Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999     

Spore Adhesion and Cell Wall Formation in Gelidium Floridanum (Rhodophyta, Gelidiales)

The attachment of spores to a substratum is essential for their germination and, therefore, to the completion of the life cycle of the red algae. In most red algae, spores are liberated without a cell wall, within a sheath of mucilage which is responsible for their primary attachment. Utilizing fluorescent-labeled lectins, we identified carbohydrate residues and their locations in the mucilage and cell walls of spores of Gelidium floridanum. Cell wall formation and mucilage composition were studied with calcofluor, toluidine blue (AT-O), alcian blue (AB) and periodic acid-Schiff (PAS). In the mucilage we identified α-D mannose, α-D glucose, β-D-galactose, N-acetyl-glucosamine and N-acetyl-galactosamine. The first two sugar residues were not found in the cell wall of the germ tube but they were present on the rhizoid’s cell wall indicating their importance to substrate adhesion. A cell wall is produced soon after the spore’s attachment, beginning with a polar deposition of cellulose and its gradual spread around the spore as indicated by calcofluor. The cell wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while cellulose microfibrills were positive to PAS. A polar disorganization of the cell wall triggers the process of germination. As spores are the natural form of propagation of Gelidium, the understanding of the mechanisms of spore attachment may contribute to the cultivation of this valuable seaweed.     

Holographic microscopy provides new insights into the settlement of zoospores of the green alga Ulva linza on cationic oligopeptide surfaces

Interaction of zoospores of Ulva linza with cationic, arginine-rich oligopeptide self-assembled monolayers (SAMs) is characterized by rapid settlement. Some spores settle (ie permanently attach) in a ‘normal’ manner involving the secretion of a permanent adhesive, retraction of the flagella and cell wall formation, whilst others undergo ‘pseudosettlement’ whereby motile spores are trapped (attached) on the SAM surface without undergoing the normal metamorphosis into a settled spore. Holographic microscopy was used to record videos of swimming zoospores in the vicinity of surfaces with different cationic oligopeptide concentrations to provide time-resolved insights into processes associated with attachment of spores. The data reveal that spore attachment rate increases with increasing cationic peptide content. Accordingly, the decrease in swimming activity in the volume of seawater above the surface accelerated with increasing surface charge. Three-dimensional trajectories of individual swimming spores showed a ‘hit and stick’ motion pattern, exclusively observed for the arginine-rich peptide SAMs, whereby spores were immediately trapped upon contact with the surface.     

Brefeldin A affects adhesion of zoospores of the green alga Enteromorpha.

Primary adhesion of zoospores of the green macroalga Enteromorpha to substrata involves a massive release of adhesive glycoproteins from Golgi-derived, membrane-bounded vesicles in the anterior region of the spore, followed by rapid curing. This process is sensitive to low concentrations (5-10 microg x ml(-1)) of the secretion-inhibiting antibiotic, brefeldin A (BFA). The proportion of cells that settled in BFA was reduced by approximately 50%, but the effect was fully reversed by washing in seawater to remove the BFA. Ultrastructural observations showed that BFA caused the breakdown of Golgi stacks in the majority of cells examined. When settled cells were subjected to shear stress, a greater proportion of those settled in the presence of BFA were detached, compared with controls, indicating reduced adhesion strength in the presence of the antibiotic. The most likely reason for this is that strong adhesion to substrata either requires the synthesis of extra adhesive materials beyond those present in the swimming spore, or the secretion of an additional component required for adhesive curing. The novel use of atomic force microscopy in force modulation mode demonstrated that the adhesive secreted by most spores in the presence of BFA did not undergo the rapid curing process typical of control spores. However, some variation between zoospores was observed, with some cells showing no ultrastructural changes and normal adhesive curing. These results are discussed in relation to variations observed in the propensity and competence of spores to settle, which may be reflected in differential requirements for de novo synthesis and secretion of materials needed for full adhesion.     

Ultrastructural,cytochemical, and enzymatic studies on the adhesive “plaques” of the brown algaeLaminaria saccharina (L.) lamour. andNereocystis luetkeana (nert.) post. et rupr. 1

Summary Newly settled zoospores of bothLaminaria saccharina andNereocystis luetkeana are surrounded by adhesive plaques. At this stage cell wall deposition has not occurred and similar structures cannot be detected in the cytoplasm. In free swimming zoospores, however, plaques are observed exclusively in small vesicles within the cytoplasm. Enzymatic extractions and cytochemical tests indicate that plaques are glycoproteid in nature. Studies on the influence of several enzymes on the attachment of zoospores show that plaques are involved in the adhesion of zoospores to the substratum.Project supported by NRC grant #A2288. Nereocystis data are part of a Ph.D. dissertation presented by this author to the University of British Columbia.     

Characterization of Laurencia arbuscula spore mucilage and cell walls with stains and FITC-labelled lectins

In most red algae, spores are liberated without a cell wall, within a sheath of mucilage that is responsible for its primary attachment. Utilizing fluorescent-labelled lectins, we identified carbohydrate residues and their location in the mucilage and cell walls of spores of Laurencia arbuscula. Cell wall formation and mucilage composition were studied with Calcofluor, Toluidine Blue (AT-O), Alcian Blue (AB) and periodic acid-Schiff (PAS). In the mucilage, we identified α-d-mannose, α-d-glucose, N-acetyl-glucosamine, N-acetyl-galactosamine and β-d-galactose. All sugar residues were found in the cell wall, in the spore body rather than in the rhizoid, which suggests that the residues may be related to initial substrate adhesion. A cell wall is produced soon after the spore's attachment, beginning with a deposition of cellulose around the spore, as indicated by Calcofluor. A polarization of the cell wall triggers the process of germination. The cell-wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while neutral polysaccharides were positive to PAS.     

PRIMARY ADHESION OF ENTEROMORPHA (CHLOROPHYTA,ULVALES) PROPAGULES: QUANTITATIVE SETTLEMENT STUDIES AND VIDEO MICROSCOPY1

Quantitative methods and associated kinetic analyses have been used for the first time to study detailed aspects of the settlement and adhesion of various types of Enteromorpha popagule. Time course experiments showed that quadri and biflagellate zoospores and zygotes adhered rapidly, but a proportion within any one population appeared to be incompetent at adhering to the substratum. Kinetic (Scatchard) analysis of adhesion experiments performed at a range of zoospore concentrations revealed density-dependent effects not previously reported, with positive cooperativity at low spore densities and negative cooperativity at high spore densities. High-resolution video microscopy was used for the first time to reveal details of the various stages in the settlement and adhesion of zoospores and zygotes. Novel observations were made of an initial, temporary phase of attachment via the apical papilla, followed by a permanent phase of commitment, characterized by discharge of adhesive-containing cytoplasmic vesicles, as the cell contracted against the surface, and adsorption of flagella. The phase of commitment was followed by exploitation of the surface through amoeboid-like movements at the interface. Gregarious settlement behavior was frequently observed leading to the formation of rafts of cells. The possible mechanisms and significance of density-dependent spore adhesion are discussed.     

THE STRUCTURE AND NANOMECHANICAL PROPERTIES OF THE ADHESIVE MUCILAGE THAT MEDIATES DIATOM‐SUBSTRATUM ADHESION AND MOTILITY1

We investigated the adhesive mucilage and mechanism of cell‐substratum adhesion of two benthic raphid diatoms, the marine species Craspedostauros australis E. J. Cox and the freshwater species Pinnularia viridis (Nitzsch) Ehrenberg. SEM images of P. viridis and C. australis cells revealed the presence of multistranded tethers that appear to arise along the raphe openings and extend for a considerable distance from the cell before forming a “holdfast‐like” attachment with the substratum. We propose that the tethers result from the elongation/stretching of composite adhesive mucilage strands secreted from raphes during the onset of cell adhesion and reorientation. Atomic force microscopy (AFM) force measurements reveal that the adhesive strands originating from the nondriving raphe of live C. australis and P. viridis are highly extensible and accumulate to form tethers. During force measurements tethers can be chemically stained and are seen to extend between the cantilever tip and a cell during elongation and relaxation. In most cases, AFM force measurements recorded an interaction with a number of adhesive strands that are secreted from the raphe. The force curves of C. australis and P. viridis revealed a sawtooth pattern, suggesting the successive unbinding of modular domains when the adhesive strands were placed under stress. In addition, we applied the “fly‐fishing” technique that allowed the cantilever, suspended a distance above the cell, to interact with single adhesive strands protruding from the raphe. These force curves revealed sawtooth patterns, although the binding forces recorded were in the range for single molecule interactions.     

Adhesion Strength of Settled Spores of the Green Alga Enteromorpha

Strengths of attachment of spores of the green fouling alga Enteromorpha to glass have been measured using a modified water jet apparatus. Surface pressures of ～250 kPa were required to quantitatively remove attached spores after 4 h contact with a surface. The development of adhesive and cohesive strength is highly time-dependent; after 8 h in contact with a surface spores did not detach, even at pressures in excess of 250 kPa. Spores settled in groups are more resistant to detachment than single spores, which suggests that the adaptive value of gregarious settlement behaviour may lie in the greater resistance of groups to detachment forces in a naturally turbulent environment. The interfacial forces exerted as water impinges on the surface and the derivation of adhesion strength values in terms of wall shear stress are discussed and compared with those obtained by other methods. A surface pressure of 250 kPa approximates to 325 Pa wall shear stress. Calculation using the power-law formula predicts that detachment forces of this magnitude are unlikely to be realized at operating speeds for most vessels and that most Enteromorpha spores would not detach from untreated hulls.     

The Use of Ferritin Labelled Antibodies in the Location of Spore and Vegetative Antigens of Bacillus cereus

S ummary . Ferritin labelled antibodies have been used to detect the presence of vegetative antigens in spores. The specificity of the labelled antibodies was demonstrated. Vegetative antigens were found along the core or cortical membrane in spore disintegrates, and after germination along the developing vegetative cell wall. The implications of these results are discussed in relation to morphological studies of ultra-thin sections.     

Engineered antifouling microtopographies: kinetic analysis of the attachment of zoospores of the green alga Ulva to silicone elastomers

Microtopography has been demonstrated as an effective deterrent to biofouling. The majority of published studies are fixed-time assays that raise questions regarding the kinetics of the attachment process. This study investigated the time-dependent attachment density of zoospores of Ulva, in a laboratory assay, on a micropatterned and smooth silicone elastomer. The attachment density of zoospores was reduced on average 70–80% by the microtopography relative to smooth surfaces over a 4 h exposure. Mapping the zoospore locations on the topography revealed that they settled preferentially in specific, recessed areas of the pattern. The kinetic data fit, with high correlation (r ² > 0.9), models commonly used to describe the adhesion of bacteria to surfaces. The grouping of spores on the microtopography indicated that the pattern inhibited the ability of attached spores to recruit neighbors. This study demonstrates that the antifouling mechanism of topographies may involve disruption of the cooperative effects exhibited by fouling organisms such as Ulva.     

The physico-chemical characterization of casein-modified surfaces and their influence on the adhesion of spores from a Geobacillus species

To gain a better understanding of the factors influencing spore adhesion in dairy manufacturing plants, casein-modified glass surfaces were prepared and characterized and their effect on the adhesion kinetics of spores from a Geobacillus sp., isolated from a dairy manufacturing plant (DMP) was assessed using a flow chamber. Surfaces were produced by initially silanizing glass using (3-glycidyloxypropyl) trimethoxysilane (GPS) or (3-aminopropyl) triethoxysilane to form epoxy-functionalized (G-GPS) or amino-functionalized glass (G-NH₂) substrata. Casein was grafted to the G-GPS directly by its primary amino groups (G-GPS-casein) or to G-NH₂ by employing glutaraldehyde as a linking agent (G-NH₂-glutar-casein). The surfaces were characterised using streaming potential measurements, contact angle goniometry, infrared spectroscopy and scanning electron microscopy. The attachment rate of spores suspended in 0.1 M KCl at pH 6.8, was highest on the positively charged (+14 mV) G-NH₂ surface (333 spores cm^?2 s^?1) compared to the negatively charged glass (?22 mV), G-GPS (?20 mV) or G-GPS-casein (?21 mV) surfaces (162, 17 or 6 spores cm^?2 s^?1 respectively). Whilst there was a clear decrease in attachment rate to negatively charged casein-modified surfaces compared to the positively charged amine surface, there was no clear relationship between surface hydrophobicity and spore attachment rate.     

Ultrastructure and chemical composition of the ballistospore wall ofConidiobolus obscurus

The ballistospores of the entomopathogenConidiobolus obscurus are spheroidal cells with a papilla corresponding to the zone of attachment on the sporophore. They are covered by a mucus responsible for the attachment of the spore to the insect. Chemical and cytochemical investigations of the nature of the wall components have been undertaken to better understand fungus-insect interactions in entomopathology. β(1→3)-Glucans and chitin together represented the main components of the wall which did not contain chitosan and uronic acids. Transmission electron microscopy revealed that the spore wall was composed of a thick electron-lucent inner layer and a thin outer electron-dense layer, the latter being absent at the papilla region. The spore is covered by a mucilaginous layer that upon spore impact on a substratum, is dispersed and forms a halo around the spore. Shadow replicas of the discharged spores showed that they are covered by rodlets except on the papilla which displayed a chitinous, microfibrillar structure. The ontogeny of the rodlets deposited on the surface of young spores was characterized by a progressive organization of separate rodlets and then a clustering of the rodlets in fascicles. Shadow replicas and chemical and enzymatic investigations of the halo surrounding discharged spores showed that the mucus was composed of long β(1→3)-glucan microfibrils embedded in amorphous proteins partly covered by rodlets discharged from the spore surface.     

Individual and Coupled Effects of Echinoderm Extracts and Surface Hydrophobicity on Spore Settlement and Germination in the Brown Alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30?min were >?400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were >?300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30?min and the settled spores allowed to subsequently germinate for 24?h. Spore germling numbers were consistently >?25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24?h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30?min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24?h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.     

VIRUSLIKE PARTICLES IN THE MARINE ALGA CHORDA TOMENTOSA LYNGBYE (PHAEOPHYCEAE)1

Polyhedral viruslike particles 170 nm in diameter were found in 3-day-old spores of the brown alga Chorda tomentosa Lyngbye grown in unsterilized seawater. In sterile seawater, spores of the same species were free of these particles and cell wall development around naked settled, zoospores and subsequent spore germination proceeded, normally. Spores containing the putative virus particles did not develop cell walls or show signs of germination. Severe lysis of cell organelles always accompanied, the presence of these particles.     

Effect of Substratum Surface Chemistry and Surface Energy on Attachment of Marine Bacteria and Algal Spores

Two series of self-assembled monolayers (SAMs) of ω-substituted alkanethiolates on gold were used to systematically examine the effects of varying substratum surface chemistry and energy on the attachment of two model organisms of interest to the study of marine biofouling, the bacterium Cobetia marina (formerly Halomonas marina) and zoospores of the alga Ulva linza (formerly Enteromorpha linza). SAMs were formed on gold-coated glass slides from solutions containing mixtures of methyl- and carboxylic acid-terminated alkanethiols and mixtures of methyl- and hydroxyl-terminated alkanethiols. C. marina attached in increasing numbers to SAMs with decreasing advancing water contact angles (θ_AW), in accordance with equation-of-state models of colloidal attachment. Previous studies of Ulva zoospore attachment to a series of mixed methyl- and hydroxyl-terminated SAMs showed a similar correlation between substratum θ_AW and zoospore attachment. When the hydrophilic component of the SAMs was changed to carboxylate, however, the profile of attachment of Ulva was significantly different, suggesting that a more complex model of interfacial energetics is required.     

Evaluation of the attachment strength of individuals of Asterina gibbosa (Asteroidea,Echinodermata) during the perimetamorphic period

Abstract

A turbulent channel flow apparatus was used to determine the adhesion strength of the three perimetamorphic stages of the asteroid Asterina gibbosa, i.e. the brachiolaria larvae, the metamorphic individuals and the juveniles. The mean critical wall shear stresses (wall shear stress required to dislodge 50% of the attached individuals) necessary to detach larvae attached by the brachiolar arms (1.2 Pa) and juveniles attached by the tube feet (7.1 Pa) were one order of magnitude lower than the stress required to dislodge metamorphic individuals attached by the adhesive disc (41 Pa). This variability in adhesion strength reflects differences in the functioning of the adhesive organs for these different life stages of sea stars. Brachiolar arms and tube feet function as temporary adhesion organs, allowing repetitive cycles of attachment to and detachment from the substratum, whereas the adhesive disc is used only once, at the onset of metamorphosis, and is responsible for the strong attachment of the metamorphic individual, which can be described as permanent adhesion. The results confirm that the turbulent water channel apparatus is a powerful tool to investigate the adhesion mechanisms of minute organisms.     

Identification of two putative adhesive polypeptides in Caulerpa prolifera rhizoids using an adhesion model system

The rhizoid section of the green alga Caulerpa prolifera (Cp) is active in attaching the developing plant to the substratum. A model system for the study of the adhesion of Cp rhizoids has been developed and identification of two putative adhesive polypeptides of Caulerpa (Vn-Cp) was revealed by immunodetection. A method for fast induction of new rhizoids was established using blade-base cutting followed by a few days of incubation. The new rhizoids were gently enclosed between two cover glasses and incubated until firm attachment developed. While analyzing protein extracts, two ∼60–70 kDa polypeptides (Vn-Cp I and Vn-Cp II) were identified by immunodetection with monoclonal antibodies to human vitronectin (Vn). The relative concentration values of the Vn-Cp proteins increased significantly in the ‘cell-wall’ fraction of the attached rhizoids during the incubation period. However, Vn-Cp proteins were not detected in non-attached rhizoids. Furthermore, the Vn-Cp proteins were also detectable on glass substratum subsequent to attached rhizoid removal. The induction and accumulation of Vn-Cp proteins on the ‘cell-wall’ of Caulerpa rhizoids and the firm attachment of the rhizoids to the glass substratum during the incubation period suggest that Vn-Cp proteins play a significant role in adhesion, which may be similar to the function of vitronectin in other adhesion systems. Furthermore, the high accumulation of Vn-Cp proteins on the glass substratum during attachment of new rhizoids suggests that the Vn-Cp proteins are secreted to the extracellular matrix and directly connect rhizoids to the glass substratum as an intermediate compound. These unique properties of Cp make it an excellent model system for the establishment of high amounts of adhesive material for future research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Encystment of Pythium aphanidermatum Zoospores is Induced by Root Mucilage Polysaccharides, Pectin and a Monoclonal Antibody to a Surface Antigen

The infection of roots by the pathogenic Oomycete Pythium aphanidermatuminvolves interactions between the fungal zoospores and rootsurface mucilage polysaccharides. After initial recognitionat the root surface the zoospores are triggered to encyst duringwhich adhesive glycoproteins are secreted followed by a fibrillarcyst wall. In this paper a simple in vitro assay has been usedto assess the ability of a variety of macromolecules to inducezoospore encystment. Mucilage polysaccharides of the cress rootsurface trigger encystment. Whole mucilage was fractionatedby gel filtration and a fraction low in uronic acid, containing5% fucose, was shown to be more effective in triggering encystmentthan a uronic acid-rich fraction. Encystment can also be inducedby commercial pectin. The lectin Con A, and PA1, one of a rangeof monoclonal antibodies specific for zoospore surface antigens,also triggered encystment. In Western blotting experiments PA1recognizes protein epitopes of a 75 kDa surface antigen. Theresults suggest that at least one mechanism of zoospore triggeringmay involve a specific zoospore surface receptor. Key words: Pythium aphanidermatum, recognition, encystment, zoospore, mucilage, root, monoclonal antibodies, polysaccharides     

Use of Yeast Spores for Microencapsulation of Enzymes

Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose. This suggests that substrate selectivity could be altered by the encapsulation.