Conservation of Mannan Synthesis in Fungi of the Zygomycota and Ascomycota Reveals a Broad Diagnostic Target

Abstract

Ascomycetes and zygomycetes account for the majority of (i) fungi responsible for cutaneous, subcutaneous, and invasive human fungal infections, (ii) plant fungal pathogens, (iii) fungi that threaten global biodiversity, (iv) fungal agents of agricultural spoilage, and (v) fungi in water-damaged buildings. Rapid recognition of fungal infection (or contamination) enables early treatment (or remediation). A bioinformatics search found homologues of Saccharomyces cerevisiae Mnn9p present in members of the Zygomycota and Ascomycota phyla and absent in members of the Chytridiomycota and Basidiomycota. Mnn9p is a component of the yeast mannan polymerization complex and is necessary for α-1,6 mannan production. A monoclonal antibody (2DA6) was produced that was reactive with purified mannans of Mucor, Rhizopus, Aspergillus, Fusarium, and Candida species. Experimentation using a 2DA6 antigen capture enzyme-linked immunosorbent assay (ELISA) and extracts of fungi from the four phyla found agreement between the presence or absence of Mnn9p homologues and production or lack of production of mannan reactive with 2DA6. Studies of cell extracts from yeast mannan mutants identified α-1,6 mannan as the epitope recognized by 2DA6. To translate this finding into a point-of-use diagnostic, a 2DA6 lateral flow immunoassay was constructed that detected mannan in (i) extracts of dermatophytes and fungi that produce trauma-related infection and (ii) tissue from plants infected with Grosmannia clavigera or Sclerotium cepivorum These studies (i) revealed that the conservation of α-1,6-linked mannan in fungi of the Zygomycota and Ascomycota can be exploited as a broad diagnostic target and (ii) have provided a means to detect that target in an immunoassay platform that is well suited for clinic or field use.IMPORTANCE A key question asked when faced with an infection, an infestation, or environmental damage is whether it is a fungus. Identification of fungi as the cause of the problem can lead to remediation or treatment. Zygomycetes and ascomycetes account for the vast majority of fungal causes of human, animal, and plant disease, large-scale biodiversity loss, agricultural spoilage, and contamination of water-damaged buildings. These studies revealed the conservation of a common cell wall structural component of zygomycetes and ascomycetes to be a diagnostic target applicable to multiple pathogenic fungi and have leveraged that insight for practical use. Monoclonal antibodies reactive with this pan-fungal structure were produced and used to construct immunoassays (including ELISA and lateral flow assay) for detection of a broad range of pathogenic fungi.

Keywords: Mnn9; diagnostics; immunodetection; invasive fungal infection; lateral flow immunoassay; mannan; point of care.

FIG 1

Reactivity of MAb 2DA6 with purified mannans of different fungal genera. Results are shown from a sandwich ELISA in which plates were (i) coated with MAb 2DA6 to enable mannan capture, (ii) incubated with serial dilutions of purified mannan (20 µg/ml starting concentration), and (iii) incubated with HRPO-labeled MAb 2DA6. (Inset) Limit of detection (in nanograms per milliliter) of the sandwich ELISA for mannans isolated from different fungal genera.

FIG 2

Reactivity of MAb 2DA6 with mannans of the wild-type and the indicated mannan mutants of S. cerevisiae. (Left) A sandwich ELISA constructed from MAb 2DA6 was used to assess reactivity of cell extracts from the wild-type and Mnn2 and Mnn9 mutants. (Right) Reactivity of purified wild-type and Mnn2 mutant mannans in the sandwich ELISA. The starting concentration for the purified mannans was 20 µg/ml. Neg, negative. (Inset) Limit of detection (LoD [in nanograms per milliliter]) of the sandwich ELISA for mannans isolated from the wild-type and Mnn2 mutant strains.

FIG 3

Effect of treatment of wild-type (WT) and Mnn2 mutant mannan with periodate and proteinase K on reactivity with MAb 2DA6 in a sandwich ELISA. Mannans were treated with each reagent or were subjected to a mock treatment where all reagents and reactions were identical to those used with the treated group but where the periodate or proteinase K was omitted. The starting concentration for the mannans was 20 µg/ml.

FIG 4

Reactivity of hot citrate extracts from various fungi in a sandwich ELISA constructed from MAb 2DA6 Pneumocystis carinii isolated from infected rat lung was used for that fungus. In all other cases, extracts were prepared from mycelia or yeasts from culture.

FIG 5

Detection of mannan in hot citrate extracts from cultures of medically relevant fungi in a lateral flow immunoassay constructed from MAb 2DA6. (Left) Extracts from cultures of Epidermophyton spp., Microsporum spp., and Trichophyton spp. that cause dermatophyte infection in humans and animals. (Right) Extracts from cultures of fungi that produce combat-related invasive fungal infection. Negative control, citrate buffer.

FIG 6

Use of LFIA constructed from MAb 2DA6 to detect mannan in hot citrate extracts from tissue of Pinus contorta (lodgepole pine) infected with Grosmannia clavigera (blue stain fungus [left]) and tissue from Allium species (onion) infected with Sclerotium cepivorum (Allium white rot [right]). Results are shown for extracts from healthy and diseased plants.