains with respect to their ECM binding ability. As this feature is important for probiotic bacteria to be able to colonize intestine, these strains should be considered for their wider use in fermented feed (or probiotic preparations) for animals.     

Chemo-enzymatic synthesis of poly-<Emphasis Type="Italic">N</Emphasis>-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces

Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized β-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human β1,4-galactosyltransferase-1 and β1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His₆CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Collagen binding toEscherichia coli strain NG7C

Escherichia coli strain NG7C was shown to bind iodine-labeled human type IV collagen (Cn). The binding was rapid and saturable. The number of binding sites was estimated to be 1.5×10⁴ sites/cell and the dissociation constant 85 nM. The binding was inhibited by unlabeled type I, type IV, and type X Cn, gelatin and, at high doses by vitronectin and fibrinogen. Heat treatment of bacteria abolished the binding. A cell sonicate of strain NG7C inhibited the binding. Heat or protease treatment of the sonicate reduced its inhibitory activity by more than 50% Cell surface extracts of strain NG7C likewise inhibited Cn binding. Cells ofE. coli NG7C also bound to type IV Cn immobilized on microtiter plates. The Cn binding appears to be mediated by cell surface protein(s). Type IV Cn binding toE. coli NG7C differed from the earlier reported Cn binding mechanisms toE. coli, i.e., binding of soluble type II Cn, and from binding of immobilized type V Cn by enterobacteria.E. coli strains can thus produce different surface proteins which mediate binding to collagens. Expression of Cn binding byE. coli may enhance colonization of subepithelial tissues.     

Adherence of Abiotrophia defectiva and Granulicatella species to fibronectin: is there a link with endovascular infections?

During a 6-year period, we isolated three Abiotrophia defectiva, six Granulicatella adiacens and two G. 'para-adiacens' strains from clinical specimens. All A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas the Granulicatella spp. strains were isolated from immunosuppressed patients with primary bacteremia. As the capacity of bacteria to adhere to the host extracellular matrix (ECM) has been implicated in the pathogenesis of endovascular infection, we investigated the ability of A. defectiva and Granulicatella spp. isolates to bind different ECM components immobilized in microtiter plates. Adherence tests showed a strong attachment of A. defectiva strains to fibronectin, whereas Granulicatella spp. strains were not adherent. The poor adherence of Granulicatella spp. strains to the ECM could be correlated with a lower propensity to induce endocarditis.     

Lectin-like binding and antibiotic sensitivity of enterococci from wild herbivores

Fifty eight enterococcal isolates from wild herbivores were tested for their antibiotic sensitivity pattern and lectin-like binding of extracellular matrix (ECM) and serum proteins. Kanamycin resistance was very frequent; many multiresistant strains were also isolated. All isolates were sensitive to rifampicin. Resistance to gentamicin, novobiocin, and tetracycline was widely distributed in the microflora of wild herbivores breeded in zoological garden in Košice.

No autoaggregating strains were detected among these 58 enterococcal isolates. Various degrees of binding of mucins, fetuin, heparin, fibrinogen, and fibronectin were observed in individual strains. However, bovine lactoferrin binding by enterococci from deers and chamoises was either negative (0) or strongly positive (3).

With regard to influence of growth media, TH agar was found to be better for the expression of lectin-like binding than blood agar, TH broth and Nutrient broth.

A significant effect (P < 0.001 or P < 0.05) of proteolytic treatment was observed in six selected strains. However, there is a difference between the effect of trypsin and pronase P.Pronase treatment more effectively decreased binding of some strains (1H, 6A, EF 1111, EC 1292), while trypsin treatment decreased more binding of other enterococcal strains (EF953 and 1E). Significant (P < 0.001) influence of metaperiodate, which cleaves the C-C bond between vicinal groups of sugars, on collagen I binding by three selected strains (1E, 1H, 6A) and bovine lactoferrin binding (by EF 1111, EC 1292, EF 953) was also observed. However, its influence was very different. In two strains (1H and EC 1292), ECM binding was decreased, while in four other strains (1E, 6A, EF 1111, EF 953) it was increased.     

Analyses of Pseudomonas aeruginosa Lectin Binding to α-Galactosylated Glycans

The specificity and binding capacity of the galactophilic lectin from the Gram negative bacterium Pseudomonas aeruginosa (PA-IL) was determined by solid phase measurements using galactosylated neoglycoproteins immobilized on microtiter plates. The bacterial lectin reacted with both short chain (monosaccharide) and long chain (pentasaccharide) glycoconjugates. Among the Galα1-XGal disaccharides, the highest affinity was observed towards the Galα1-3Gal structure. Raising the incubation temperature enhanced the lectin-polysaccharide agglutination, and it is suggested that binding to certain conformations of polysaccharides could vary between lectins with the same monocarbohydrate specificity and that this activity may, in part, be temperature dependent. Histochemical examination of lectin binding to different porcine tissues suggests a differential glycosylation of the carbohydrate antigens on endothelial cells in various parts of the vascular system. In the pancreas, PA-IL also adhered to the excretory ducts. These observations on PA-IL binding could be of importance both to determine infection foci in P. aeruginosa-mediated vacuities and to determine its role for pancreatic involvement in cystic fibrosis.     

Specific interactions between Porphyromonas gingivalis fimbriae and human extracellular matrix proteins

The interactions of the extracellular matrix (ECM) proteins (laminin, elastin, fibronectin, type I collagen, thrombospondin and vitronectin) with the fimbriae of Porphyromonas gingivalis were analyzed based on surface plasmon resonance (SPR) spectroscopy using a biomolecular interaction analyzing system (BIAcore). The BIAcore profiles demonstrated that fimbriae specifically bound to all of the ECM proteins with significant association constants (Ka). Vitronectin showed the highest affinity to fimbriae (Ka = 3.79 x 10(6) M-1), while the affinity of laminin was lowest (Ka = 2.15 x 10(6) M-1). A synthetic peptide which is a potent inhibitor of fimbrial binding to salivary proteins was not significantly effective on the fimbrial interactions with the ECM proteins. Using polystyrene microtiter plates revealed that P. gingivalis fimbriae bound markedly to immobilized fibronectin and type I collagen, while the interaction of fimbriae with the other ECM proteins was not clearly demonstrated. These results suggest that interactions between fimbriae and the ECM proteins occur with specific affinities which are not mediated by mechanisms identical to those of salivary proteins. It was also shown that SPR spectroscopy is a useful method to analyze these specific interactions.     

An integrated high throughput strategy to screen mutants of Paenibacillus polymyxa with high polymyxin E-productivity

An integrated high-throughput screening (HTS) strategy was developed to screen large numbers of polymyxin E-producing mutants of Paenibacillus polymyxa. Various types of mutants were transferred onto the surfaces of solidified agar in 96-well microtiter plates, and then inoculated to 96-deep-well microtiter plates for micro-cultivation. The culture conditions were optimized for the production of polymyxin E. The supernatants from the micro-culture plates were transferred to 96-well bioassay microtiter plates containing Escherichia coli JM109 for high-throughput bioassay. By using this high-throughput screening (HTS) procedure, one best producer P. polymyxa PE 5.808 was identified from a large NTG mutated library with about 5,000 isolates. The volumetric productivity of polymyxin E of P. polymyxa PE 5.808 was 1,200 μg/ml in shake flasks, about 140% improvement compared with that of the wild type strain.     

Binding of Extracellular Matrix Proteins by Enterococci

Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of ¹²⁵I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity. Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin, human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding of ¹²⁵I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers such as dextran sulfate (M _r 5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine, but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface. Received: 22 April 1996 / Accepted: 29 June 1996     

Screening ofExiguobacterium acetylicum from soil samples showing enantioselective and alkalotolerant esterase activity

About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed hgh apparent enantioselectivity (E _app>100) for (R)-2PB-O-res and were identified asExiguobacterium acetylicum. The JH13 strain showed high esterase activity onp-nitrophenyl acetate (pNPA), but showed low lipase activity onp-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.     

The Mycoplasma genitalium MG352‐encoded protein is a Holliday junction resolvase that has a non‐functional orthologue in Mycoplasma pneumoniae

Recombination between repeated DNA elements in the genomes of Mycoplasma species appears to lie at the basis of antigenic variation of several essential surface proteins. It is therefore imperative that the DNA recombinatorial pathways in mycoplasmas be unravelled. Here, we describe the proteins encoded by the Mycoplasma genitalium MG352 and Mycoplasma pneumoniae MPN528a genes (RecU_Mge and RecU_Mpn respectively), which share sequence similarity with RecU Holliday junction (HJ) resolvases. RecU_Mge was found to: (i) bind HJ substrates and large double‐stranded DNA molecules and (ii) cleave HJ substrates at the sequence 5′‐^G/_TC↓^C/_TT^A/_GG‐3′ in the presence of Mn²⁺. Interestingly, RecU_Mpn(from M. pneumoniae subtype 2 strains) did not possess obvious DNA binding or cleavage activities, which was found to be caused by the presence of a glutamic acid residue at position 67 of the protein, which is not conserved in RecU_Mge. Additionally, RecU_Mpn appears not to be expressed by subtype 1 M. pneumoniae strains, as these possess a TAA translation termination codon at position 181–183 of MPN528a. We conclude that RecU_Mge is a HJ resolvase that may play a central role in recombination in M. genitalium.     

A Colorimetric DNA Diagnostic Method for Falciparum Malaria and Vivax Malaria: A Field Trial in the Solomon Islands

Abstract

We have developed a colorimetric assay, “microtiter plate-hybridization”, for the detection of malaria parasites Plasmodium falciparum and P. vivax in human blood, in which the target DNA sequences (18s small subunit ribosomal RNA gene) amplified by polymerase chain reaction (PCR) are hybridized with the species-specific probes immobilized on a microtiter well. This assay system was tested in Guadalcanal, Solomon Islands, where malaria is highly endemic. We obtained blood samples by finger puncture from 130 asymptomatic donors. Among the 130 samples, 30 (23 %) were P. falciparum positive, 28 (22 %) were P. vivax positive, and 8 (6 %) were mixed infections. The results of our DNA diagnostic method showed good correlation with those of acridine orange microscopy.

     

Particle agglutination assays to identify fibronectin and collagen cell surface receptors and lectins in Aeromonas and Vibrio species.

A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.     

Particle agglutination assays to identify fibronectin and collagen cell surface receptors and lectins in Aeromonas and Vibrio species

A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.     

A novel microtiter plate based method for identification of B‐cell epitopes

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

不同生境外生菌根真菌对铝胁迫的响应

以南北方不同生境下的10株外生菌根真菌为研究对象,采用液体培养的方法,研究了铝对不同菌根真菌的生物量、有机酸分泌及养分含量的影响,以期筛选出抗铝性强的优良菌株,并探讨其抗铝机理。结果表明:外生菌根真菌Sl 08抗铝性最强;Pt 715、Ld 03、Bo 11、Sl 01、Bo 15也具有不同程度的耐铝性;Sl 14、Gc 99、Cg 04抗铝性较差;Sg 11抗铝性最差。来自南方酸性森林土壤的菌株总体抗铝性强于来自北方石灰性土壤的菌株,这表明外生菌根真菌的铝耐受能力与其原始生境有着密切的联系。外生菌根真菌能分泌多种有机酸,且不同菌株分泌的有机酸种类不同。其中,受铝胁迫分泌量增加最多的是草酸。研究中,铝胁迫能增加大多数铝抗性菌株的草酸分泌量,其中铝抗性最强的Sl 08表现最为明显。但铝胁迫并没有促进具备一定铝抗性的Bo 11和Sl 01草酸的分泌量,同时在铝敏感的菌株中均观察到了草酸分泌量的增加。这表明分泌草酸可能并不是外生菌根真菌抵抗铝毒的唯一途径。对各菌株铝胁迫下对氮,磷及钾的吸收研究表明,除铝敏感菌株Sl 14外,铝胁迫均能促进各供试菌株对氮,磷或钾的吸收。综上,在一定铝浓度下,一些外生菌根真菌可通过增加草酸分泌来抵御铝毒。此外,铝胁迫下外生菌根真菌还可通过调控氮、磷、钾等营养元素的吸收来抵抗铝毒,即通过增加对营养元素的吸收来增强其在铝胁迫下的生存能力,这可能是其抵御铝胁迫的应激反应之一。     

Concanavalin A and wheat germ agglutinin binding to sea urchin embryo basal laminae

Summary The basal laminae and inner extracellular matrices of Lytechinus pictus and Arbacia punctulata embryos were characterized on the basis of lectin binding. Developmental stage specific patterns of lectin binding were observed after microinjection of Con A-FITC and WGA-FITC. Lectin-specific patterns differed between control, sulfate free sea water (SFSW) and tunicamycin treated embryos. Con A injection resulted in the rounding-up of cells in the epithelium and was most pronounced in embryos cultured in the presence of tunicamycin. Basal laminae were isolated by Triton X-100 extraction of whole embryos. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose and incubated in biotinylated lectins. Lectin-binding glycoproteins were detected with avidin peroxidase. The electrophoretic pattern of Con A-binding proteins in early developmental stages of Arbacia was similar with several low molecular weight species appearing at gastrulation in control and SFSW embryos. WGA-binding in Arbacia and Lytechinus control embryos was limited to a 125,000 Mr glycoprotein (gp125). In addition to gp125, several high molecular weight WGA-binding glycoproteins were also detected in SFSW embryos. The evidence suggests that mesenchyme migration and gastrulation are correlated with changes in the molecular composition of the ECM.