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The Journal of Immunology, 2006, 177: 4718-4726.
Copyright © 2006 by The American Association of Immunologists, Inc.

Galectin-3 Induces Death of Candida Species Expressing Specific beta-1,2-Linked Mannans1

Luciana Kohatsu*, Daniel K. Hsu{dagger}, Armin G. Jegalian*, Fu-Tong Liu{dagger} and Linda G. Baum2,*

* Department of Pathology and Laboratory Medicine, Jonsson Comprehensive Cancer Center, School of Medicine, University of California, Los Angeles, CA 90095; and {dagger} Department of Dermatology, School of Medicine, University of California Davis, Sacramento, CA 95817


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lectins play a critical role in host protection against infection. The galectin family of lectins recognizes saccharide ligands on a variety of microbial pathogens, including viruses, bacteria, and parasites. Galectin-3, a galectin expressed by macrophages, dendritic cells, and epithelial cells, binds bacterial and parasitic pathogens including Leishmania major, Trypanosoma cruzi, and Neisseria gonorrhoeae. However, there have been no reports of galectins having direct effects on microbial viability. We found that galectin-3 bound only to Candida albicans species that bear beta-1,2-linked oligomannans on the cell surface, but did not bind Saccharomyces cerevisiae that lacks beta-1,2-linked oligomannans. Surprisingly, binding directly induced death of Candida species containing specific beta-1,2-linked oligomannosides. Thus, galectin-3 can act as a pattern recognition receptor that recognizes a unique pathogen-specific oligosaccharide sequence. This is the first description of antimicrobial activity for a member of the galectin family of mammalian lectins; unlike other lectins of the innate immune system that promote opsonization and phagocytosis, galectin-3 has direct fungicidal activity against opportunistic fungal pathogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infections with opportunistic fungi are a critical health concern; thus, elucidation of different innate immune strategies to control fungal infections is an important goal. A number of endogenous lectins participate in innate immune responses and are important for control of microbial infections, especially those caused by fungal pathogens. As pattern-recognition receptors (PRRs),3 lectins recognize unique carbohydrate ligands, or pathogen-associated molecular patterns (PAMPs), present on the surface of the pathogen but absent in the host (1, 2, 3, 4, 5). Fungal oligosaccharide PAMPs are recognized by lectin PRRs to induce rapid and broad host defense responses such as opsonization, activation of complement, activation of coagulation cascades, phagocytosis, inflammation, and direct microbial killing (3). Engagement of PRRs on macrophages and dendritic cells can also attract, activate, and regulate T cells that are critical for an acquired immune response to fungal pathogens (6).

Several types of lectins function as PRRs during the host response to fungal infections, including pentraxin-3, dectin-1, and the collectin family members surfactant proteins A and D (1, 7, 8, 9, 10, 11). Recently, it has become apparent that the galectin family of lectins can also participate in the innate immune defense against pathogens (12). Galectins are present in all multicellular organisms, including worms, sponges, multicellular fungi, and insects (13, 14, 15, 16, 17, 18, 19). Galectins are PRRs in several types of organisms, and many mammalian pathogens express saccharide structures that are recognized by galectins. Galectin-1 binds saccharide ligands on envelope glycoproteins of Nipah virus and HIV (20, 21). Galectin-3 and galectin-9 bind Leishmania major and galectin-9 promotes L. major-macrophage interactions (22, 23). Galectin-3 binds mycolic acids, a major component of the cell envelope of Mycobacterium tuberculosis (24), and participates in clearance of late mycobacterial infections (25). Galectin-3 also binds Pseudomonas aeruginosa, Klebsiella pneumoniae, and Neisseria gonorrhoeae (26, 27, 28), as well as GalNAc1–4GlcNAc sequences in Schistosoma mansoni soluble egg Ag (29). Galectin-3 mediates adhesion of Trypanosoma cruzi to human vascular smooth muscle cells (30), and expression of human galectin-1 and galectin-3 is up-regulated in APCs and gastric epithelial cells infected with T. cruzi and Helicobacter pylori (31, 32, 33). Several studies indicate that galectin-3 specifically participates in innate immunity, as galectin-3 is expressed in a variety of cell types including dendritic cells, macrophages, and NK cells, as well as activated T and B cells (34, 35, 36, 37, 38). However, while galectin-3 can recognize specific PAMPs, no direct microbicidal function for galectin-3, or any galectin, has been reported.

Galectins possess a conserved carbohydrate-recognition domain (CRD) and typically bind to glycans containing beta-galactosides (39). However, subtle structural differences among galectin CRDs result in distinct binding affinities for specific glycan ligands (40). Galectin-3 has an extended carbohydrate-binding pocket compared with galectin-1 (41). This structural difference allows galectin-3 to bind to a wider range of oligosaccharide structures, including structures containing mannose (41). Galectin-3 also differs structurally from the other members of the galectin family. The 14 mammalian galectins are divided into three subgroups, monomeric, tandem repeat, and chimeric, based on domain structure. Galectin-3 is the only member of the chimera-type galectin subgroup, with a CRD in the C terminus and a distinct N-terminal domain that mediates oligomerization of the lectin into pentamers upon binding multivalent saccharide ligands (13, 42, 43). As mentioned above, galectin-3 is highly expressed in macrophages and immature dendritic cells; expression is up-regulated in activated macrophages and is down-regulated during dendritic cell maturation (37, 44). Galectin-3 is also expressed in many types of epithelial cells and stromal cells, including fibroblasts after activation or adhesion (45, 46). Like all galectins, galectin-3 is made as a monomer in the cytosol and is secreted from the cytosol via a nonclassical secretion mechanism; secreted galectin-3 binds to multivalent saccharide ligands on cells and extracellular matrix in the immediate milieu (47, 48, 49, 50).

Surprisingly, Fradin et al. (51) demonstrated that galectin-3 binds to beta-1,2-linked oligomannosides, an uncommon PAMP present on the surface of the pathogenic fungus Candida albicans but absent on Saccharomyces cerevisiae. Three types of beta-1,2-linked oligomannans have been identified in different species of the genus Candida (52, 53, 54). We now demonstrate that galectin-3, but not galectin-1, binds in a carbohydrate-dependent manner to Candida species bearing beta-1,2-oligomannosides in the yeast cell wall, and that galectin-3 binding to C. albicans is inhibited by C. albicans mannans but not by S. cerevisiae mannans. Moreover, binding of galectin-3 resulted in death of yeast expressing specific beta-1,2-oligomannoside structures. Thus, we report a novel fungicidal activity for galectin-3, suggesting that galectin-3 participates in host protection against opportunistic fungal infections.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Yeast species and reagents

C. albicans serotype A (ATCC 10231), C. albicans serotype B (ATCC 60193), Candida glabrata (ATCC 2001), and Candida guilliermondii (ATCC 6260) were obtained from the American Type Culture Collection. Cells were cultured on Sabouraud dextrose agar plates at 37°C, and subcultured for three passages in Sabouraud dextrose broth for 24 h before each experiment. S. cerevisiae BY4741a (gift from Dr. G. Payne, University of California, Los Angeles, CA (UCLA)) was passaged three times in YPD medium (1% Becto yeast extract, 2% galactose, and 2% dextrose) at 30°C for 24 h before each experiment. For all experiments, cells were washed three times in cold PBS and adjusted to a final OD450 of 1.0 (0.5 x 107 yeast/ml). Recombinant human galectin-3 was made as previously described (55).

Tissue immunostaining

Human tissue was obtained from the Division of Anatomic Pathology, UCLA Department of Pathology, with appropriate institutional review and approval for use. Five-micron sections of paraffin-embedded tissue were heated at 60°C for 30 min, deparaffinized with xylene and alcohol, and fixed with 3% H2O2 for 5 min. Slides were blocked with 1% BSA in PBS for 20 min before addition of anti-galectin-3 rabbit serum or nonimmune rabbit serum diluted 1/1000 in 1% BSA/PBS. Bound primary Ab was detected with HRP-conjugated goat anti-rabbit IgG 1/1000 in PBS, and developed with chromogen reagent (Peroxidase Chromogen kit; Biomeda) for 10 min at room temperature. Slides were counterstained with hematoxylin for 1–2 min at room temperature and washed with dH2O. Parallel sections were stained with periodic acid-Schiff reagent. Images were analyzed on a BX41 light microscope (Olympus).

Detection of galectin-3 binding to yeast

Galectin-1, -3, and control BSA were biotinylated using the manufacturer’s recommended protocol (EZ-Link Sulfo-NHS-Biotin kit; Pierce). A total of 5 x 105 C. albicans ATCC 60193 in 50 µl of dH2O were heat fixed on slides, blocked with 1% BSA/0.1 M sucrose, and blocked with blocking reagent (Avidin/Biotin Blocking kit; Vector Laboratories). Cells were incubated with 30 µg of (20 µM) biotinylated galectin-3, 30 µg of biotinylated galectin-3 plus 0.1 M lactose, or buffer alone. Bound protein was detected using streptavidin (SA)-HRP (Zymed Laboratories) and developed as above for 10 min at room temperature (RT). Images were analyzed on a Zeiss Axioskop2 plus microscope and photographed with a Zeiss Axiocam digital camera.

Alternatively, triplicate samples of 5 x 105 Candida or S. cerevisiae were incubated for 45 min at 4°C with 15 µg (20 µM) biotinylated galectin-1, 18–30 µg (12.5 to 20 µM) biotinylated galectin-3, or 50 µg of biotinylated BSA. Bound protein was detected with 1.8 µg of SA-(4,6-dichlorotriazinyl) aminofluorescein (SA-DTAF) (Jackson ImmunoResearch Laboratories) by flow cytometry on a BD-LSR I Analytic Flow Cytometer and data were analyzed with CellQuest software (BD Biosciences). For inhibition assays, 22.5 µg of galectin-3 was preincubated on ice with 0.1 M beta-lactose (Sigma-Aldrich), 0.1 M sucrose (Fisher Scientific), 1 mg/ml S. cerevisiae cell wall mannan (Sigma-Aldrich), or 1 mg/ml C. albicans serotype A cell wall mannan Takara MG001 (Takara Mirus Bio), before binding to live yeast.

Serotyping of cell surface expression of beta-1,2-oligomannan antigenic factors 5 and 6

Candida check rabbit antisera against antigenic factors 5 (F5) and 6 (F6) (Iatron Laboratories) that specifically identify distinct Candida beta-1,2-linked oligomannans were used. Rabbit antisera against F5 recognizes (Man-beta1,2-Man)n≥2 sequences on either acid labile or acid stable phosphodiesterified N-glycans (45). Rabbit antisera against F6 recognizes (Man-beta1,2-Man)n≥1-{alpha}1,2-Man sequences only on acid labile phosphodiesterified N-glycans (53, 54). A total of 5 x 105 Candida or S. cerevisiae cells were incubated on ice for 1 h with 1/25 dilution of primary antiserum to F5 (IF5, Candida Check Iatron RM302-3), 1/50 dilution of primary antiserum to F6 (IF6, Candida Check Iatron RM302-4), or 1/25 dilution of preimmune rabbit serum. Bound Ab was detected with FITC-labeled goat anti-rabbit IgG (diluted 1/50) (Jackson ImmunoResearch Laboratories) by flow cytometry and data analyzed with CellQuest software as described above.

Assessment of yeast viability

For initial assessment of CFU, 0.5 x 105 C. albicans ATCC 10231 were incubated with the indicated concentration of galectin-3 or buffer alone in a final volume of 50 µl at 37°C for 2 h. For cell sorting, 5 x 105 C. albicans ATCC 10231 were incubated with the indicated concentration of galectin-3 or buffer alone in a final volume of 50 µl at 37°C for 2 h. Six tubes of treated yeast were pooled for flow cytometric cell sorting using a FACSAria cell sorter (BD Biosciences). The small, granular population of cells in galectin-3-treated samples were sorted from the cell population that demonstrated normal morphology and each population was collected under sterile conditions in separate tubes. The number of cells collected in each tube was recorded, and volumes were adjusted to yield 1.8 x 104 cells/ml/tube. Samples were diluted 1/10 and 10-µl samples were plated on Sabouraud dextrose agar and cultured at 37°C for 24 h. Colony images were captured using a model 2.1.1 camera and a Chemilmager 5500 digital imaging system (Alpha Innotech).

Quantitative assessment of yeast viability by Fun-1

Cell viability was analyzed using the dye Fun-1 (Molecular Probes). The Fun-1 dye initially produces a diffuse green cytoplasmic staining of yeast. To confirm that flow cytometric analysis of Fun-1 staining could discriminate live (FL-1bright) and dead (FL1dim) cells, 20 µM Fun-1 was added to live or killed yeast for 30 min at 37°C, to avoid the conversion of the dye in live cells to yellow-orange that occurs by 1 h, according the manufacturer’s insert. Analysis of cells in the FL1 and FL2 channels at various time points after addition of dye demonstrated greater discrimination in FL1 than in FL2, consistent with the emission data in the manufacturer’s insert, so that all samples were analyzed in FL1 to detect green fluorescence. As controls for discrimination of dead and live cells, cells were killed with heat (80°C for 5 min and stored on ice) or Fungizone (4 µg/ml; Invitrogen Life Technologies). Triplicate samples were analyzed by flow cytometry using a BD-LSR I Analytic Flow Cytometer equipped with an argon-ion laser. The excitation laser was set at 488 nm and Fun-1 green fluorescence was collected with a 530/28-nm bandpass filter. A total of 10,000 events were acquired per analysis. To assess the effects of galectin-3 on yeast cell viability, 5 x 105 cells of each Candida strain were incubated with 7.5–105 µg (5–70 µM) of galectin-3 in a final volume of 50 µl; for inhibition studies, 105 µg of galectin-3 was preincubated on ice with 0.1 M beta-lactose (Sigma-Aldrich), or 0.1 M sucrose (Fisher Scientific) before addition to the cells. Samples were treated at 37°C for 2 h and cell viability analyzed with Fun-1 as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Galectin-3 is present in fungal granulomata

Galectin-3 has been reported to bind to beta-1,2-mannans isolated from C. albicans serotype A (51). Because galectin-3 has been found in liver granulomata from hamsters infected with Schistosoma mansoni (29), we asked whether galectin-3 was present in human tissues infected with C. albicans. As shown in Fig. 1, galectin-3 was detected in lung tissue from a patient with systemic candidiasis. Galectin-3 was detected throughout granulomata in the infected lung tissue, especially in the walls of fungal abscesses, as well as in macrophages and stromal cells in the granulomata and surrounding tissue, suggesting that galectin-3 could interact with C. albicans in infected tissues. In noninfected tissues, galectin-3 expression was only observed in macrophages, consistent with previous work demonstrating expression of galectin-3 in the cytoplasm and on the surface of monocytes and macrophages (38). We observed no reactivity in tissues stained with control nonimmune serum (data not shown).


Figure 1
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  		FIGURE 1. Galectin-3 expression in lung tissue from a patient with disseminated C. albicans infection. Serial sections were stained with Periodic acid Schiff (A) to detect C. albicans, or anti-galectin-3 antiserum (B) and counterstained with hematoxylin. A, Periodic acid-Schiff staining demonstrates fungal cells in the center of granulomatous inflammation in lung (left, x100; right, x400). B, Galectin-3 (red) was detected in the stroma surrounding the fungi (arrowhead, right panel), as well as in individual cells (arrows) in the tissue surrounding the granuloma (left, x100; right, x400). Inset, Galectin-3 expression in a macrophage (x1000).

 
Galectin-3 binds to C. albicans in a carbohydrate-specific manner

As mentioned above, galectin-3 binds to beta-1,2-oligomannans isolated from C. albicans (51). However, direct binding of galectin-3 to intact yeast has not been examined. As a semiquantitative assessment of galectin-3 binding, we examined galectin-3 binding to fixed yeast by immunohistochemistry. A suspension of C. albicans was air-dried and heat-fixed onto glass slides. Biotinylated BSA or biotinylated galectin-3 was added to the cells, and bound protein was detected with streptavidin-HRP. As shown in Fig. 2A, we detected significant galectin-3 binding to C. albicans, and galectin-3 binding was reduced in the presence of lactose, demonstrating that galectin-3 binding to yeast was carbohydrate dependent.


Figure 2
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  		FIGURE 2. Galectin-3 directly binds to C. albicans, and not to S. cerevisiae in a carbohydrate-dependent manner. A, Biotinylated galectin-3 (center, right) or biotinylated BSA as a control (left) was added to heat killed C. albicans and bound protein detected with streptavidin-HRP (red). Galectin-3 bound to C. albicans (center) and binding was inhibited by lactose (right), demonstrating that binding was carbohydrate dependent. B, Biotinylated galectin-3 (red) or BSA (gray) was added to C. albicans (left) or S. cerevisiae (right) and bound protein detected by flow cytometry using SA-DTAF. One representative from one of four independent experiments is shown. C, Biotinylated galectin-3 was bound to C. albicans in the absence (red) or presence (dotted line) of saccharide competitors and binding detected as above. Lactose (left), but not sucrose (right), inhibited galectin-3 binding) to the level of background binding seen with biotinylated BSA. Gray, Biotinylated BSA. One representative from three independent experiments is shown. D, Biotinylated galectin-3 was added to C. albicans in the absence (red) or presence (dotted line) of yeast cell wall mannans and binding detected as above. C. albicans mannans that contain beta-1,2-linked oligomannans inhibited binding (left), while S. cerevisiae mannans that lack beta-1,2-linked oligomannans had minimal inhibitory effect (right). Gray, Biotinylated BSA. Mean fluorescence intensity of histograms are depicted below. One representative of two independent experiments is shown.

 
Direct binding of galectin-3 to live C. albicans was measured by flow cytometric analysis. beta-1,2-oligomannans that have been described as ligands for galectin-3 are present on C. albicans but absent on S. cerevisiae (51). To assess specific binding of galectin-3 to yeast containing beta-1,2-oligomannans, we compared binding of biotinylated galectin-3 to C. albicans and S. cerevisiae. Galectin-3 bound to C. albicans, but we detected no binding of galectin-3 to S. cerevisiae above the level of background binding of biotinylated BSA (Fig. 2B). Galectin-3 binding to C. albicans was carbohydrate dependent, as binding could be competed to the level of background BSA binding with lactose, a disaccharide that binds galectin-3, but not with the nonspecific disaccharide inhibitor sucrose (Fig. 2C). Moreover, preincubation of galectin-3 with C. albicans serotype A cell wall mannans significantly reduced galectin-3 binding to C. albicans cells, while S. cerevisiae cell wall mannans only minimally reduced galectin-3 binding to C. albicans (Fig. 2D). These observations indicate that galectin-3 preferentially binds to oligosaccharide ligands present in C. albicans mannans that are not present in the cell wall mannans of S. cerevisiae.

Galectin-3 binds to Candida species expressing beta-1,2-mannans

There are three known types of beta-1,2-linked oligomannans in the cell walls of different species of the genus Candida. These structures have been termed F5, F6, and F9 (Fig. 3A), and different Candida species express one or more of these structures (Table I). As mentioned above, galectin-3 bound to beta-1,2-linked oligomannans isolated from C. albicans serotype A strain VW32 (51), that contains both F5 and F6 types of beta-1,2- linked oligomannans. To investigate whether galectin-3 binding to cells required the presence of a specific type of beta-1,2-linked oligomannans, we assayed galectin-3 binding to four different Candida species that display specific beta-1,2-linked oligomannans on the cell surface.


Figure 3
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  		FIGURE 3. Cell surface expression of beta-1,2-linked oligomannans in Candida species. A, Schematic of beta-1,2-linked oligomannans F5, F6, and F9 present in the cell wall of various Candida species. B, Different fungal species express different combinations of beta-1,2-linked oligomannans. Binding of specific rabbit antisera to F5 and F6 or preimmune rabbit serum (control) was detected with FITC-labeled goat anti-rabbit IgG and analyzed as in Materials and Methods.

 

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  		Table I. Surface expression of beta-1,2-oligomannans F5, F6, and F9 on selected yeast

 
We first confirmed expression of different beta-1,2-linked oligomannans on C. albicans serotypes A and B, C. guilliermondii, and S. cerevisiae by flow cytometry using specific antisera to the F5 and F6 mannan structures. As shown in Fig. 3B, C. albicans serotype A is F5+F6+, C. albicans serotype B is F5+F6–, C. guilliermondii is F5–F6–, and S. cerevisiae is F5–F6–, corresponding to the previously reported patterns of expression shown in Table I.

We examined binding of biotinylated galectin-3 or galectin-1 to four different Candida species (Fig. 4). Galectin-3 bound to C. albicans serotype A and serotype B, C. glabrata and C. guilliermondii. Thus, galectin-3 bound to Candida species expressing surface F5, F6, or F9 beta-1,2-linked oligomannans (Table I). In contrast, galectin-1 did not bind to any of the four Candida species, demonstrating that recognition of saccharides on the yeast cell wall is a specific function of galectin-3.


Figure 4
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  		FIGURE 4. Galectin-3, but not galectin-1, binds to Candida species bearing a variety of beta-1,2-linked oligomannans. Biotinylated galectin-3 but not biotinylated galectin-1 bound to C. albicans serotype A, C. albicans serotype B, C. glabrata, and C. guilliermondii. Biotinylated BSA was used as a negative control. Bound proteins were detected with streptavidin FITC and experiments analyzed as indicated in Materials and Methods. One representative of three independent experiments is shown.

 
Galectin-3 induces C. albicans death

During binding analyses, we observed that binding of purified, recombinant galectin-3 resulted in distinct morphological changes in the cells. We observed a population of yeast in galectin-3-treated samples with reduced forward and increased side scatter by flow cytometric analysis (Fig. 5A). The morphological changes resulting from addition of galectin-3 were inhibited in the presence of 0.1 M lactose, indicating that the effect was mediated by carbohydrate-dependent binding of galectin-3. We quantified viability in galectin-3 and control-treated cells, and found that there was a 38% reduction in CFU for galectin-3 vs control-treated cells (Fig. 5B). Moreover, the decrease in CFU for cells treated with galectin-3 was comparable to the fraction of cells that demonstrated morphologic changes after galectin-3 binding, ~30% in Fig. 5A. This morphologic change was not observed when cells were incubated with recombinant galectin-1, consistent with the lack of galectin-1 binding shown in Fig. 4 (data not shown). As the altered morphology of cells in galectin-3-treated samples was similar to the flow cytometry characteristics of dying cells (decreased cell size and increased cell granularity), we directly investigated the viability of the two populations of cells. We sorted the two distinct cell populations generated by galectin-3 treatment by flow cytometry, and analyzed each population for the ability to grow on agar plates (Fig. 5C). The small, granular population of cells that appeared after galectin-3 treatment demonstrated almost no growth, while the larger cells that were morphologically similar to untreated yeast demonstrated normal colony growth. Both sorted populations were plated at serial dilutions to rule out a fungistatic, rather than fungicidal, effect. As we observed minimal colony growth of the small, granular cells at 1/10, 1/100, and 1/1000 dilutions (Fig. 5C), the galectin-3 effect appeared to be fungicidal.


Figure 5
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  		FIGURE 5. Galectin-3 induces C. albicans cell death. A, Galectin-3 binding to C. albicans results in distinct morphological changes. Flow cytometric analysis of C. albicans cells after galectin-3 binding revealed a population of cells (middle) with decreased forward scatter and increased side scatter, compared with buffer control (left). Galectin-3 induced morphologic changes were inhibited in the presence of 100 mM lactose (right), indicating a carbohydrate-dependent effect. B, Colony-forming assay for cells treated in the absence and presence of galectin-3. Cells were treated as described in Materials and Methods, and 10 µl plated at the indicated dilutions. Cells treated with galectin-3 ({blacksquare}) demonstrated decreased CFU compared with control cells ({square}). One of seven replicate experiments is shown (range: 16–45% inhibition). C, Decreased viability of the small granular cells. C. albicans were treated with 30 µg of galectin-3 and the large and small cell populations were isolated by FACS. The cell number in each sample was adjusted to 1.8 x 104 cells/ml and 10 µl of 1/10 serial dilutions were plated as indicated (bottom). The small, granular cells (right) yielded significantly reduced colony formation compared with the larger cells (left).

 
We developed a high throughput method to measure galectin-3 induced fungal death using the dye Fun-1. Developed for fluorescent microscopic analysis of yeast viability, Fun-1 initially produces green fluorescence in cells that take up the dye. We reasoned that we could discriminate live and dead cells by the intensity of Fun-1 fluorescence. To establish the assay, we analyzed Fun-1 staining of live yeast vs yeast killed by heat or Fungizone. As shown in Fig. 6A (left), Fungizone- or heat-treated dead cells were Fun-1dim, while live cells were Fun-1bright. As a control, a mixture of 50% live and 50% dead yeast demonstrated two peaks, a Fun-1bright and a Fun-1dim, demonstrating that this Fun-1 assay could discriminate live and dead yeast populations by flow cytometry (Fig. 6A, right).


Figure 6
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  		FIGURE 6. The small C. albicans cells resulting from galectin-3 treatment are Fun-1dim. A, Left, Live cells are Fun-1bright and dead cells are Fun-1dim. Cells were killed with heat (dotted line) or Fungizone (gray line) and the ability of live and dead cells to retain the vital dye Fun-1 was analyzed by flow cytometry. Cells treated with buffer alone remained Fun-1bright (black line), while dead cells were Fun-1dim. Right, A mixture of 50% live and 50% heat-killed yeast yielded discreet Fun-1bright and Fun-1dim peaks, demonstrating that the assay could discriminate live and dead cells in the same sample. One representative from 1 of 4 independent experiments is shown. B, Left, Treatment of C. albicans with galectin-3 (black line), but not with buffer control (gray line), yielded a Fun-1 dim population. (center). When galectin-3 treated cells were analyzed by forward vs side scatter as in Fig. 5, the small nongranular cells (R1 gate) were Fun-1dim (right). One representative from 1 of 14 independent experiments is shown.

 
We assessed yeast viability after galectin-3 binding using the Fun-1 assay. As shown in Fig. 6B, galectin-3 treatment of C. albicans resulted in a distinct Fun-1dim population that corresponded to the small, granular population detected by forward vs side scatter analysis of the same cells. Thus, as shown in Fig. 5, galectin-3 directly reduced the viability of the cells. Furthermore, we performed a dose-response analysis to determine the minimum concentration of galectin-3 required for the fungicidal effect. We observed minimal galectin-3 fungicidal activity at concentrations below 5 µM, while the maximal effect was observed at >20 µM (Fig. 7A). To confirm that the fungicidal effect detected by Fun-1 uptake was carbohydrate dependent, we performed inhibition assays using lactose and sucrose, as in Fig. 2. In the presence of lactose, we observed >65% decrease in the Fun-1dim population compared with the samples treated with galectin-3 alone, while sucrose had no inhibitory effect (Fig. 7B).


Figure 7
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  		FIGURE 7. Specificity of galectin-3 induced C. albicans cell death. A, Galectin-3 induced C. albicans cell death is dose dependent. Cells were treated with the indicated concentration of galectin-3 at 37°C and stained with Fun-1. The mean percent ± SD. Fun-1dim cells from triplicate samples is shown on the y-axis. A small increase in the Fun-1dim population was observed at 5 µM galectin-3, while maximal cell death was observed at >20 µM. B, Galectin-3-mediated cell death of C. albicans is carbohydrate dependent. Cells treated with the highest concentration of galectin-3 in A (80 mM), and percent Fun-1dim cells determined. The percent Fun-1dim cells was reduced in the presence of lactose, but not sucrose. The mean percent ± SD Fun-1dim cells from triplicate samples is shown on the y-axis.

 
Galectin-3-mediated Candida species death requires specific beta-1,2-linked oligomannans on the yeast cell surface

As we observed galectin-3 binding to four different species of Candida expressing one or more of the three types of beta-1,2-linked oligomannans (Table I), we asked whether galectin-3 binding to the yeast cell surface was sufficient to induce death, or whether a specific type of beta-1,2-linked oligomannan was required for galectin-3 to kill the cells. C. albicans serotypes A and B, C. glabrata, and C. guilliermondii were treated with galectin-3 and cell viability assessed by Fun-1 uptake. As shown in Fig. 8, flow cytometric analysis revealed a Fun-1dim population in galectin-3-treated C. albicans serotype A (F5+F6+F9–), C. albicans serotype B (F5+F6–F9–), and C. glabrata (F5–F6+F9–). However, like S. cerevisiae, galectin-3-treated C. guilliermondii (F5–F6–F9+) did not have a significant Fun-1dim population (Fig. 8). Thus, the fungicidal effect of galectin-3 appeared to be specific for Candida species bearing cell surface F5 or F6 beta-1,2-linked oligomannans, while expression of F9 beta-1,2-linked oligomannans in the absence of F5 or F6, although sufficient for galectin-3 binding, was not sufficient to render the cells susceptible to galectin-3-mediated death.


Figure 8
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  		FIGURE 8. Galectin-3-mediated Candida cell death requires specific beta-1,2-oligomannosides on the yeast cell surface. C. albicans serotype A (F5+F6+), C. albicans serotype B (F5+F6–), C. glabrata (F5–F6+), C. guilliermondii (F5–F6–) or S. cerevisiae were treated with galectin-3 (open histogram) or buffer control (filled histogram) at 37°C and stained with Fun-1. A significant fraction of C. albicans serotype A, C. albicans serotype B and C. glabrata cells became Fun-1dim, while galectin-3 treatment of C. guilliermondii and S. cerevisiae did not demonstrate an increase in the percent Fun-1dim cells. One representative from two independent experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Infections with opportunistic fungi are a significant concern, especially in immunocompromised patients. Thus, identification of novel antifungal activities against C. albicans will allow development of new therapeutic strategies to combat fungal infections. Galectin-3 has been suggested to act as a PRR for several microbial pathogens (26, 27), including the yeast C. albicans (51). Although galectins were originally defined by the ability to bind to beta-galactosides, some members of the galectin family, e.g., galectin-10 (42), can recognize mannose with high affinity. Furthermore, galectin-3 binds to beta-1,2-oligomannosides isolated from the cell wall of C. albicans serotype A (51), and synthetic analogs of beta-1,2-oligomannosides administered orally to mice prevented colonization of the intestine by C. albicans (56). However, the effect of galectin-3 binding to intact yeast was not explored, and no direct microbicidal activity for galectin-3 has been described.

We found that galectin-3 bound to four Candida species expressing different combinations of beta-1,2-oligomannans, but did not bind S. cerevisiae that lacks beta-1,2-oligomannans. Consistent with the binding specificity, binding of galectin-3 to C. albicans was inhibited by C. albicans cell wall mannans but not by S. cerevisiae cell wall mannans. Thus, galectin-3 is a PRR that recognizes specific oligosaccharides on various Candida species.

Surprisingly, we found that galectin-3 binding to C. albicans was directly fungicidal. We observed a unique population of cells with morphologic characteristics of dead or dying cells after treatment with galectin-3, and the direct fungicidal activity of galectin-3 was confirmed by demonstrating markedly reduced colony forming capability of this population. Although lectins can participate in the host defense against fungal pathogens by promoting opsonization by complement and/or fungal clearance by host macrophages, the fungicidal activity of galectin-3 is complement independent, as we observed the effect with purified recombinant human galectin-3 in the absence of serum. And, although galectin-3 is known to participate in phagocytosis of microbial PAMP-coated beads (29), we observed the fungicidal effect of galectin-3 in the absence of macrophages or other cells of the mammalian immune system. The mechanism of galectin-3 fungal death is not clear at this point; however, recent studies of oligosaccharide-based vaccines for C. albicans demonstrated a direct fungicidal activity for Abs that recognize beta-glucans in the yeast cell wall (57). These observations suggest that cross-linking of oligosaccharide components in the fungal cell wall, either by multivalent galectins such as galectin-3 or by Abs, can directly trigger death.

Although we consistently observed galectin-3-induced death of C. albicans, we never observed morphologic changes or death of the entire population of cells, although all the cells bound galectin-3. In all assays we performed, we detected death of ~30–70% of cells (Figs. 7 and 8). Thus, the reduction in CFU was most apparent when we sorted the two morphologically distinct populations (Fig. 5). We determined that incomplete susceptibility was not due to gross variation in cell cycle in an asynchronous population, as all assays were performed on cells 24 h after dilution from stationary cultures (see Materials and Methods). We also asked whether longer exposure to galectin-3 would increase the fraction of susceptible cells. In time course experiments, we observed negligible death after galectin-3 binding at 30 min, with dead cells becoming apparent at 60–90 min and maximal death observed after 120 min binding of galectin-3 (data not shown). At longer time points (4 and 6 h) the effects of galectin-3 were confounded by proliferation of the surviving cells (data not shown), so we limited the assays to 2 h. Intriguingly, in studies of galectin-3- and galectin-1-induced death of mammalian lymphocytes and lymphoid cell lines, 100% cell death is also not observed, although all cells in the populations examined bind the galectins (13, 58, 59). As glycosylation is not templated, and factors such as residence time in the Golgi apparatus will affect the type and abundance of glycans added to a glycoprotein (60), subtle differences in glycosylation or density of critical ligands on the surface of cells may regulate the susceptibility of the cells to galectin-3. The mechanism of galectin-3-mediated fungal death is currently being investigated in our laboratories.

Although galectin-3, but not galectin-1, bound to all Candida species that contained one or more types of beta-1,2-linked oligomannans, galectin-3 killing of Candida appeared to require the presence of the specific beta-1,2-linked oligomannans F5 or F6 (Figs. 3A and 8). In contrast, the beta-1,2-linked oligomannan F9 did not appear to be sufficient to mediate galectin-3 induced death, as C. guilliermondii (F5–F6–F9+) bound galectin-3 but did not die (Figs. 4 and 8). Thus, direct fungicidal activity of galectin-3 may be restricted to specific Candida species. It is of interest to note that the F5 and F6 beta-1,2-linked oligomannan epitopes are both on the same phosphomannan-containing branch structure, while the F9 beta-1,2-linked oligomannan epitope is on a different N-glycan structure (Fig. 3A); this suggests that the phosphomannan-containing oligosaccharides on specific Candida species may be important for galectin-3 death. Gow and colleagues (61) recently reported that outer chain N-glycans, such as those to which the F5 and F6 epitopes can be attached, are essential for cell wall integrity of C. albicans.

Induction of Candida death is a unique function of galectin-3, because galectin-1 did not bind to any of the Candida species examined (Fig. 4), nor did galectin-1 have a fungicidal effect on C. albicans serotype B at concentrations up to 35 µM (data not shown). Galectin-3 binding and induction of Candida death are carbohydrate specific, as both functions were blocked by the disaccharide ligand lactose, but not by the disaccharide sucrose that is not recognized by galectin-3 (Figs. 2A, 2C, 5A, and 7B). Furthermore, galectin-3 binding to C. albicans was markedly inhibited by C. albicans mannans, with little inhibition by S. cerevisiae mannans (Fig. 2D), demonstrating that galectin-3 specifically binds to ligands present in the cell wall of Candida species but absent on S. cerevisiae. Our results indicate that galectin-3 binding and induction of death of Candida species is a specific, lectin-mediated activity of galectin-3.

Galectin-3 is expressed by epithelia lining the body surfaces that are exposed to Candida colonization, such as oral mucosa, corneal, and conjunctival epithelia, and intestinal epithelia (62, 63, 64, 65), that constitute the first line of host defense against Candida invasion. Although relatively high concentrations of soluble galectin-3 (20 µM) were required to trigger Candida death in vitro, abundant galectin-3 has been detected in the local milieu surrounding inflammatory cells in human tissues (58), and galectin binding to extracellular matrix proteins has been shown to concentrate galectin (59). Moreover, total galectin-3 concentration in homogenized splenic tissue from various mammals can reach 80 mg/kg tissue, or roughly 2 µM (66). Thus, in infected tissues, the local concentration of galectin-3 surrounding macrophages and stromal cells could be quite high. Our observation of galectin-3 expression in granulomata in lung tissue from a patient with systemic candidiasis (Fig. 1) supports a role for galectin-3 in host protection against Candida infection. Candida species are the fourth leading cause of nosocomial bloodstream infections, with a mortality rate of 40% in the United States (67), and vulvovaginal candidiasis affects 75% of women of childbearing age (68). Thus, understanding the contribution of human lectins such as galectin-3 to resistance to Candida may contribute to control of infections in both immunocompetent and immunocompromised patients. Intriguingly, a recent report demonstrated that C. albicans infection of a macrophage cell line in vitro reduced expression of galectin-3 ~3-fold, suggesting a mechanism for the pathogen to evade the fungicidal effect of galectin-3 (69). However, increased galectin-3 production by activated but noninfected macrophages and stromal cells may contribute to control of Candida infections in vivo.

Antimicrobial activity may be a common feature of galectins in different species. AJL-1 is a member of the galectin family of lectins expressed in the skin mucus of the Japanese eel Anguilla japonica. High expression of AJL-1 correlates with resistance to infection in the eel. Furthermore, AJL-1 has a carbohydrate- and species-specific agglutinating activity for the pathogenic Gram-positive bacteria Streptococcus difficile (70), suggesting that agglutination of invading bacteria on the eel skin surface traps the pathogen. As mentioned above, mammalian galectin-3 also interacts with L. major (22, 23) and T. cruzi (30, 71), as well as bacterial and mycobacterial pathogens (24, 25, 26, 27, 28). Thus, galectin-3 may have multiple functions in human host protection against microbial pathogens. Importantly, recent evidence demonstrates that a robust response to fungal pathogens involves several host lectin PRRs, including the macrophage mannose receptor and dectin-1, recognizing different glycans on the fungal cell surface (72). Galectin-3, specifically recognizing beta-1,2-linked mannans, may be an additional lectin PRR that contributes a unique, direct fungicidal activity to the innate immune defense against Candida infection.


   Acknowledgments
 
We thank Dr. Elizabeth Wagar, Dr. Joseph Hernandez, and Esteban Fernandez for helpful discussions, and Mabel Pang for technical assistance. We also thank Michael Gulrajani and the staff of the UCLA Flow Cytometry Core Facility at the Jonsson Comprehensive Cancer Center (UCLA CFAR CA-16042).


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health (NIH) Grant AI07323 (Microbial Pathogenesis Training Grant) (to L.K.), NIH Grant R01 GM63281 (to L.G.B.), and R01 AI20958 and R01 AI39620 (to F.-T.L.). Back

2 Address correspondence and reprint requests to Dr. Linda G. Baum, Department of Pathology and Laboratory Medicine, School of Medicine, University of California Los Angeles School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: lbaum{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: PRR, pattern recognition receptor; PAMP, pathogen-associated molecular pattern; CRD, carbohydrate recognition domain; SA, streptavidin; DTAF, (4,6-dichlorotriazinyl) aminofluorescein. Back

Received for publication February 6, 2006. Accepted for publication July 19, 2006.


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 Introduction
 Materials and Methods
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