Department of Inland Water Research Magdeburg, UFZ Centre for Environmental Research, Leipzig-Halle, Brueckstrasse 3A, 39114 Magdeburg, Germany1
National Water Research Institute, Saskatoon, Saskatchewan, Canada2
Author for correspondence: Thomas R. Neu. Tel: +49 391 8109 800. Fax: +49 391 8109 150. e-mail: neu{at}gm.ufz.de
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ABSTRACT |
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Keywords: biofilms, lectins, extracellular polymeric substances, glycoconjugates, confocal laser scanning microscopy
Abbreviations: CLSM, confocal laser scanning microscopy; CY5, cyanine dye; EPS, extracellular polymeric substances; FITC, fluorescein isothiocyanate; ICBA, intensity-corrected binding area; LSD, least significant difference; TRITC, tetramethyl rhodamine isothiocyanate; UEA-I, Ulex euroaeus lectin I
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INTRODUCTION |
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Labelled lectins have been successfully used in many microbial pure culture studies to probe for cell-surface structures. They were employed in transmission electron microscopy studies of the bacterial capsule (Vasse et al., 1994 ), cell surface localization of specific carbohydrates (Jones et al., 1986
; Morioka et al., 1987
), detection of slime in biofilms (Sanford et al., 1995
) and characterization of adhesive surface appendages e.g. holdfasts (Merker & Smit, 1988
; Hood & Schmidt, 1996
). Fluor-conjugated lectins have been used in flow cytometry studies to compare the cell surface chemistry versus the cell agglutination behaviour (Yagoda-Shagam et al., 1988
). Most investigations using fluor-conjugated lectins were done using binding assays and subsequent epifluorescence microscopy. Thereby, the special features of the Caulobacter holdfast (Merker & Smit, 1988
; Ong et al., 1990
), the adhesive capsule of Hyphomonas (Quintero & Weiner, 1995
) and microbial footprints (Neu & Marshall, 1991
) were examined. Furthermore, lectins have been employed as an enzyme-linked lectin-sorbent assay to quantify biofilm development (Thomas et al., 1997
). Finally, there are the first reports on the application of lectins in complex environments e.g. marine habitats (Michael & Smith, 1995
) as well as freshwater systems (Neu & Lawrence, 1997
; Lawrence et al., 1998a
; Neu, 2000
) and multi-species biofilm communities (Wolfaardt et al., 1998
). Thus lectins may represent a useful probe for in situ techniques to three-dimensionally examine the distribution of glycoconjugates in fully hydrated environmental biofilm systems (Neu & Lawrence, 1999
).
For the evaluation of lectin-binding analysis, the structural complexity of microbial polysaccharides must be considered. The potential of carbohydrates to encode information in terms of saccharides is even larger than that of amino acids and nucleotides. The latter two compounds can only build one dimer whereas one type of monosaccharide can form 11 different disaccharides. Further, four monosaccharides, which is a common number in the repeating unit of polysaccharides, may form 35560 different disaccharides (Sharon & Lis, 1989 ). This demonstrates the enormous variety of polysaccharides that may be expected from the diversity of microbial species that may colonize and form biofilms at environmental interfaces. In addition, it explains why specificity in nature is written in the form of saccharides. However, this may also indicate the possible difficulties and limitations in the applicability of lectin-binding analysis for the complex matrices common to environmental biofilm systems. The key interactions of lectins with carbohydrates are stereochemically via hydrogen bonds, metal coordination, and van der Waals and hydrophobic interactions (Liener et al., 1986
; Weiss & Drickamer, 1996
; Elgavish & Shaanan, 1997
). Generally, lectins interact with their target through multiple binding sites, which increases affinity and specificity. These basic principles also apply to lectin interactions within complex interfacial microbial communities in the environment.
In this study lectin-binding analysis is described as a technique to probe for glycoconjugate distribution in environmental model biofilms grown with river water as the sole source of carbon and nutrients. The biofilms were stained with various fluor-conjugated lectins and three-dimensionally examined by confocal laser scanning microscopy (CLSM). The staining technique was critically assessed using several control experiments in order to refine lectin-binding analysis as an in situ tool in biofilm research.
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METHODS |
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CLSM and image analysis.
Examination of all stained and control materials was carried out using an MRC 1000 confocal laser scanning microscope (Bio-Rad) attached to a Microphot SA microscope (Nikon). For observation, the following water-immersible lenses were used: 63x0·9 NA (Zeiss) and 40x0·55 NA (Nikon). Signals were recorded in the green channel (excitation 488 nm, emission 522/32 nm), red channel (excitation 568 nm, emission 605/32 nm) and far-red channel (excitation 647 nm, emission 680/32 nm). Image analyses were performed using NIH Image version 1.61 (http://rsb.info.nih.gov/nih-image/). Additional information on CLSM and image analysis may be found in Lawrence et al. (1998b ). Files were printed from Photoshop 5.5 (Adobe) using a UP-D8800 digital printer (SONY).
Experimental design, sampling and statistical analysis.
A series of replicated experiments were carried out to assess the effects of incubation time, concentration, the nature of the fluorescent conjugate (FITC, TRITC or CY5), the order of addition of the lectins, interactions between lectins, the presence of inhibiting carbohydrates and the effect of the biofilm matrix. A series of CLSM images were taken at five random locations on all control and treatment slide pieces. Controls consisted of unstained slide pieces which were examined and used to determine the laser intensity, pinhole, gain and voltage settings of the CLSM at which no signal was detected. Settings were then adjusted using stained material and these levels held constant throughout the collection of experimental image series to provide a basis for comparison of treatment effects.
Statistical analyses were done by using a one way analysis of variance (ANOVA). Pair wise comparisons following a significant ANOVA were conducted using Fisher least significant difference (LSD) test. Differences were considered to be significant at P0·05.
Quantitative estimation of lectin binding.
Although it is complicated to determine the exact thickness of an optical section, our observations indicate that when imaging with a 40x0·55 NA water-immersible lens, the optical thin section is approximately 5 µm. Calculations of the theoretical thickness of an optical section taken with this lens indicate a thickness of 4 µm (Xiao & Kino, 1987 ). Therefore any single optical section in our calculation was assumed to be 5 µm in thickness.
Image analysis was used to define the area of the biofilm binding a specific lectin. In addition, the mean grey value of the defined area was determined. These two parameters were used to quantify the area binding a specific lectin according to equation (1).
![]() | (1) |
% ICBA, intensity-corrected binding area; TA, thresholded area of lectin binding; AGV, average grey value within thresholded area; 255=grey value of saturated pixels; 393216=number of pixels in a full image (768x512).
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RESULTS |
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DISCUSSION |
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Quantitative analyses of lectin binding
Assessment of the time for complete lectin binding to occur is essential to a practical reproducible assay. The results of our study indicated that an interval of 20 min was optimal for staining of complex lotic river biofilm materials. Previous studies (Merker & Smit, 1988 ; Michael & Smith, 1995
; Hood & Schmidt, 1996
) also used 20 min, whereas Quintero & Weiner (1995)
used a 30 min incubation. Thus the incubation time for the essential assay is well established. The influence of lectin concentration is also a methodological consideration. In this study we were unable to find a saturating concentration for any lectin tested. This was in part due to the lack of dynamic range of the CLSM photomultipliers; once the photomultiplier is saturated no further effect of increasing lectin binding can be detected. However, by readjusting the gain, pinhole and laser intensity it was possible to obtain an image with a normal distribution of pixel intensity (0255) regardless of the lectin concentration added. Therefore we selected a standard concentration of lectin for the experiments.
Usually, two different types of signals are obtained in lectin-binding analyses of biofilms. Fluor-conjugated lectins stain cell surface carbohydrate features of prokaryotes and eukaryotes thereby giving a signal which defines the outline of the cell (Fig. 3b, c
). In addition, lectins stain excreted and released carbohydrates present in the form of polysaccharides as part of the EPS matrix in biofilm systems. This type of signal has a more cloud-like appearance and is usually associated with microcolonies and cell clusters (Fig. 3a
, e
).
In general, the specificity of lectins is ascribed to the manufacturer (i.e. Sigma, Molecular Probes, EY Laboratories) and is based on the influence of lectins on blood cell agglutination. Therefore, lectin specificities are indicated in most publications as for example, Tri. vulgaris and Banderia simplicifolia are lectins specific for N-acetylglucosamine, and U. europeaus is specific for L-fucose (see for example, Merker & Smit, 1988 ; Michael & Smith, 1995
; Quintero & Weiner, 1995
; Hood & Schmidt, 1996
; Lawrence et al., 1998a
; Neu & Lawrence, 1997
; Wolfaardt et al., 1998
). In an almost unique case, Quintero & Weiner (1995)
used a haemagglutination assay with a purified bacterial EPS to demonstrate the specific binding of Bauhinia purpurea lectin to the capsule of the bacterial strain MHS-3. However, such confirmation may be virtually impossible for complex biofilm matrices. In the present study we applied a simple assay using a relatively high concentration of inhibitory carbohydrate (Table 3
). The concentrations were within the range used in haemagglutination assays and those used by other authors to assess inhibition of lectin binding in other microbial systems (Merker & Smit, 1988
; Quintero & Weiner, 1995
; Michael & Smith, 1995
). Although a range of evaluation techniques have been used to assess lectin binding in microbial studies such as flow cytometry, electron microscopy and enzyme-linked immunoassays, the most common method of examining lectin binding has been standard epifluorescence microscopy and visual scoring of binding (Merker & Smit, 1988
; Michael & Smith, 1995
). We adapted the quantitative method of Wolfaardt et al. (1998)
involving the application of CLSM coupled with fluor-conjugated lectins to rapidly and quantitatively probe the spatial relationships of EPS components within these lotic biofilm communities. In this system there were sufficient quantities of glycoconjugates to be easily visualized using CLSM. The advantages of this approach are greater sensitivity than the dark-adapted eye, and the images can be collected under standardized conditions of specimen preparation (lectin concentration, incubation conditions), image collection (laser power, voltage, gain, pinhole) and digital image analysis techniques. We also introduced the concept of a standardized method for quantitative analysis and expression of lectin binding as percentage of ICBA which includes both binding and intensity of binding, allowing statistical evaluation of treatment effects on lectin binding.
Influence of the fluor conjugate
Although it is generally assumed that conjugation with a fluor does not influence the specificity of a lectin, it is apparent from the manufacturers data sheets that affinities as indicated by level of carbohydrate required for inhibition are influenced by both the presence and the nature of the fluor. In the present study we showed that all three fluorescent labels (FITC, TRITC and CY5) tested are not equally well suited for lectin-binding analysis in complex environmental systems. CY5-labelled lectins are not ideal due to their emission in the far-red part of the spectrum which overlaps with the autofluorescence signal of chlorophyll-containing organisms, e.g. algae (Lawrence et al., 1998a ). For TRITC-labelled lectins used in the inhibition experiments the expected decrease in signal with increasing sugar concentrations was not found. This was in contrast to experiments with FITC-labelled lectins where a clear decrease in signal was found when selected sugar concentrations were increased. This has, to the authors knowledge, not been reported in the literature as other authors have attempted to confirm the identity of glycoconjugate binding sites through fairly limited inhibition studies. For example, Merker & Smit (1988)
demonstrated inhibition of binding of Tri. vulgarisFITC lectin to Caulobacter holdfasts by N-acetylglucosamine monomers and oligomers. They determined that trimers were most effective in inhibition of lectin binding and suggested that this indicated the presence of stretches of contiguous N-acetylglucosamine residues in holdfast material. Michael & Smith (1995)
demonstrated inhibition of C. ensiformisFITC with
-methyl D-mannoside. Our observations indicated that FITC-conjugated lectins exhibited binding patterns closer to the predicted outcomes when used in the biofilm systems. Thus, particularly with regard to interpretation of EPS chemistry, FITC-conjugated lectins may be the better choice to stain carbohydrate structures in biofilm systems. The interpretation of binding by CY5- and TRITC-labelled lectins may be confounded by the fact that the molecules are charged and that TRITC is zwitterionic in nature. This ionic character may have an effect on the lectin binding interaction with biofilm components. For example, the TRITC lectins often show extensive general binding to biofilm material with little apparent localization, consistent with non-specific binding as noted in a pure culture biofilm study (Johnsen et al., 2000
).
Lectin specificity in the biofilm matrix
The effect of carbohydrate inhibition on lectin binding was tested with carbohydrates that have been indicated by the supplier to inhibit lectin binding. Lectin specificity has been largely based on the results of inhibition of haemagglutination with a specific range of carbohydrates. As biofilms contain a broad range of polysaccharide compounds including a large variety of chemical structures containing carbohydrates with the following features: neutral sugars, uronic acids, amino sugars, acetylated and methylated as well as many other residues (Kenne & Lindberg, 1983 ; Sutherland, 1996
; Lindberg, 1990
; Sanford et al., 1995
), additional inhibition studies were deemed necessary. A variety of other carbohydrates were selected to test their effect on lectin binding in the biofilm matrix. The data showed a complex pattern of inhibition, enhancement and no effect of the inhibiting carbohydrate, which was further confounded by the nature of fluor conjugated to the lectin molecule (Tables 1
, 2
, 4
and 5
). Carbohydrate inhibition patterns for specific lectins were in general broader than indicated by the existing database. For example, although U. europeausFITC binding was inhibited by fucose as expected, significant reductions were also observed in the presence of arabinose, fructose, glucuronic acid, melibiose, methyl
-pyrannoside, raffinose, glucose, mannose, galactose and rhamnose. Furthermore, with several of the non-lectin-specific carbohydrates a surprising enhancement of lectin binding was found. Similar observations were made during experiments using lectins and carbohydrates in ELISA tests (T. Bog-Hanson, personal communication). The enhancement of lectin binding may be simply based on the inadequacy of existing databases regarding the range of carbohydrates showing interactions with lectins. Alternatively, the enhancement of lectin binding may be due to the presence of multiple carbohydrate binding sites. The addition of a specific carbohydrate may focus the second binding site and thereby may result in a higher binding affinity for another carbohydrate. This may provide a partial explanation for the observations summarized in Table 6
showing the changes in impact of carbohydrates between the 1997 and 1998 biofilms, where the same carbohydrate was observed to inhibit lectin binding in one year and enhance it in another. These shifts presumably reflect changes in EPS chemistry present in biofilms of different age, history etc. Consequently, lectin binding will be different and thus inhibiting mono-carbohydrates may act differently during inhibition tests. Furthermore, the effect may be influenced by the non-carbohydrate binding site of the lectin which may recognize protein in the biofilm matrix (Ochoa et al., 1981
; Barondes, 1988
; Sharon & Lis, 1989
; Elgavish & Shaanan, 1997
). Our observations on the binding of Tetragonolobus and Ulex lectins, both with specificity for fucose, indicate that relative affinity for binding sites may be an important factor during multiple lectin staining. In this case Tet. purpureasTRITC lectin has a 101000-fold higher affinity according to the suppliers data sheet and in our study substantially alters the binding pattern of UEA-IFITC. These observations indicate that a test series using an extensive range of relevant carbohydrates is required to fully evaluate the binding characteristics of a lectin prior to selection of a panel of lectins for analysis of biofilms. The determination of a relevant or focused carbohydrate inhibition panel may also require more extensive chemical characterization of the EPS matrix prior to in situ analyses using lectin techniques.
The results of the carbohydrate inhibition experiments also indicated that the lectins from Tri. vulgaris, A. hypogaea and L. polyphemus (given the wide range of carbohydrates that inhibit their binding in biofilms) may be useful as general EPS stains. Lawrence et al. (1998a ) previously suggested that Tri. vulgarisFITC or TRITC may act as a general EPS probe for river biofilms.
Role of interactions between lectins
CLSM provides the opportunity to carry out multiple staining. Therefore it is important to assess the effects of interactions between lectins prior to multiple staining experiments. In experiments with dual-lectin staining, the order of lectin addition as well as the specific fluorescent labels were varied. Experiments evaluating the effect of order of addition indicated that the presence of one lectin may impact positively, negatively or have no effect on the binding of any subsequent lectin. These observations may be explained by a number of factors: (i) the differences in molecular mass and thereby the different mobility of the lectins (Lawrence et al., 1994 ; De Beer et al., 1997
); (ii) the net charge of the lectin/fluorochrome combination may also exclude or provide additional binding sites for subsequent lectins; (iii) the binding of the first lectin may also result in the masking of specific regions of mono-/di-/oligo-saccharides in the matrix by creating new targets that are recognized by subsequent lectins; (iv) the first lectin may simply provide binding sites for the subsequent lectin. For example, we observed formation of precipitates when C. ensiformisTRITC was combined with Abrus precatoriusFITC lectin, indicating a strong reaction between the probes. In the case of enhancement of binding of U. europeausFITC by the presence of Tet. purpureasTRITC (Tables 1
and 2
), it may be that the TRITC-labelled lectin had created an altered target for the second lectin. In addition, the number and types of binding sites (carbohydrate and protein) and the relative binding affinity of the lectins for certain mono-/di-/oligo-saccharides may also have an effect on multiple-lectin binding. There are only a few cases where the interaction of the lectin with its carbohydrate target is well understood (e.g. Gohier et al., 1996
). Thus it is important to assess the potential for lectin interactions prior to any multiple-labelling experiments. Indeed in some cases, lectin interactions may preclude their combined application.
Lectin selection
The lectins described in the literature are isolated mainly from plants and other eukaryotic organisms (Brooks et al., 1997 ; Van Damme et al., 1997
; Singh et al., 1999
). These types of lectins may not be the ideal lectin source for probing the EPS matrix within environmental microbial biofilm systems. In general it may be more appropriate to apply bacterial- or algal-specific lectins, which may have direct access to the typical bacterial or algal glycoconjugates present in biofilm systems. These may be lectins specific for certain unique monosaccharides, a typical di-/oligosaccharide sequence within a repeating unit or even for a certain type of polysaccharide e.g. alginate or emulsan (Sutherland, 1996
).
To utilize lectin-binding analysis for chemical characterization of biofilms, the complete carbohydrate inhibition spectrum of a given lectin should be known. However, this is generally not the case as the standard catalogue lectins are, according to the data sheet, tested only against a few carbohydrates. These carbohydrates are usually those which are important for cell biological research and they may not necessarily be present in the EPS of natural biofilms. In addition, other specific glycoconjugates may be unique to bacteria and biofilms such as, for example, alginate (Christensen, 1999 ). As a consequence, each biofilm system should be tested against a well selected range of lectins with well defined inhibition patterns for the matrix under study. Furthermore, knowledge about the secondary carbohydrate binding specificity of the lectin may be important for the understanding of their binding behaviour. This binding specificity may be for the same or another carbohydrate (Barondes, 1988
; Reeke & Becker, 1988
; Weiss & Drickamer, 1996
).
In conclusion, lectin-binding analysis may be a suitable in situ technique to probe microbial biofilm systems for glycoconjugate distribution. However, each system has to be tested against a set of different lectins to exclude some of the above-mentioned uncertainties. Apart from the carbohydrate specificity, the secondary specificity for non-carbohydrates (e.g. proteins) should also be known. Additional uncertainties arise from the nature of the complex biofilm matrix including the absence of a true target for the lectin employed, the enormous variety of potential monosaccharide combinations, the presence of many cell surface types in biofilms, diversity of eukaryotic and prokaryotic EPS types and the presence of confounding biofilm components such as humic compounds. Further uncertainties arise from the properties of the lectin; these include the effect of the fluorescent label, the actual chemistry of the fluorescent label, the influence of fluor conjugation on the active site of the lectin and the possibility of either charged, hydrophobic or other non-specific interactions. Nevertheless, in opposition to the wet chemistry approach, the lectin approach is able to examine the fully hydrated EPS matrix in environmental biofilm systems for chemical heterogeneities without the need of isolating single polysaccharides. Clearly there is also a need for the chemical characterization of complex biofilm matrices. In this respect, the lectins currently available may be a valuable tool to estimate the glycoconjugate distribution in biofilm systems in complex natural as well as in artificial environments. This information is important for understanding the sorption properties of the EPS in relation to, for example, nutrients as well as elemental and organic contaminants. The isolation and characterization of new lectins, as suggested, may provide in the future the ultimate probes to unequivocally detect biofilm specific glycoconjugate distribution in fully hydrated and complex biofilm systems.
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ACKNOWLEDGEMENTS |
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Received 22 May 2000;
revised 9 October 2000;
accepted 1 November 2000.