Conidial Hydrophobins ofAspergillus fumigatus

Abstract

The surface of Aspergillus fumigatus conidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. The RODA gene was previously characterized, and ⌬rodA conidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). The RODB gene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp of A. nidulans. ⌬rodB conidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of ⌬rodA conidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the ⌬rodA ⌬rodB double mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.The surface of many fungal conidia is covered by a thin layer of regularly arranged rodlets. This structure, which favors air buoyancy and dispersion of the conidia by air currents (2), is mainly proteinaceous (3,(8)(9)(10)16). The proteins present in the cell wall of aerial structures of fungi responsible for this rodlet configuration are the hydrophobins, a family of small, moderately hydrophobic proteins characterized by the conserved spacing of eight cysteine residues (42, 44). For the human opportunistic pathogen Aspergillus fumigatus, the presence of a rodlet layer has been visualized and the RODA gene has been previously shown to be involved in the formation of the rodlets of its conidia (41). In plants, hydrophobins have been associated with the virulence of phytopathogenic fungi (38). Although it has been repeatedly shown that cell wall and associated structures help human fungal pathogens to resist host defense reactions (22), to date no studies have analyzed the role of the rodlet layer in the resistance of the conidia to phagocytosis. Even though the rodlet layer of the conidia of Neurospora crassa, Beauveria bassiana, and Magnaporthe grisea contained a single hydrophobin (5, 39, 40), A. nidulans, a species phylogenetically close to A. fumigatus, has two conidial hydrophobins, RodAp and DewAp (35,36). These data have prompted us to reexamine the surface layer of the conidia of A. fumigatus with a view to (i) analyzing exhaustively hydrophobins present on the surface of the conidia and (ii) studying their role in resistance to phagocytosis. A. fumigatus is a good model for the later study, since conidia, which are a main component of the airborne thermophilic fungal florae (1), are all engulfed and killed by lung alveolar macrophages (AM) following their inhalation (11; B. Philippe, O. Ibrahim-Granet, M. C. Prévost, M. A. Gougerot-Pocidalo, J. Roes, M. SanchezPerez, A. Van der Meer...

Innate Immunity toAspergillusSpecies

“…Conidial maturation triggers a profound morphological change that involves the loss of the proteinaceous hydrophobic layer and exposure of the inner cell wall (120,163). This cell wall is composed mainly of polysaccharides consisting of ␤-glucan, mannan, chitin, and galactomannan (89 (64,74,157), and optimal CD4 ϩ T-cell responses appear to occur only in response to live conidia (136).…”

Dectin-1 and TLRs Permit Macrophages to Distinguish between Different Aspergillus fumigatus Cellular States

“…We therefore examined the ability of dectin-1 to recognize A. fumigatus at different stages of maturation. During activation and maturation into germ tubes, conidia swell, in the process losing a cell wall that consists largely of hydrophobic proteins (rodlets) and pigments, both of which confer resistance to macrophage killing (11,19). We produced sDectin in HEK293 cells (16) and used it to identify dectin-1 binding sites on A. fumigatus resting conidia, swollen conidia, germ tubes, and hyphae.…”

“…During maturation, conidia undergo a morphologic change that involves loss of a thin proteinaceous hydrophobic cell wall before development of hyphal forms, the cell walls of which are composed primarily of ␤(1,3)-glucan, chitin, and galactomannan (11,12). We specifically examined the role of dectin-1, a type II transmembrane receptor that triggers phagocytosis of ␤-glucan-containing particles (13) and collaborates with TLR2 to coordinate inflammatory responses, including cytokine secretion and production of reactive oxygen species (ROS) (14,15).…”

Parallels in Fungal Pathogenesis on Plant and Animal Hosts

“…Conidial germination and infectious growth are not altered (79). Hydrophobins in conidial walls protect the animal pathogen A. fumigatus from being killed by macrophages during initial invasion events (62).…”