Fast and Highly Specific Histological Detection of Pathogenic Fungi in Human Tissue

By: Monika Baeumle, Bernhard Schoenenberger, Jakob Zbaeren, Microbiology Focus Edition 1.3

Monika Baeumle, PhD, Product Manager Biochemistry ... monika.baeumle@sial.com
Bernhard Schoenenberger, PhD, Supervisor R&D
Jakob Zbaeren, Thrombose Laboratory, Inselspital Bern

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 pathogenic 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.

The recently developed Atto-dye labeled lectins have many applications, including carbohydrate, mitogenic and histochemical studies. Atto-dyes have very bright fluorescent signals and high photo stability, 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 (Figure 1).

Figure 1: Direct one step binding of fluorescent labeled lectins.


Lectin histology was performed on both polymer and paraffin embedded human skin tissue. The lectin conjugate used was Phytolacca americana- Atto 488 (Cat. No. 39905). The conjugate was diluted 100 times in PBS buffer (pH 7.4) before incubating with each specimen for 30 min. After washing to remove any unbound lectin and counterstaining the nuclei with DAPI (Cat. 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.

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.

Figure 2: Fluorescence microscopy of human skin tissue section (paraffin fixation) with fungal infection. The target carbohydrate subunit chitotriose [(GlcNAc)3] 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. Zbaeren, Inselspital Bern, Switzerland.


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.

The Atto-dye lectin conjugates below are now available from Sigma Life Science. Additional lectins and lectin conjugates from Sigma Life Science may be found on at sigma-aldrich.com/enzymeexplorer.

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Materials

     

References

  1. Lectin Methods and Protocols; J. M. Rhodes; J. D. Milton; Humana Press, Totowa, New Jersey, 1997

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