Surface Structure Characterization of Aspergillus fumigatus Conidia Mutated in the Melanin Synthesis Pathway and Their Human Cellular Immune Response

Abstract

gIn Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface.

Secondary metabolite arsenal of an opportunistic pathogenic fungus

“…Initial genome sequence predicted a total of 22 BGCs [11], but additional bioinformatic analysis now suggests over 30 BGCs are present in the genome of A. fumigatus [12]. The DHN-melanin cluster, first characterized in 1999 [13], has garnered considerable attention as a virulence factor in this organism [14].The second BGC to be characterized in A. fumigatus produced gliotoxin, which, like DHN-melanin, impacts virulence [15][16][17][18]. Following a reoccurring theme in studies of the roles of SM in fungal biology, both DHN-melanin and gliotoxin exhibit bioprotective properties well beyond a role as simply a virulence factor.…”

Fungal melanin stimulates surfactant protein D–mediated opsonization of and host immune response to Aspergillus fumigatus spores

“…The binding of GM or GAG to SP-D is mediated by its CRD in a calcium-dependent manner, while melanin binding is through the collagen-like domain of SP-D that is independent of calcium. There exists a layer of superficial rodlet and a underlying melanin layer on the dormant A. fumigatus conidial surface (28,29,34,35); however, at places, this melanin layer protrudes out of the rodlet layer, and hence the punctate binding pattern of SP-D on the dormant conidial surface. During germination, these rodlet and melanin layers are destroyed, exposing the cell wall polysaccharides, including GAG and GM, resulting in a uniform binding of SP-D.…”

“…At late interaction time (20h), MDMs cultured with SP-D-opsonized conidia produced significantly higher TNF-α, IL-6 and IL-8 than unstimulated MDMs and those co-cultured with un-opsonized conidia ( Figure 5B). fumigatus dormant conidial surface melanin pigment and two cell-wall polysaccharides, galactomannan (GM) that is masked by the surface melanin-rodlet layers in dormant conidia (28,29) and galactosaminogalactan (GAG), which is only synthesized during germination (27). The binding of GM or GAG to SP-D is mediated by its CRD in a calcium-dependent manner, while melanin binding is through the collagen-like domain of SP-D that is independent of calcium.…”

“…GM is found in the cell-wall of all the morphotypes of A. fumigatus, but masked by the surface rodlet and melanin layers in the dormant conidia (28,29). Therefore, we focused our study on melanin, as it is present on the surface of the dormant conidia, the major infective morphotype of A. fumigatus.…”

“…The assay was linear over a range of 0.1-1.0 EU/ml (10 EU = 1 ng of endotoxin). Preparation of the cell-wall components GM, GAG, chitin, RodAp and melanin ghosts were purified or prepared from conidia/mycelia as described earlier (27)(28)(29)56,57). β-(1,3)-Glucan and α-(1,3)-glucan were isolated from the alkali-insoluble (AI) and soluble (AS) cellwall fraction, respectively, as described earlier with few modifications (57).…”