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FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components

     Life and Health Sciences, Aston University, Birmingham, B4 7ET, United Kingdom

Roger A. Bird

     The Medical School, University of Birmingham, Birmingham, B15 2TJ, United Kingdom

Steven L. Kelly

     Wolfson Laboratory of P450 Biodiversity, Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, SY23 3DA, Wales, United Kingdom

Kazuko Nishimura

     Research Centre for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1–8–1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260–8673 Japan

David Poyner

     Life and Health Sciences, Aston University, Birmingham, B4 7ET, United Kingdom

Stephen Taylor

     Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, SY23 3DA, Wales, United Kingdom

Stephen N. Smith ¹

     Life and Health Sciences, Aston University, Birmingham, B4 7ET, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Flow cytometry and confocal microscopy were used to quantify and visualize FITC-lectin binding to cell-surface carbohydrate ligands of log and stationary phase acapsular and capsular Cryptococcus neoformans strains. Cell populations demonstrated marked avidity for terminal -linked mannose and glucose specific FITC-Con A, mannose specific FITC-GNL, as well as N-acetylglucosamine specific FITC-WGA. Exposure to other FITC-lectins specific for mannose, fucose and N-acetylgalactosamine resulted in little cell-surface fluorescence. The nature of cell-surface carbohydrates was investigated further by measurement of the fluorescence from surfaces of log and stationary phase cell populations after exposing them to increasing concentrations of FITC-Con A and FITC-WGA. Cell fluorescence increased significantly with small increases in FITC-Con A and FITC-WGA concentrations attaining reproducible maxima. Measurements of this nature supported calculation of the lectin binding determinants EC 50, Hn, Fmax and relative Bmax values. EC50 values indicated that the yeast-cell surfaces had greatest affinity for FITC-WGA, however, relative Bmax values indicated that greater numbers of Con A binding sites were present on these same cell surfaces. Hn values suggested a co-operative lectin-carbohydrate ligand interaction. Imaging of FITC-Con A and FITC-WGA cell-surface fluorescence by confocal microscopy demonstrated marked localization of both lectins to cell surfaces associated with cell division and maturation, indicative of dynamic carbohydrate ligand exposure and masking. Some fluorescence was associated with entrapment of FITC-Con A by capsular components, but FITC-Con A and FITC-WGA readily penetrated the capsule matrix to bind to the same cell surfaces labelled in acapsular cells.

Key words: confocal microscopy, Cryptococcus, FITC-lectins, flow cytometry

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Fungal cell-wall composition, architecture and cell-surface nature have received considerable attention. A typical ascomycete or basidiomycete fungal cell wall is composed predominantly of N-acetyl glucosamine, in the form of chitin microfibrils, overlain with gel-like glycoproteins, many of a mannose nature (Gooday 1995). Chitin microfibrils are responsible for the architecture and structural integrity of cell walls, while matrix-forming glycoproteins in outer wall layers are associated with a number of cell-surface phenomena (Gooday 1995).

However, as noted by Casadevall and Perfect (1998), cryptococcal cell walls remain largely uncharacterized. The intimate association of cryptococcal cell walls with a distinctive enveloping capsule hinders isolation and purification of cell-wall constituents, thereby compromising an understanding of cryptococcal cell-wall composition, structure and associated biochemistry. Studies focussed on the acapsular strain Cap 67, indicated that Cryptococcus cell walls differ from other basidiomycetes in composition and structure. James et al (1990) demonstrated that cell walls of Cap 67 were composed primarily of water-soluble and water-insoluble glucopyrannans. In contrast to other yeasts, little N-acetyl glucosamine or mannoprotein were isolated from the cell walls of acapsular Cap 67. However, mannose forms a major part of the unique Cryptococcus capsule and has been shown to elicit considerable interleukin synthesis and concomitant interferon release potentially modulating an immune response in vertebrates (Pitzurra et al 2000, Pietrella et al 2001). Most capsular mannose exists as the polysaccharide glucoronoxylomannan (GXM), forming up to 88% of capsular material (Cherniak and Sundstrom 1994). GXM is composed of a core of repeating mannan residues, which do not readily bind Con A (Cherniak et al 1982), indicating that no mannose residues are terminally exposed.

Smith et al (1999, 2001) have used flow cytometry, image analysis protocols and panels of FITC-lectins to characterize carbohydrate exposure on filamentous and unicellular fungal cells. Mannose, N-acetyl galactosamine and N-acetyl glucosamine residues were exposed in varying amounts on the surfaces of maturing Coniothyrium conidia and in different patterns on germling surfaces, while yeasts and filamentous Candida surfaces demonstrated marked avidity for mannose, glucose and N-acetyl glucosamine binding lectins. Furthermore, yeast, pseudohyphal and hyphal regions varied significantly in fluorescence when exposed to FITC-Con A, thereby furnishing some insight into the nature of cell-surface mannose and glucose exposure with Candida morphology. Employing a similar approach of flow cytometry, confocal microscopy and a panel of FITC-lectins, this study investigated Cryptococcus neoformans cell-surface lectin avidity and carbohydrate exposure.

	MATERIALS AND METHODS

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Isolates and culture methods – Four isolates of C. neoformans, designated CN IRM 5815, CN IRM 5854, CN IRM 45922 and 52817 (Cap 67) were obtained from the American Type Culture Collection (ATCC), Manassas, Virginia, and the Research Centre for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Japan. These isolates were selected by such criteria as presence or absence of capsule as visualized with Indian ink stain, productivity and ease of harvesting. Cap 67 is a defined acapsular strain derived by chemical mutagenesis (Jacobson and Tingler 1994). The other isolates were defined by the Mycology Reference Laboratory, Bristol Public Health Laboratory, Bristol, U.K., as: CN IRM 5815 variant gattii, serotype unknown, large capsule; CN IRM 5854 and CN IRM 45922 variant neoformans, serotype A. Isolate stocks were maintained in Sabouraud dextrose broth (Lab M, U.K.) supplemented with glycerol (10% v/v) and stored at -70 C. Subcultures were established at monthly intervals by inoculating isolates onto Sabouraud dextrose agar (Oxoid, U.K.), which in turn were incubated at 30 C for 3–4 d and subsequently stored at 4 C as long as required to establish broth cultures. Log and stationary phase cultures of test isolates were obtained by inoculating 25 mL Sabouraud dextrose broth with 100 µL overnight Sabouraud dextrose broth cultures, which then were incubated for appropriate periods, as previously determined for each strain, at 37 C with 200 rpm^-1 agitation.

Determination of FITC-lectin binding to cryptococcal cell populations – Depending on the isolate under investigation and incubation timescale, replicate 2–5 mL aliquots of log phase cultures were transferred to sterile centrifuge tubes and cells were pelleted by centrifugation at 1000 g for 10 min. Supernatants were discarded and pelleted cells resuspended in 10 mL filter sterilized 10 mM HEPES buffer (pH 7.5) supplemented with 100 µM CaCl₂ and 10 µM MnCl₂, a procedure repeated twice more before cells were suspended in 20 mL supplemented HEPES buffer. Before transfer of 1.5 mL aliquots of cell suspension to sterile Eppendorf tubes, cell concentration was adjusted to 1 x 10⁷ cell mL^-1. Eppendorf tubes subsequently were centrifuged (6500 g) for 10 min, supernatants discarded and pelleted cells resuspended in 1 mL sterile supplemented HEPES buffer.

Cell suspensions of 200 µL from each test strain gently were mixed with 200 µL aliquots of fluorescein-isothiocyanate conjugated lectin (30 µg lectin mL^-1 final concentration, chosen after preliminary trials) in sterile supplemented HEPES buffer and incubated at 4 C in the dark for 30 min. We used these FITC-conjugated lectins: concanavalin A (Con A), 6.5 mol FITC mol^-1lectin; Galanthus nivalis lectin (GNL), 3.5 mol FITC mol^-1lectin; Lens culinaris agglutinin (LCA), 5.0 mol FITC mol^-1lectin; Lotus tetragonolobus lectin (LTL), 2.5 mol FITC mol^-1lectin; Pisum sativum agglutinin (PSA), 2.2 mol FITC mol^-1lectin; soya bean agglutinin (SBA), 4.2 mol FITC mol^-1lectin; Ulex europaeus agglutinin (UEA-I), 3.0 mol FITC mol^-1lectin and wheat germ agglutinin (WGA), 5.4 mol FITC mol^-1lectin (Vector Laboratories, U.S.A.). Residual FITC-lectin was removed after centrifugation of samples (6500 g) for 20 min. Cell pellets twice were resuspended in sterile 10 mM HEPES (pH 7.5) and again pelleted by centrifugation (6500 g) for 20 min before resuspension in 0.6–1.0 mL 1% w/v paraformaldehyde/10 mM HEPES buffer (pH 7.5) to ensure a minimum 1 x 10⁶ cells mL^-1 before determination of cell fluorescence by flow cytometry. Additional experiments involving a range of FITC-Con A and FITC-WGA concentrations (0–100 µg mL^-1 in supplemented HEPES) also were conducted with both log and stationary phase cells.

Stationary phase cells from all four test strains, prepared in the manner outlined above, were incubated with respective inhibitory haptens to determine whether nonspecific adhesion of FITC-lectin was a major contributor to cell fluorescence. Aliquots of FITC-Con A and FITC-WGA to give a concentration of 60 µg mL^-1 lectin were incubated with 200 mM methyl--D-mannopyranoside/200 mM methyl--D-glucopyranoside (Sigma, U.S.A.) and 500 mM N-acetyl-glucosamine respectively, for 30 min at 4 C in the dark, before addition of 200 µL of stationary cell suspension. Samples further were incubated and prepared for flow cytometry.

After a brief vortexing of samples before their introduction to sheath fluid, individual fluorescence from 10 000 cells was determined with a Becton Dickinson FACS 440 (fluorescence activated cell sorter), using an argon laser (300 mW), exitation wavelength 488 nm, emitted light detector 530 nm (± 15 nm), adjusted to a fixed channel using standard Brite Beads (Coulter, U.S.A.).

Analysis of lectin concentration binding data was achieved in this manner: Autofluorescence values were subtracted from each data point and the resulting data fitted to a sigmoidal equation with variable Hill coefficient, of the form

where Fmax is the maximum fluorescence value when all binding sites have been saturated, L is the concentration of lectin and EC50 is the concentration of lectin that produces 50% of the maximum saturation. Hn is the Hill coefficient, a measure of the co-operativity in the system (Rang et al 1999). Data fitting was carried out with GraphPad Prism version 3.0 for Windows (GraphPad Software, San Diego, California). The binding curve from each experiment was analyzed to obtain EC50, Fmax and Hn values. The relative abundance of binding sites for each lectin was determined from mean Fmax values, which were divided by the specific activity of each lectin, to produce a maximum fluorescence per unit of protein. These values then were normalized relative to the abundance of FITC-WGA binding sites on logarithmic growth phase cultures of strain CN IRM 5815.

Characterization of cryptococcal cell FITC-lectin avidity – Aliquots of 7.5 µL log and stationary phase cells, exposed to a final concentration of 30 µg mL^-1 FITC-lectins in supplemented HEPES buffer, prepared in the manner outlined above, in 1% w/v paraformaldehyde/10 mM HEPES buffer (pH 7.5) in turn were dispensed into wells of separate multispot microscope slides (Hendley, U.K.). Cell suspensions then were air dried and fixed by the addition of 7.5 µL cold acetone, which subsequently was allowed to evaporate under warm air. Wells then were sealed with 5 µL Vectashield (Vector Laboratories, U.S.A.), a cover slip firmly affixed with Tippex correction fluid and stored at 5 C in the dark until fluorescence characterization by confocal microscopy. Images were acquired with a Zeiss Axiovert/Biorad MRC 1024 OS laser scanning confocal microsope facility, using Laser Sharp 2000 software (Bio-Rad Laboratories). An argon laser (100 mW), excitation wavelength 488 nm moderated by a series of neutral density filters, gain, offset and zoom functions was used to induce and optimize image fluorescence intensity, contrast and composition. Slides were viewed with an oil immersion Zeiss Neuflor 1.3 NA objective and subjective material captured as 512 x 512 pixel images in turn converted from Bio-Rad PICT to TIFF format.

Statistical analysis – Data were analyzed with two factor ANOVA (Microsoft Excel 2000, Microsoft Corp., Seattle). These comparisons were analyzed: (i) differences in yeast cell fluorescence between strain and growth phase for test lectins FITC-Con A and FITC-WGA; (ii) differences in yeast cell fluorescence between strain and lectin concentration for test lectins FITC-Con A and FITC-WGA; (iii) the influence of respective inhibitory haptens on FITC-lectin binding to stationary phase yeast cells of each strain. Standard errors associated with EC 50, Fmax and Hn determinations were calculated with Graphpad Prism software.

	RESULTS

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FITC-lectin binding to cryptococcal cell populations – A preliminary assessment of cryptococcal cell lectin avidity (Table I) shows that lectin binding to test strains varied markedly after adjustment for autofluorescence and lectin/FITC molar ratios. Populations of test strains fluoresced most when exposed to mannose and glucose binding FITC-Con A, mannose specific FITC-GNL and N-acetylglucosamine specific FITC-WGA. Among remaining FITC-lectins, greatest fluorescence was obtained from cells exposed to mannose binding FITC-PSA and to a lesser extent FITC-LCA. In contrast, little fluorescence was detected from cells exposed to the fucose-binding lectins FITC-LTL, FITC-UEA I, or the N-acetylgalactosamine specific FITC-SBA. The specificity of the cryptococcal cell FITC-lectin avidity is confirmed by data in Table II, as pre-incubation of FITC-Con A and FITC-WGA with their respective inhibitory haptens significantly reduced FITC-Con A and FITC-WGA binding to stationary phase cell populations (P < 0.001). A similar degree of hapten blocking also was observed for Candida (Smith et al 2001), suggesting that some nonspecific binding of FITC-lectins might occur among test Candida and Cryptococcus species.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Fluorescence of four log phase C. neoformans strains; , 5815; , 5854; , 45922; •, 52817; after incubation with increasing concentrations of FITC-Con A

View larger version (21K): [in this window] [in a new window]   FIG. 2. Fluorescence of four stationary phase C. neoformans strains; , 5815; , 5854; , 45922; •, 52817; after incubation with increasing concentrations of FITC-Con A

View larger version (20K): [in this window] [in a new window]   FIG. 3. Fluorescence of four log phase C. neoformans strains; , 5815; , 5854; , 45922; •, 52817; after incubation with increasing concentrations of FITC-WGA

View larger version (20K): [in this window] [in a new window]   FIG. 4. Fluorescence of four stationary phase C. neoformans strains; , 5815; , 5854; , 45922; •, 52817; after incubation with increasing concentrations of FITC-WGA

FITC-Con A and FITC-WGA binding to cryptococcal cells – Representative confocal microscopy images clearly demonstrated localization of FITC-lectin mediated cell fluorescence. FITC-Con A bound markedly to nascent cell surfaces and sites associated with daughter cell initiation and separation of log and stationary phase cells (Fig. 5a, b). In turn such an occurrence makes sense in the light of differing population fluorescence determinations from log and stationary phase cell populations.

View larger version (137K): [in this window] [in a new window]   FIG. 5. Confocal microscopy images of FITC-lectin (30 µg mL-1) binding to C. neoformans cell surfaces; 6a, Log phase capsular 5815 exposed to FITC-Con A; 6b, stationary phase capsular 5815 exposed to FITC-Con A; 6c, log phase acapsular 5854 exposed to FITC-WGA; 6d, stationary phase acapsular 52817 exposed to FITC-WGA

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The nature of FITC-Con A and FITC-WGA exposure appears to be a defining Cryptococcus characteristic. Fluorescence emanating from single cells exposed to FITC-Con A, although of varying intensity, was comparatively uniform in distribution across surfaces of conidia (Smith et al 1999), individual S. cerevisiae and yeast phase Candida albicans (Smith et al 2001). In contrast, confocal images of representative capsular and acapsular C. neoformans cells exposed to FITC-Con A and FITC-WGA showed marked localization of cell fluorescence around sites of cell division. Other studies employing panels of fluorescent lectins and diverse fungi have demonstrated that particular fungal lectin avidity may vary with hyphal morphology and nature (Freytag and Mendgen 1991, O'Connell et al 1996, Bircher and Hohl 1997). However, carbohydrate ligand exposure among C. neoformans in common with adhesin exposure by budding Rhodosporidium toruloides (Buck and Andrews 1999) appears more influenced by cell-wall synthesis and maturation.

Despite the marked localization of fluorescence, flow cytometry determinations remained of value in contrasting and defining lectin avidity for Cryptococcus cell surfaces. The lack of marked fluorescence from cells exposed to FITC-LTL, FITC-UEA I and FITC-SBA indicates little C. neoformans cell-surface fucose and N-acetyl galactosamine exposure. In contrast, intense cell fluorescence was detected with FITC-Con A, FITC-GNL and FITC-WGA, which primarily bind to mannose and terminal linked glucose, mannose and N-acetyl glucosamine respectively (van Damme et al 1998). Although comparative Candida values were markedly greater (Smith et al 2001) the localized cryptococcal FITC-Con A, and FITC-WGA carbohydrate ligands were present in large numbers. Hill coefficients in excess of unity supported the assumption that both FITC-Con A and FITC-WGA bound to their respective ligands in a more complex relationship than that described for Candida strains (Smith et al 2001). Further evaluation of isolate cell populations exposed to FITC-Con A and FITC-WGA indicated that FITC-WGA in particular bound with marked affinity to members of log and stationary populations, which exposed varying numbers of FITC-Con A and FITC-WGA carbohydrate ligands. Abundance of particular carbohydrates exposed and the nature of cell wall and capsule carbohydrate ligand exposure by C. neoformans therefore appears strain and growth phase related.

Passage of FITC-Con A and FITC-WGA to respective cell-wall binding sites did not seem inhibited by capsule presence, the nature and integrity of which appeared little influenced by cell divisional status when visualized through Indian ink staining (S.N. Smith, unpubl). Furthermore although mannose predominates in such capsule polysaccharides as glucuronoxylomannan and galactoxylomannan, only diffuse capsular fluorescence was observed after exposure to FITC-Con A because no mannose residues are terminally exposed (Cherniak and Sundstrom 1994). Entrapment of FITC-Con A might account for the diffuse fluorescence observed in both representative and additional archived confocal images, however similar fluorescence was not detected in cells exposed to FITC-WGA, in turn confirming that N-acetylglucosamine residues remain firmly attached to cell-wall surfaces. The FITC-Con-A mediated fluorescence observed among capsular cells, therefore might result from FITC-Con A conjugation with mannoprotein, an important modulator of host immunity (Pietrella et al 2001), which diffused, albeit in relatively small amounts, through the capsular matrix, according to Cherniak and Sundstrom (1994).

	FOOTNOTES

 
¹ Corresponding author. E-mail: S.N.Smith{at}aston.ac.uk

Accepted for publication May 15, 2003.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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