Attachment of Mycoplasma salivarium and Mycoplasma orale to glass surfaces

Mycoplasma salivarium ATCC 23064 and 24 other oral strains, and Mycoplasma orale ATCC 15539 and 22 other oral strains were examined quantitatively for attachment to glass surfaces by using ELISA method. Although all of the tested strains attached to glass surfaces, M. salivarium attached far more readily than M. orale. The results suggested that electrostatic bonds were involved in the attachment and that bivalent metal ions also played a role.     

Dextran T 500--induction of spreading in Ehrlich ascites tumour cells on glass surface

The experiments were carried out in order to find factors which could induce attachment and spreading of Ehrlich ascites tumour (EAT) cells on solid substrata. In normal culture media, serum-free as well as serum-containing, these cells did not spread and very weakly attached onto glass. It was found that after coating the cell surfaces with dextran T 500 the EAT cells strongly attached and spread extensively on glass. This spreading could be inhibited or reversed by washing out the dextran or adding calf serum. Dextran T 500 caused rapid spreading also in chick embryo fibroblasts and mouse lymphocytes. Some aspects of these results in connection with contemporary views concerning the processes of cell attachment and spreading are discussed.     

Movement on the cell surface of the gliding bacterium,Mycoplasma mobile,is limited to its head-like structure

Mycoplasma mobile cells glide on solid surfaces such as glass with a fast and continuous motion in the direction of the membrane protrusion (head-like structure) at one cell pole. To examine its cell-surface movement, a latex bead was attached to a cell and behavior in gliding was monitored. The bead was carried without movement relative to the cell body, suggesting that the cell does not roll around the cell axis and the surface movement is limited to a small area. A small percentage of cells showed an elongated head-like structure in an old batch culture. The head-like structure moved forward, sometimes leaving the cell body in one position, resulting in a stretching of this head-like structure. These results indicate that the head-like structure drags the cell body, leading us to conclude that the force for gliding is generated at the head-like structure.     

RGD-directed attachment of isolated rat osteoclasts to osteopontin, bone sialoprotein, and fibronectin

Osteoclasts isolated from the long bones of 5-day-old rats were seeded onto glass surfaces coated with osteopontin, bone sialoprotein, or fibronectin. Cell binding was promoted by all three proteins and inhibited in a dose-dependent manner by an RGD-containing peptide, while an RGE-containing peptide was ineffective. Immunocytochemistry of bone tissue showed enhanced concentration of osteopontin in bone opposite the clear zone of the osteoclasts, whereas immunolocalization of bone sialoprotein and fibronectin showed no accumulation on bone surfaces facing cells. The observations corroborate previous findings that the osteoclast is attached via an integrin to osteopontin on the bone surface. Although bone sialoprotein and fibronectin can mediate osteoclast binding in vitro, such a role in vivo is not supported by the immunocytochemical observations.     

Role of curli fimbriae in mediating the cells of enterohaemorrhagic Escherichia coli to attach to abiotic surfaces

AIMS: The objectives of this study were to evaluate the role of curli in assisting the cells of enterohaemorrhagic Escherichia coli (EHEC) in attaching to abiotic surfaces and to determine the influence of cell-surface contact time on the efficiency of the attachment. METHODS AND RESULTS: Three pairs of EHEC cultures, each with a curli-expressing and a noncurli-expressing variant (O111:H- 7-57C+ and O157:H7 5-9C-, O157:H7 5-11C+ and 5-11C-, as well as O103:H2 7-52C+ and 7-52C-), were allowed to interact with polystyrene, glass, stainless steel and rubber surfaces at 28 degrees C for 24 h (short-term attachment) or 7 days (long-term attachment). The quantities of the cells that attached to the surfaces were measured daily in the long-term attachment study, and in 4 h intervals in the short-term attachment study. Quantification of the cells that attached to the surfaces was accomplished with a crystal violet binding assay. The results of the long-term attachment study indicated that 7-57C+ attached to the polystyrene and glass surfaces more efficiently (P < 0.05) than did 5-9C-. The curli-expressing variant of 5-11 possessed a better ability to adhere to the polystyrene and glass surfaces than did its noncurli-expressing counterpart (P < 0.05). The differences in attachment between 7-52C+ and 7-52C- on polystyrene and stainless steel surfaces were statistically significant (P < 0.05). However, the attachment of the pair on the glass surfaces was statistically insignificant (P > 0.05). In addition, the two members of all three EHEC pairs attached equally well to rubber surfaces (P > 0.05). In the short-term attachment study, only the pair of 7-52 attached differently on glass and stainless steel surfaces (P < 0.05). CONCLUSIONS: These results suggest that curli could be an important cell surface component to mediate the attachment of some EHEC cells to certain abiotic surfaces. Cell-surface contact time could have a significant influence on EHEC attachment to abiotic surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The study signifies a possible role of curli in assisting the cells of EHEC in attaching to food-contact surfaces. It underlines the importance of cleaning and sanitizing food-contact surfaces regularly and thoroughly, and of identifying chemical agents that can effectively remove the attached EHEC cells from these surfaces.     

Identification of a 349-kilodalton protein (Gli349) responsible for cytadherence and glass binding during gliding of Mycoplasma mobile

Several mycoplasma species are known to glide in the direction of the membrane protrusion (head-like structure), but the mechanism underlying this movement is entirely unknown. To identify proteins involved in the gliding mechanism, protein fractions of Mycoplasma mobile were analyzed for 10 gliding mutants isolated previously. One large protein (Gli349) was observed to be missing in a mutant m13 deficient in hemadsorption and glass binding. The predicted amino acid sequence indicated a 348,758-Da protein that was truncated at amino acid residue 1257 in the mutant. Immunofluorescence microscopy with a monoclonal antibody showed that Gli349 is localized at the head-like protrusion's base, which we designated the cell neck, and immunoelectron microscopy established that the Gli349 molecules are distributed all around this neck. The number of Gli349 molecules on a cell was estimated by immunoblot analysis to be 450 +/- 200. The antibody inhibited both the hemadsorption and glass binding of M. mobile. When the antibody was used to treat gliding mycoplasmas, the gliding speed and the extent of glass binding were inhibited to similar extents depending on the concentration of the antibody. This suggested that the Gli349 molecule is involved not only in glass binding for gliding but also in movement. To explain the present results, a model for the mechanical cycle of gliding is discussed.     

Centipede and inchworm models to explain Mycoplasma gliding

The twelve Mycoplasma species known to glide on solid surfaces all lack surface flagella or pili, and no genes homologous to known motility systems have been found in the five genomes sequenced to date. Recent studies on the fastest of these species, M. mobile, examined novel proteins involved in the gliding mechanism, binding targets on the solid surfaces, energy sources and mechanical characteristics of the movements. Accordingly, I propose a working model for the gliding mechanism, called the centipede (power stroke) model, in which the 'leg' proteins repeat a cycle of binding to and release from the solid surface, using energy from ATP. Another 'inchworm model' suggested from the structural studies of a human pathogen, M. pneumoniae, is also discussed.     

Amino Acid Assimilation and Electron Transport System Activity in Attached and Free-Living Marine Bacteria

Amino acid assimilation and electron transport system activity of a marine Pseudomonas sp. was evaluated to determine whether the activity of bacteria attached to solid surfaces differed from that of free-living bacteria or bacteria which had been attached but subsequently desorbed from the substratum (detached bacteria). Bacteria were allowed to attach to glass and to a range of plastic surfaces (Thermanox, polyvinylidene fluoride, polyethylene, polytetrafluoroethylene). Microautoradiography and staining with a tetrazolium salt to demonstrate electron transport system activity were used to compare the activity of these organisms with that of free-living or detached cells. The water-wettability of the surfaces was evaluated by measuring the advancing contact angle (θ_A) of water on each surface, to determine whether there was a relationship between activity and substratum hydrophilicity. There was an increase in the proportion of leucine-assimilating attached bacteria and in the proportion of attached cells demonstrating electron transport system activity with an increase in substratum θ_A, but the relationship between activity of attached and free-living cells depended on the substratum. Activity appeared to promote firm attachment, and detached bacteria assimilated fewer amino acids than did attached cells. There was no general effect of surfaces on attached bacterial activity, and attached cells may be more, or less, active than free-living cells, depending on the amino acid, its concentration, and substratum properties.     

A cell-binding, immunoglobulin-like protein from human plasma. II. Cell-binding activity

Cell adhesion to plastic surfaces coated with a new high-molecular-mass immunoglobulin-like protein from normal human plasma was studied. Mouse subdermal fibroblasts, hamster kidney cells, human umbilical vein endothelial cells, and human skin fibroblasts were found to become attached to the surface, but cancer cells derived from human stomach cancer and human breast cancer did not. The appearance of the attached cells differed from that of cells attached to surfaces coated with fibronectin or concanavalin A. The cell adhesion to the surfaces coated with the protein was inhibited by goat anti-human IgM. Furthermore, the binding of the protein to the cell surfaces was demonstrated by the indirect immunofluorescence method. It is concluded that this protein is a new cell-binding protein.     

Attachment of mycobacteria to fibronectin-coated surfaces

This report investigates the extent of the expression of fibronectin (FN) binding properties among the mycobacteria and provides preliminary characteristics of the bacterial molecule(s) mediating attachment. Eight BCG substrains, three Mycobacterium tuberculosis strains and four other mycobacterial species all expressed FN-binding capacity. Treatment of organisms with detergent prior to the binding assay destroyed the FN-binding capacity of BCG but not that of Staphylococcus aureus. Trypsin pretreatment eliminated the FN-binding capacity of both BCG and S. aureus. [35S]Methionine-labelled material in supernatants from BCG and M. tuberculosis cultures attached to FN-coated surfaces. These culture supernatants inhibited the attachment of BCG but not S. aureus to FN-coated surfaces. This inhibitory activity of the supernatants was removed by affinity chromatography on FN-Sepharose but was not affected by similar passage over a control column (human serum albumin attached to Sepharose). These results demonstrate that the ability to bind FN is present in all mycobacterial species tested and suggest that attachment is mediated by trypsin-sensitive cell-surface component(s).     

Mycoplasma biofilms ex vivo and in vivo

Biofilms are communities of microorganisms that are encased in polymeric matrixes and grow attached to biotic or abiotic surfaces. Despite their enhanced ability to resist antimicrobials and components of the immune system in vitro , few studies have addressed the interactions of biofilms with the host at the organ level. Although mycoplasmas have been shown to form biofilms on glass and plastic surfaces, it has not been determined whether they form biofilms on the tracheal epithelium. We developed a tracheal organ-mounting system that allowed the entire surface of the tracheal lumen to be scanned using fluorescence microscopy. We observed the biofilms formed by the murine respiratory pathogen Mycoplasma pulmonis on the epithelium of trachea in tracheal organ culture and in experimentally infected mice and found similar structure and biological characteristics as biofilms formed in vitro . This tracheal organ-mounting system can be used to study interactions between biofilms formed by respiratory pathogens and the host epithelium and to identify the factors that contribute to biofilm formation in vivo .     

The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis.     

The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.     

Influence of substrate diffusion on degradation of dibenzofuran and 3-chlorodibenzofuran by attached and suspended bacteria.

Dibenzofuran uptake-associated kinetic parameters of suspended and attached Sphingomonas sp. strain HH19k cells were compared. The suspended cells were studied in a batch system, whereas glass beads in percolated columns were used as the solid support for attached cells. The maximum specific activities of cells in the two systems were the same. The apparent half-maximum uptake rate-associated concentrations (Kt') of attached cells, however, were considerably greater than those of suspended cells and depended on cell density and on percolation velocity. A mathematical model was developed to explain the observed differences in terms of substrate transport to the cells. This model was based on the assumptions that the intrinsic half-maximum uptake rate-associated concentration (Kt) was unchanged and that deviations of Kt' from Kt resulted from the stereometry and the hydrodynamics around the cells. Our calculations showed that (i) diffusion to suspended cells and to single attached cells is efficient and therefore only slightly affects Kt'; (ii) diffusion to cells located on crowded surfaces is considerably lower than that to single attached cells and greatly increases Kt', which depends on the cell density; (iii) the convective-diffusive transport to attached cells that occurs in a percolated column is influenced by the liquid flow and results in dependency of Kt' on the flow rate; and (iv) higher specific affinity of cells correlates with higher susceptibility to diffusion limitation. Properties of the experimental system which limited quantitative proof of exclusively transport-controlled variations of Kt' are discussed.     

Coated pits and asialoglycoprotein receptors redistribute to the substratum during hepatocyte adhesion to galactoside surfaces

Rat hepatocytes bind in a sugar-specific and concentration-dependent manner to flat polyacrylamide matrices containing covalently attached galactosyl (Gal) groups. Previous studies (Weigel, P.H., J. Cell Biol. 87, 855, 1980) concluded that binding was likely mediated by the asialoglycoprotein receptor. Here we confirm that adhesion is mediated by this receptor, since cell binding is inhibited by antireceptor antibody and a threshold binding response is also observed when hepatocytes adhere to surfaces coated with asialoorosomucoid, a ligand for this receptor. Cells that had bound to a Gal surface and were then sheared from the surface left a membrane patch behind on the substratum. The cytoplasmic side of these plasma membrane patches was visualized on the substratum by indirect immunofluorescence using antireceptor antibody or anticlathrin antibody. The density of punctate coated pits, visualized with the latter antibody, was enriched in a circular membrane region of about 4 microns 2 area that mediated cell binding. This zone also contained concentrated receptors, although the staining pattern with antireceptor antibody was more uniform and less punctate. The results show that both asialoglycoprotein receptors and coated pits are redistributed at the substratum interface on hepatocytes bound to Gal surfaces.     

Surface attachment of nitrifying bacteria and their inhibition by potassium ethyl xanthate

Ion exchange resins and glass microscope slides were used to investigate factors affecting attachment of nitrifying bacteria to solid surfaces and the effect of attachment on inhibition ofNitrobacter by potassium ethyl xanthate. The ammonium oxidizerNitrosomonas attached preferentially to cation exchange resins while the nitrite oxidizerNitrobacter colonized anion exchange resins more extensively. Colonization was always associated with growth, and the site of substrate (NH₄ ⁺ or NO₂ ^–) adsorption was the major factor in attachment and colonization. The specific growth rate of cells colonizing either ion exchange resin beads or glass surfaces was greater than that of freely suspended cells, butNitrobacter populations colonizing glass surfaces were more sensitive to the inhibitor potassium ethyl xanthate. The findings indicate that surface growth alone does not protect soil nitrifying bacteria from inhibition by potassium ethyl xanthate and explain different patterns of inhibition for ammonium and nitrite oxidizers in the soil.     

Analysis of DNA-microarrays produced by inverse in situ oligonucleotide synthesis.

5'-Phosphoramidites protected by 2-nitrophenylethyl (NPE) and 2-(4-nitrophenyl)ethoxy carbonyl (NPEOC) functions were employed for in situ synthesis of oligonucleotides in 5'-->3' direction on flat glass surfaces. By this inverse synthesis format, the oligonucleotides are attached to the solid support via their 5'-ends while the free 3'-hydroxyl groups are available as substrates for enzymatic reactions such as elongation by polymerases, thereby adding another feature to the portfolio of chip-based applications. Having a fluorescence dye present at the first base during synthesis, the quality of the oligonucleotides was analysed quantitatively by capillary electrophoresis after release from the solid support. With about 95% yield per condensation, it was found to be equivalent to synthesis results achieved on CPG support. The chip-bound oligonucleotides could be extended enzymatically upon hybridisation of a DNA-template. Surprisingly, however, only 63% of the oligonucleotides were elongated in polymerase reactions, while oligonucleotides that were released from the support behaved normally in standard PCR amplifications. This rate of 63% nevertheless compares favourably with an extension rate of only 50%, which was achieved under identical conditions, if pre-fabricated oligonucleotides of identical sequence had been spotted to the glass support.     

Extracellular polymeric substances responsible for bacterial adhesion onto solid surface

The influence of extracellular polymeric substances (EPS) on bacterial cell adhesion onto solid surfaces was investigated using 27 heterotrophic bacterial strains isolated from a wastewater treatment reactor. Cell adhesion onto glass beads was carried out by the packed-bed method and the results were discussed in terms of the amount of each EPS component produced and cell surface characteristics such as zeta potential and hydrophobicity. Protein and polysaccharides accounted for 75-89% of the EPS composition, indicating that they are the major EPS components. Among the polysaccharides, the amounts of hexose, hexosamine and ketose were relatively high in EPS-rich strains. For EPS-poor strains, the efficiency of cell adhesion onto glass beads increased as the absolute values of zeta potential decreased, suggesting that electrostatic interaction suppresses cell adhesion efficiency. On the other hand, the amounts of hexose and pentose exhibited good correlations with cell adhesiveness for EPS-rich strains, indicating that polymeric interaction due to the EPS covering on the cell surface promoted cell adhesion. It was concluded that, if the EPS amount is relatively small, cell adhesion onto solid surfaces is inhibited by electrostatic interaction, and if it is relatively large, cell adhesion is enhanced by polymeric interaction.     

The application of an improved solid phase synthetic technique to the delineation of an antigenic site of apolipoprotein A-II

Previous studies have shown that the antigenic sites of human plasma high-density apolipoprotein A-II (apoA-II) are separate from their lipid-binding determinants in human high density lipoproteins (HDL). A specific radioimmunoassay has shown that three distinct antigenic sites are located in residues 4-23, 31-46, and 56-77; these studies suggested that an antigenic site might be restricted to residues 60-77 in the 56-77 fragment. To further delineate this site, we have developed a solid phase radioimmunoassay technique using an improved solid support on which selected sequences of peptides were synthesized, deprotected with HF, and the resulting peptidyl-resins tested for their capability of binding purified 125I-anti-apoA-II antibodies. Amino acid analyses and solid phase sequence analyses were performed to verify the sequence of the synthetic peptide on the solid support. Using this technique, 125I-anti-apoA-II antibodies had achieved 50% of maximal binding when residues 61-77 were attached to the solid support. The maximal binding was achieved by the addition of one more residue, Leu60, thus confirming our suggestion that a major antigenic site is located in residues 60-77. The binding to the peptidyl-resin was inhibited by a synthetic fragment corresponding to residues 60-77 indicating that the antibodies were specifically bound to the resin.