Effect of anti-glycosphingolipid monoclonal antibodies in pathogenic fungal growth and differentiation. Characterization of monoclonal antibody MEST-3 directed to Manpalpha1-->3Manpalpha1-->2IPC

Abstract

Background: Studies carried out during the 1990's demonstrated the presence of fungal glycoinositol phosphorylceramides (GIPCs) with unique structures, some of them showed reactivity with sera of patients with histoplasmosis, paracoccidioidomycosis or aspergillosis. It was also observed that fungal GIPCs were able to inhibit T lymphocyte proliferation "in vitro", and studies regarding the importance of these molecules to fungal survival showed that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis.

Results: In this paper, we describe a detailed characterization of an IgG2a monoclonal antibody (mAb), termed MEST-3, directed to the Paracoccidioides brasiliensis glycolipid antigen Pb-2 (Manpalpha1-->3Manpalpha1-->2IPC). mAb MEST-3 also recognizes GIPCs bearing the same structure in other fungi. Studies performed on fungal cultures clearly showed the strong inhibitory activity of MEST-3 on differentiation and colony formation of Paracoccidioides brasiliensis, Histoplasma capsulatum and Sporothrix schenckii. Similar inhibitory results were observed when these fungi where incubated with a different mAb, which recognizes GIPCs bearing terminal residues of beta-D-galactofuranose linked to mannose (mAb MEST-1). On the other hand, mAb MEST-2 specifically directed to fungal glucosylceramide (GlcCer) was able to promote only a weak inhibition on fungal differentiation and colony formation.

Conclusions: These results strongly suggest that mAbs directed to specific glycosphingolipids are able to interfere on fungal growth and differentiation. Thus, studies on surface distribution of GIPCs in yeast and mycelium forms of fungi may yield valuable information regarding the relevance of glycosphingolipids in processes of fungal growth, morphological transition and infectivity.

Figure 1

Reactivity of fungal GIPCs with MEST-3. Fungal GIPCs were purified by a combination of chromatography in DEAE-Sephadex, silica gel 60, HPLC and preparative HPTLC. HPTLC was developed in solvent A. Panel A, stained with orcinol/H₂SO₄ and panel B, immunostaining with MEST-3. Lane 1, GIPC Pb-2 from mycelium form of P. brasiliensis; lane 2, acidic GSLs from mycelium form of P. brasiliensis; lane 3, acidic GSLs from yeast form of P. brasiliensis (Pb); lane 4, acidic GSLs from hyphae of A. fumigatus (Af); lane 5, acidic GSLs from hyphae of A. nidulans (An); lane 6, acidic GSLs from mycelium form of H. capsulatum (Hc); lane 7, acidic GSLs from yeast form of H. capsulatum; lane 8, acidic GSLs from mycelium form of S. schenckii (Sc); lane 9, acidic GSLs from yeast form of S. schenckii; lane 10, acidic GSLs from the edible mushroom Agaricus blazei (Ab). Arrows indicates chromatographic migration of Pb-2, Af-2, An-2, Hc-Y2 and Ss-Y2. Panel C, GIPCs (first well 0.1 μg) were serially double diluted in ethanol and adsorbed on a 96-well plate. MEST-3 (100 μl) was added and incubated overnight at 4°C. The amount of antibody bound to GSLs was determined by incubation with rabbit anti-mouse IgG (2 h) and 10⁵ cpm of ¹²⁵I-labeled protein A in 1% BSA. Pb-2 from yeast (closed square) and from mycelium (closed triangle) forms of P. brasiliensis; Ss-Y2 (open circle) from yeast form of S. schenckii; Af-2 (open triangle) from A. fumigatus, Hc-Y2 (open inverted triangle) from yeast forms of H. capsulatum, Pb-3 (closed inverted triangle) from yeast and Pb-3 (closed diamond) from mycelium forms of P. brasiliensis and Ss-M2 (open diamond) from mycelium forms of S. schenckii.

Figure 2

Inhibition of mAb MEST-3 binding to Pb-2. 96-well plates were adsorbed with GIPC Pb-2 from mycelium forms of P. brasiliensis. Methyl-glycosides, disaccharides and GIPC-derived glycosylinositols (first well 100 mM) were serially double diluted with PBS and preincubated with MEST-3, and the inhibition assay was carried out as described in Materials and Methods. The effects of the methyl-glycosides, disaccharides and glycosylinositols are expressed as percentages of inhibition of MEST-3 binding to Pb-2. (closed square) Manpα1→2Manp, (closed circle) Manpα1→3Manp, (closed triangle) Manpα1→6Man, (open diamond) methyl-α/β-D-glucopyranoside; (open circle) methyl-α/β-D-galactopyranoside; (open triangle) methyl-α/β-D-mannopyranoside, (closed diamond) Manα1→3Manα1→2Ins, (open square) Manα1→3Manα1→6Ins.

Figure 3

Indirect immunofluorescence. Indirect immunofluorescence of yeast forms of P. brasiliensis (Pb), H capsulatum (Hc) and S. schenckii (Ss), with mAb MEST-3. A- fluorescence. B- phase contrast.

Figure 4

Effect of monoclonal antibodies on fungal growth. Panel A, Yeast forms of P. brasiliensis, H. capsulatum and S. schenckii were incubated for 24 h with mAbs, or a control IgG or left alone, at 37°C. Yeasts were transferred to a petri dish containing PGY or BHI-agar medium, and incubated for 2 days at 37°C. Colony forming units (CFUs) were counted, and expressed as percentage of those incubated with an irrelevant mAb, considered as 100% of CFU. Panel B, MTT assay of fungi after incubation with mAbs MEST-1, -2, and -3. Yeast forms of P. brasiliensis, H. capsulatum and S. schenckii were incubated with mAbs, a control IgG or left alone. After incubation for 48 h, MTT solution was added to each well and was further incubated for 3 h at 37°C. The absorption was measured at 580 nm. MEST-1 (closed square), MEST-2 (closed circle) and MEST-3 (closed triangle). * p < 0.05.

Figure 5

Effect of monoclonal antibodies on yeast to mycelium transformation. Yeast forms of P. brasiliensis, H. capsulatum and S. schenckii were incubated for one hour with different concentration of MEST-1, -2 and -3, and control IgG at 37°C. After that the yeast cultures were transferred to a 25°C incubator, and kept for 2 days. Three hundred yeasts were counted, and the number of yeast showing hyphae growth was evaluated. In control experiment 100% of yeast showed hyphae formation; the results represent the percentage of those incubated with an irrelevant mAb, considered as 100% of yeast to mycelium transformation. MEST-1 (closed square), MEST-2 (closed circle) and MEST-3 (closed triangle). * p < 0.05.

Figure 6

Effect of mAbs on mycelia formation. Yeasts were suspended in 1 ml of PGY or BHI medium. This suspension was added to a 24-well plate and supplemented with mAb MEST-1 or -3 (50 μg/ml), after one hour at 37°C cells were placed at 25°C. After 24 and 48 h of incubation yeast differentiation into mycelia forms was observed in an inverted microscope. Controls experiments were performed identically in presence of irrelevant immunoglobulins (normal mouse total Ig).

Figure 7

Effect of mAb MEST-3 on yeast formation. P. brasiliensis hyphae fragments were suspended in 1 ml of PGY medium and supplemented or not with mAb MEST-3 (50 μg/ml). Cells were placed on a 24-well plate at 37°C, and after 96 h of incubation, hyphae differentiation into yeast (M→Y) forms was observed under microscope. Panel A shows M→Y differentiation in free-mAb medium, Panel B shows M→Y differentiation in medium containing MEST-3, and Panel C shows the mycelia growth of hyphae fragments maintained at 23°C for 96 h in free-mAb medium.