Research Article: Conidial surface proteins at the interface of fungal infections

Date Published: September 12, 2019

Publisher: Public Library of Science

Author(s): Matthew G. Blango, Olaf Kniemeyer, Axel A. Brakhage, Donald C Sheppard.


Partial Text

Spores are small, usually unicellular reproductive units produced to propagate genetic material by prokaryotic and eukaryotic microbes, including algae and protozoa, lower vascular plants, and even a subset of animals [1]. In prokaryotes, the endospores generated by some members of the phylum Firmicutes evolved for stress resistance and long-term survival of extreme environments [2]. In the lower vascular plants like ferns and some mosses, unicellular spores located underneath the leaves of nonflowering plants, similar to multicellular seeds in fruits or flowers, transfer genetic material to the next generation and often into new environments [3]. In eukaryotic microbes, the unicellular slime molds and fungi produce spores during their life cycle or in response to environmental stress [4, 5]. Although often overlooked, fungi play an essential role in the environment as saprotrophs, mycorrhizal symbionts, and even, in some cases, as parasites or pathogens. Fungi are typically nonmotile, but production and dissemination of spores by both marine and terrestrial fungi offer a mechanism for wider genetic dispersal [6]. Water currents, plants, and animals all disperse fungal spores, but the most commonly considered form of spore transport is wind, where astonishingly high fungal spore fluxes have been observed in terrestrial ecosystems (513 spores per m2 s1) [6, 7]. Locally higher fluxes are even present during meteorological events, like thunderstorms or high wind events, and in particular ecosystems, like cropland, which show measured fluxes of approximately 2,500 spores per m2 s1 [6, 7]. The presence of spores in the air column is commonly linked to respiratory diseases, as in the case of thunderstorm asthma [8, 9]. Fungal spores are not only a problem in humans but are also a major source of disease in insects, plants, and other animals. Our shifting climate is expected to lead to increasing exposure to spores and subsequent fungal infections due to the ubiquity of fungi in the environment [10].

The best-studied examples of conidial surface proteins are the widely conserved hydrophobins of filamentous fungi. Conidial hydrophobins are cysteine-containing functional amyloid proteins that drive hydrophobicity and promote air buoyancy [11, 12]. Biophysical characterizations have revealed two classes of hydrophobins; Class I hydrophobins form a characteristic rodlet structure often present on conidia, whereas Class II hydrophobins assemble amphiphilic films at air–water interfaces [13]. The hydrophobins are found in a variety of fungal genera, including both saprophytes and pathogens of the Ascomycetes (Class I and II hydrophobins) and Basidiomycetes (Class I hydrophobins), such as Aspergillus, Cladosporium, Penicillium, Neurospora, Magnaporthe, Schizophyllum, Phanerochaete, and Beauveria [11, 12, 14, 15]. The cellular localization of hydrophobins is quite variable. In some cases, these proteins are found only on the conidia, while in other organisms, they are present on mycelia or even secreted [16, 17]. In the important human pathogen Aspergillus fumigatus, the hydrophobins are tightly regulated, with the hydrophobic barrier dismantled during germination to aid in nutrient exchange and growth [18]. The A. fumigatus hydrophobins, along with closely related orthologs, have been shown to contribute to immune evasion of conidia by masking host Dectin-1– and Dectin-2–dependent immune recognition of fungal spores [19, 20]. The A. fumigatus hydrophobins, in particular, the RodA protein, also inhibit platelet activation during infection, providing an advantage for the fungus in establishing infection in an immunocompromised host [21]. In line with these findings, the frequency of human antigen–specific T cells that recognize conidial proteins is lower than for those that target mycelial antigens, again reiterating the capacity of A. fumigatus conidia to evade the immune response [22]. Interestingly, this saprophytic mold is thought to have developed these immune evasion strategies in the environment and not in the host [23], potentially in response to predation by soil-dwelling amoebae [24–26]. Conversely, the human host had to evolve to efficiently remove these ubiquitous conidia while limiting hyperreactivity that would damage host tissues [19].

The hydrophobins are not alone on the conidial surface, and in fact, many other proteins contribute to substrate adhesion. The best examples come from human pathogens, for which multiple studies have described A. fumigatus proteins contributing to adhesion. Interestingly, in A. fumigatus hyphae, the exopolysaccharide galactosaminogalactan is the major mediator of hyphal adhesion; however, this molecule is absent from conidia, suggesting that other factors likely contribute to adhesion [30, 31]. Early studies linked adherence to the hydrophobin, RodA, and the allergen, AspF2 (reviewed in [32]), but it was quickly realized that other proteins must also contribute to adhesion. The glycophosphatidylinositol-anchored protein CspA was next shown to aid in surface adhesion [33], likely through indirect effects on cell wall architecture [32, 34]. The FleA lectin is another example of a conidial surface protein that mediates adhesion to the host, particularly to airway mucins [35]. Detection of FleA by the host is essential for proper clearance of conidia by macrophages and resolution of the infection [35]. In more recent work using comparative phenotypic and transcriptomic analyses, additional adhesion molecules were predicted, including a haemolysin-like protein that potentially has a moonlighting function on the conidial surface [36]. These predictions remain preliminary, and further experimentation will be required to prove that these proteins are both on the surface and contributing to adhesion, but collectively, these studies assert that a large number of proteins contribute to A. fumigatus adhesion in the host. In the mucoralean fungus Rhizopus oryzae, the CotH proteins found on the surface of spores promote adhesion and invasion by acting as ligands for glucose-regulated protein 78 (GRP78) on the surface of endothelial cells, similar to the examples from A. fumigatus [37].

We have already learned that surface proteins often contribute to adherence, stress resistance, and immune evasion, so it is perhaps not surprising that there are also many cases in which conidial proteins directly influence the outcome of infection. The R. oryzae CotH protein, important for adherence, has also been linked to virulence. Heterologous production of the CotH protein in nonpathogenic Saccharomyces cerevisiae facilitated invasion of host cells via the GRP78 receptor, indicating that CotH is genetically sufficient to confer invasion to a nonpathogenic organism [37]. In addition, an R. oryzae cotH deletion strain exhibited decreased invasion, reduced epithelial cell damage, and partially attenuated virulence in a mouse model of mucormycosis [37]. Intriguingly, antibodies targeting CotH were shown to be protective against infection in the mouse model, suggesting potential as an immunotherapeutic agent in the future [37].

The ultimate goal of defining the conidial surface proteome is to improve our understanding of fungal pathogenesis and identify novel targets for early detection or immunotherapy. In particular, detection of fungal conidia from environmental samples might provide an early warning to those suffering from lung conditions like asthma or chronic obstructive pulmonary disease, in which patients show a heightened susceptibility to allergic exacerbations due to fungal sensitization [46]. The hydrophobins are one putative class of proteins with potential diagnostic value, along with specific proteins like the A. fumigatus CcpA protein or the R. oryzae CotH proteins, for example [37, 43]. A key to diagnosis will be finding biomarkers that are surface-localized under a diverse array of conditions, a feature that might prove difficult. Recent work in A. fumigatus suggests that the surface proteome of conidia is quite dynamic and environment dependent, making diagnosis through a single surface biomarker, like the hydrophobins, extremely challenging [43]. Proteins like A. fumigatus CalA are of great interest, as they are on the surface of multiple morphotypes of the fungi, including swollen conidia and hyphae [45]. We also have to take into account the ubiquity of fungal conidia, which makes contamination of highly sensitive diagnostics from the local environment an ever-present issue.




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