BioFiles Volume 5, Number 1 — Glycobiology

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Histological Detection of Pathogenic Fungi in Human Tissue

Lectins are ubiquitous proteins or glycoproteins that can be isolated from plant and animal sources and can bind to specific carbohydrate moieties. Due to their high affinity to sugar residues, lectins have become important tools for sensitive detection of cellular carbohydrates, revealing subtle alteration in glycosylation between otherwise indistinguishable cells. This allows identification of cellular surface structures, e.g. cell surface, cytoplasm, and nuclear structures. Furthermore, lectin affinity binding allows for the detection of degeneration of tissue as well as pathogenic infestations such as fungi.

Histochemical studies are of importance in the histological and pathological investigation of tissue in clinical research. Lectin histochemistry can be performed on living cells in suspension, on cell smears, tissue imprints, fixed tissue sections, or fresh cryostat sections. Atto-dye labeled lectins have many applications, including carbohydrate, mitogenicity, and histochemical studies. Atto-dyes have very bright fluorescent signals and high photostability, which enable a direct one-step tissue-binding protocol. Time-consuming multistage amplification procedures are not required for Atto-dye lectin conjugates. Here, we demonstrate a highly specific identification of pathogenic fungi on human tissue via direct fluorescence detection using fluorescently labeled lectin (see Figure 1).

Lectin histology was performed on both polymer and paraffin embedded human skin tissue. The lectin conjugate used was Phytolacca americana-Atto 488 (Product No. 39905). The conjugate was diluted 100 times in PBS buffer, pH 7.4, before incubating with each specimen for 30 minutes. After washing to remove any unbound lectin and counterstaining the nuclei with DAPI (Product No. 32670), the samples were examined using a microscope equipped for epifluorescence with a 450-490 nm excitation bandpass filter and a 520-560 nm barrier (emission) filter.

 


Figure 1.Schematic of direct binding of fluorescent Atto dye conjugated lectins.

The images obtained show a very specific labeling of pathogenic fungi infecting human tissue (see Figure 2). The image demonstrates the fine filaments of the fungi containing typical mycelium, and individual fungi cells are clearly visible. A slightly higher fluorescence is observed in the separating cross-walls between two cells (septa), which are due to a higher concentration of target carbohydrates. Very low background is observed.

Fungal cell walls contain chitin, a polymer of β-(1→4) linked N-acetyl-D-glucosamine, while animal and plant cells do not synthesize chitin. The lectin Phytolacca americana targets the fungal carbohydrate fragment chitotriose [(β-N-Acetyl-D-glucosamine)3, (GlcNAc)3] shown in green (λex 485 nm). Due to the lack of the target carbohydrate chitotriose in the skin tissue, no specific interaction between the lectin Phytolacca americana and the tissue is observed.

The bright and stable fluorescence properties of the Atto 488 dye provide a strong fluorescent signal without requiring additional amplification steps. Further experiments with staining different fungal infected tissues were carried out. Similar results confirm this approach to be a successful and reliable way to detect fungi. This application may encourage scientists to investigate further histological phenomena by using lectin interactions.

 


Figure 2. Fluorescent microscopy of human skin tissue section (paraffin fixation) with fungal infection. The target carbohydrate chitotriose of the pathogenic fungi are specifically bound to lectin from Phytolacca americana Atto 488 conjugate (green). The nuclei are counterstained with DAPI (blue). Image by J. Zbären, Inselspital, Bern.

Atto-Dye Lectin Conjugates

Additional lectins and lectin conjugates available from Sigma Life Science may be found at sigma-aldrich.com/enzymeexplorer

 

Description λexem (nm) Carbohydrate Specificity Product No.
Concanavalin A-Atto 565 conjugate 563 / 592 nm in PBS (α-D-Mannose; α-D-Galactose) 69535
Lectin from Artocarpus integrifolia-Atto 594 conjugate 601 / 627 nm in PBS (α-Galactose-O-Me) 76158
Lectin from Phaseolus vulgaris-Atto 488 conjugate 501 / 523 nm in PBS (Oligosaccharide) 75319
Lectin from Phaseolus vulgaris-Atto 550 conjugate 554 / 576 nm in PBS (Oligosaccharide) 90852
Lectin from Phaseolus vulgaris-Atto 647N conjugate 644 / 669 nm in PBS (Oligosaccharide) 77363
Lectin from Phytolacca americana-Atto 488 conjugate 501 / 523 nm in PBS ((GlcNAc)3) 39905
Lectin from Phytolacca americana-Atto 550 conjugate 554 / 576 nm in PBS ((GlcNAc)3) 94816
Lectin from Phytolacca americana-Atto 647N conjugate 644 / 669 nm in PBS ((GlcNAc)3) 3065
Lectin from Triticus vulgaris-Atto 488 conjugate 501 / 523 nm in PBS ((GlcNAc)2, NeuNAc) 16441
Lectin from Triticus vulgaris-Atto 532 conjugate 532 / 558 nm in PBS ((GlcNAc)2, NeuNAc) 68917
Lectin from Ulex europaeus-Atto 488 conjugate 501 / 523 nm in PBS (α-L-Fucose) 19337
Lectin from Ulex europaeus-Atto 550 conjugate 554 / 576 nm in PBS (α-L-Fucose) 94165
Lectin from Ulex europaeus-Atto 594 conjugate 601 / 627 nm in PBS (α-L-Fucose) 73873

 

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