Purification and Characterization of a Mitogenic Lectin from Cephalosporium, a Pathogenic Fungus Causing Mycotic Keratitis

Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.


Introduction
Ophthalmic infections caused by fungi rather than bacteria are of serious concern in many developing countries as they lead to blindness. Ocular fungal infections referred to as ophthalmic mycoses are leading to keratitis of the cornea [1]. Several fungal species responsible for mycotic keratitis and other forms of eye infections have been reviewed recently [2,3]. Fusarium and Cephalosporium (Acremonium) are wide spread fungal species that belong to hyaline filamentous group which are implicated for ophthalmic mycoses [3].
The key factors involved in the pathogenesis of the mycotic keratitis include adherence, invasiveness, morphogenesis, and toxigenicity [4]. Fungal lectins are gaining importance as they are implicated in the process of specific recognition between fungal parasites and their host cells, involving protein-carbohydrate interactions leading to infection [5]. Lectins constitute a heterogeneous group of proteins of nonimmune origin with noncatalytic binding sites capable of recognizing and binding reversibly to specific carbohydrate moieties [6]. Although several reports are available on the lectins from higher fungi [7], very few lectins from animal and plant pathogenic fungi are reported [5,[8][9][10][11][12][13]. Further, fungal lectins are drawing greater attention as many of them exhibit interesting physiological effects such as lymphomitogenic activity, immunomodulatory effect, suppression of cell proliferation, and antitumor activity [14].
The present paper reports the purification, characterization, and mitogenic activity towards human PBMCs of a lectin from Cephalosporium, a pathogenic fungus, isolated from the infected eye of a keratitis patient.  [15]. After 10 days, the mycelial mat was harvested and washed with distilled water on cheese cloth. The mycelial mass was subsequently freeze dried and powdered in a glass mortar, and the fine powder was stored at −20 • C and used for the purification of lectin.

Materials.
Mucin (porcine stomach, type III), fetuin (fetal calf serum), PHA-L, Histopaque 1077, and fine sugars used in hapten inhibition studies were purchased from Sigma Chemical Co., St. Louis, USA. Sepharose 4B was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Asialofetuin was prepared as described by Spiro and Bhoyroo [16]. Asialofetuin-Sepharose 4B, affinity matrix, was prepared by coupling asialofetuin to cyanogen bromideactivated Sepharose 4B according to the method of March [17]. Tritiated thymidine was procured from BRIT (Board of Radiation and Isotope Technology), India. The tissue culture flasks and 96 well plates were procured from NUNC (Denmark). Human blood samples were obtained from Health Center, Karnatak University, Dharwad, India, with approval from Institutional Review Board. All other chemicals used were of analytical reagent grade.

Isolation and Purification of CSL. Dried
Cephalosporium mycelial powder was homogenized in 50 ml (1 : 50 w/v) of 50 mM sodium phosphate buffer, pH 7.2, containing 154 mM NaCl (PBS) for 5 minutes and stirred overnight at 4 • C. The extract was centrifuged (9500 xg) for 30 minutes at 4 • C. The resulting supernatant was membrane filtered (0.45 μm) and used for the purification of the lectin. The crude extract was subjected to affinity chromatography on asialofetuin-Sepharose 4B column (10 × 1.3 cm) that had been equilibrated with PBS and 3 ml fractions were collected at a flow rate of 15 ml/hr. The washing of the column with PBS was continued, until the absorbance of the eluting fractions reads zero at 280 nm (double beam spectrophotometer, Hitachi 2800). The bound lectin was eluted with glycine-HCl buffer (100 mM, pH 2.0) containing 500 mM NaCl. Fractions containing lectin activity were pooled and dialyzed extensively against PBS and stored at −20 • C for further studies.

Hemagglutination Assay and Hapten Inhibition Studies.
Hemagglutination activity of CSL during various stages of purification was determined by the serial twofold dilution method using trypsinized human erythrocytes in 96 well, "U" bottom micro titer plates [18]. The highest dilution of the extract causing visible hemagglutination was regarded as the titre and the minimum concentration of the protein was required for agglutination (MCA) as one unit of hemagglutinating activity. The specific hemagglutination activity was expressed as unit mg −1 protein. The sugar specificity of the purified lectin was determined by a hapten inhibition assay. Inhibition assays were carried out by incubating the lectin sample (with hemagglutinating titre of 4) with serially diluted sugar/glycoprotein prior to the addition of erythrocytes in a total volume of 50 μl, and the hemagglutination was visually observed. The minimum inhibitory concentration of the sugar/glycoprotein was taken as the inhibitory titre of the hapten.
Protein concentrations were determined routinely by Lowry's method [19] and total sugar content of the lectin was estimated by phenol-sulfuric acid method [20].

Molecular Mass Estimation.
The molecular mass of the purified lectin was determined by SDS-PAGE in 15% gel, according to the method of Laemmli [21] and by gel filtration chromatography on a superdex G-75 column (1.5 × 80 cm, in PBS) precalibrated with standard proteins of known molecular weight.

Binding of CSL to Human
PBMCs. Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by density gradient using Histopaque-1077 (Sigma) and resuspended in complete medium (RPMI 1640 + 10% FCS). Fluorescein isothiocyanate-conjugated CSL (FITC-CSL) required for flow cytometry was prepared using the protocol of Goldman [22]. The binding of CSL to PBMCs was determined by flow cytometry. Cells (1 × 10 5 ) were incubated with FITC-CSL (2 μg/100 μl) for 1 hour on ice and were washed thoroughly with 50 mM PBS and then fixed with 2% freshly prepared paraformaldehyde. Data were acquired for 10,000 events using an FACS Vantage (Becton Dickson) equipped with a 488 nm argon laser and analyzed with Cellquest-pro software for determining % positivity and mean fluorescence intensity (MFI). Unstained cells that had been processed similarly were used as negative control.
Receptor-mediated CSL binding to PBMCs was determined by preincubating FITC-CSL with mucin, fetuin and asialofetuin (100 μg/ml) for 1 hour at room temperature. This lectin-sugar complex was added to the PBMCs preparation and analyzed by flow cytometry.

Mitogenic Activity by Tritiated Thymidine Incorporation
Assay. Freshly isolated PBMCs were suspended in RPMI-1640 containing 10% FCS and 1 × 10 5 cells/100 μl/well were plated in 96 well tissue culture plate (NUNC, Denmark) and incubated with CSL concentration ranging from 0.625 μg/ml to 10 μg/ml for 72 hours at 37 • C in 5% CO 2 . PBMCs stimulated with PHA-L (0.156 μg/ml to 2.5 μg/ml, Sigma Chemicals) were used as positive control. Cells were pulsed with tritiated thymidine (1 μCi /well, BRIT, India) 18 hour prior to harvesting and incorporation was measured as counts per minute (CPM).

Statistical
Analysis. Statistical analysis was performed using student's t-test and Mann-Whitney rank sum test. A P-value < .05 was considered to be statistically significant.

Isolation and Purification of the Lectin from the Mycelium of Cephalosporium.
The lectin was purified to homogeneity in a single step by affinity chromatography on asialofetuin-Sepharose 4B column (Figure 1). The fold purification and the total % recovery of the purified lectin from 1 g of the dry mycelial powder are summarized in Table 1; the minimum concentration of the protein required for agglutination (MCA) was found to be 1.145 μg for the crude extract and 0.021 μg for the purified lectin. The eluted lectin was found to be homogenous as revealed by single band on SDS-PAGE in 15% gel (Figure 1, Inset). Subunit molecular mass of 14 kDa was estimated for the lectin by SDS-PAGE, whereas molecular mass of 57 kDa was estimated by gel filtration chromatography (Figure 2), suggesting tetrameric nature of the lectin. Purified CSL agglutinated the human erythrocytes of all blood groups, indicating that it has a blood group nonspecific nature. Hapten inhibition studies showed that the hemagglutinating activity of CSL was inhibited by mucin, fetuin, and asialofetuin, with mucin being the most potent inhibitor with minimum inhibitory concentration (MIC) of 0.785 μg/50 μl ( Table 2).

Binding of CSL to Human PBMCs.
To study the interaction of CSL with PBMCs, cells were stained with FITC-CSL and its binding was analysed by flow cytometry. As depicted in Figure 3(a), 99.62% of the cells were found to be positive for CSL binding, with mean fluorescence intensity (MFI) of 411.27, in comparison with control unstained cells which were set to 1% positivity and the MFI of this 1% cells was 76.89. The receptor-mediated lectin binding to PBMCs was confirmed by studying the binding of CSL after preincubation of the lectin with different competing glycoconjugates or haptens. Flow cytometry histograms of CSL binding to PBMCs before and after blocking with mucin, fetuin, and asialofetuin are presented in Figures 3(a), 3(b), 3(c), and 3(d), respectively. Mucin at 100 μg/ml concentration was the most effective inhibitor of CSL followed by fetuin and asialofetuin at 100 μg/ml.

Mitogenic Activity of CSL.
Mitogenic effect of CSL on human PBMCs was determined by tritiated thymidine incorporation assay. CSL stimulated the uptake of thymidine by human PBMCs in a dose-dependent manner, with maximum incorporation occurring at 10 μg/ml of

Discussion
In the present study, the lectin from human pathogenic fungus Cephalosporium isolated from mycotic keratitis patient was purified to homogeneity in a single step by affinity chromatography on asialo fetuin-Sepharose 4B. CSL, a tetramer, without blood group specificity, has complex sugar specificity and mitogenic activity on human PBMCs. In the past decade, sizeable numbers of fungal lectins are reported and majority of them are from higher fungi, mostly from fruiting bodies of mushrooms [7,23]. However, very few lectins are known from the mycelia forming lower fungi, particularly the pathogenic fungi, although fungal lectins are implicated in host parasite interactions [5].
There is a growing realization that the fungal lectins like bacterial lectins also play an important role in adhesion and infection process. Several hyaline filamentous fungi are considered as opportunistic human pathogens [3]. Most of these pathogens express surface factors, lectins that mediate binding to extra cellular matrix glycoproteins of host cells. Such a binding would facilitate the fungal adherence and can induce immunomodulatory effect on the host cells. In this regard, the CSL exerts mitogenic activity on human PBMCs. So far, very few lectins from human pathogenic fungi are reported including Candida albicans, Candida glabrata, Histoplasma capsulatum, Aspergillus fumigatus, and Trycophyton rubrum [5,10,24,25]. The lectin from C. albicans which recognizes Fucα1-2Galβ, occurring on all blood group substances of ABO types, is implicated in the adherence to human buccal epithelial cells [9]. A 32 kDa lectin purified from A. fumigatus, with sialic acid specificity is suggested to play an important role in the attachment of conidia to extra cellular matrix components of host cells [10]. A. fumigatus is a known opportunistic pathogen responsible for variety of ophthalmic infections such as dacryocystitis, scleritis and endophthalmitis [3]. An extracellular agglutinin from Trycophyton rubrum, a well-known dermatophyte, has specificity towards sialo oligosaccharides is implicated for the adhesion to host cells [25].
There is no direct evidence available confirming the role of lectins from these pathogens in mediating host parasite interactions and also in their immunomodulatory effects on the host cells. On the contrary, several lectins from higher fungi are known to exert interesting immunomodulatory effects. Lectins from V. volvacea and Boletus satanas Lenz exhibit mitogenic effects on human PBMCs [26,27], whereas Agrocybe cylindracea and Schizophyllum commune exerts mitogenic effect on mouse splenocytes [28,29]. Some of the mitogenic lectins like those from V. volvacea, F. velutipes, and Ganoderma lucidum were shown to exhibit immunomodulatory effects [30][31][32], and such mitogenic lectins have potential applications as important diagnostic and experimental tool to study the various aspects of cell growth and differentiation. The receptor mediated binding of CSL to human PBMCs was clearly demonstrated, as it can be effectively blocked by competing glycoconjugates such as mucin, fetuin and asialofetuin. These results substantiate our observations by hapten inhibition studies that CSL has preferential affinity towards mucin compared to fetuin or asialofetuin. The mitogenic potential of CSL was demonstrated by the observed dose-dependent increase in proliferation of PBMCs.
In summary, the lectin with complex sugar specificity purified from the pathogenic fungus Cephalosporium, which is responsible for ophthalmic mycoses, exhibits mitogenic and hemagglutinating activity. It is one of the few lectins characterized from pathogenic fungi reported and the information provides valuable account for understanding the lectin mediated host-parasite interaction in fungal infections.