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Localization of melanin in conidia of Alternaria alternata using phage display antibodies
Raffaella Carzaniga1, Daniela Fiocco, Paul Bowyer
1IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, UK.
Abstract:
Melanins derived from 1,8-dihydroxynaphthalene (DHN) are important for the pathogenicity and survival of fungi causing disease in both plants and animals. However, precise information on their location within fungal cell walls is lacking. To obtain antibodies for the immunocytochemical localization of melanin, 83 phage antibodies binding to 1,8-DHN were selected from a naive semisynthetic single-chain Fv (scFv) phage display library. Sequence analysis of the heavy chain binding domains of 17 antibodies showed a high frequency of positively charged amino acids. One antibody, designated M1, was characterized in detail. M1 bound specifically to 1,8-DHN in competitive inhibition enzyme-linked immunosorbent assays, showing no cross-reaction with nine structurally related phenolic compounds. Epitope recognition required two hydroxyl groups in a 1,8 configuration. M1 also bound to naturally occurring melanin isolated from mycelia of Alternaria alternata, suggesting that epitopes remain accessible in polymerized melanin. Transmission electron microscopy-immunogold labeling, using M1 in the form of soluble scFv fragments, showed that melanin was located in the septa and outer (primary) walls of wild-type A. alternata conidia, but not those of an albino mutant, AKT88-1. The M1 antibody provides a new tool for detecting melanized pathogens in plant and animal tissues and for precisely mapping the distribution of the polymer within spores, appressoria, and hyphae.
Insights
Researchers developed a specific antibody (M1) to locate 1,8-dihydroxynaphthalene (DHN) melanins in fungal cell walls. This melanin is crucial for fungal pathogens, and M1 helps map its distribution in plant and animal disease-causing fungi.
Area of Science:
- Mycology
- Biochemistry
- Immunology
Background:
- Fungal melanins, derived from 1,8-dihydroxynaphthalene (DHN), are vital for the pathogenicity and survival of fungi causing plant and animal diseases.
- The precise location of these melanins within fungal cell walls remains largely uncharacterized.
Purpose of the Study:
- To develop specific antibodies for immunocytochemical localization of DHN-melanin within fungal structures.
- To investigate the distribution of melanin in the cell walls of pathogenic fungi.
Main Methods:
- Selection of 83 phage antibodies binding to 1,8-DHN from a single-chain Fv (scFv) phage display library.
- Characterization of antibody M1 using competitive inhibition enzyme-linked immunosorbent assays and epitope analysis.
- Immunogold labeling with transmission electron microscopy to visualize melanin location in *Alternaria alternata*.
Main Results:
- Antibody M1 specifically recognized 1,8-DHN, requiring a 1,8-dihydroxy configuration, and bound to naturally occurring fungal melanin.
- Melanin was localized to the septa and outer walls of wild-type *A. alternata* conidia.
- No melanin signal was detected in an albino mutant, confirming antibody specificity.
Conclusions:
- The M1 antibody is a specific tool for detecting 1,8-DHN melanin in fungal pathogens.
- This antibody enables precise mapping of melanin distribution in fungal spores, appressoria, and hyphae.
- M1 facilitates the study of melanized fungal pathogens in plant and animal tissues.
