Biochemical and structural studies of target lectin SapL1 from the emerging opportunistic microfungus Scedosporium apiospermum

Scedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in humans. Host–pathogen interactions often implicate lectins that have become therapeutic targets for the development of carbohydrate mimics for antiadhesive therapy. Here, we present the first report on the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 was found using bioinformatics as a homolog to the conidial surface lectin FleA from Aspergillus fumigatus known to play a role in the adhesion to host glycoconjugates present in human lung epithelium. In our strategy to obtain recombinant SapL1, we discovered the importance of osmolytes to achieve its expression in soluble form in bacteria. Analysis of glycan arrays indicates specificity for fucosylated oligosaccharides as expected. Submicromolar affinity was measured for fucose using isothermal titration calorimetry. We solved SapL1 crystal structure in complex with α-methyl-L-fucoside and analyzed its structural basis for fucose binding. We finally demonstrated that SapL1 binds to bronchial epithelial cells in a fucose-dependent manner. The information gathered here will contribute to the design and development of glycodrugs targeting SapL1.

The Role of RodA-Conserved Cysteine Residues in the Aspergillus fumigatus Conidial Surface Organization

Immune inertness of Aspergillusfumigatus conidia is attributed to its surface rodlet-layer made up of RodAp, characterized by eight conserved cysteine residues forming four disulfide bonds. Earlier, we showed that the conserved cysteine residue point (ccrp) mutations result in conidia devoid of the rodlet layer. Here, we extended our study comparing the surface organization and immunoreactivity of conidia carrying ccrp-mutations with the RODA deletion mutant (∆rodA). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp-mutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆rodA conidia selectively bound to β-(1,3)-glucan; the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆rodA was similar to the parental strain conidia. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants showed more similarities with the parental strain, but were significantly different from the ∆rodA. Ccrp-mutant conidia were less immunostimulatory compared to ∆rodA conidia. Our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of the rodlet layer on the conidial surface, and (ii) targeted point mutation could be an alternative approach to study the role of fungal cell-wall genes in host–fungal interaction.

HYD3, a conidial hydrophobin of the fungal entomopathogen Metarhizium acridum induces the immunity of its specialist host locust

Conidial hydrophobins in fungal pathogens of plants1,2, insects3,4, and humans5,6 are required for fungal attachment and are associated with high virulence. They are believed to contribute to the pathogenesis of infection by preventing immune recognition5,6. Here, we refute this generalisation offering a more nuanced analysis. We show that MacHYD3, a hydrophobin located on the conidial surface of the specialist entomopathogenic fungus Metarhizium acridum, activates specifically the humoral and cellular immunity of its own host insect, Locusta migratoria manilensis (Meyen) but not that of other non-host insects. When topically applied to the cuticle, purified MacHYD3 improved the resistance of locusts to both specialist and generalist fungal pathogens but had no effect on the fungal resistance of other insects, including Spodoptera frugiperda and Galleria mellonella. Hydrophobins extracted from the generalist fungal pathogens M. anisopliae and Beauveria bassiana had no effect on the resistance of locusts to fungal infection. Thus, the host locust has evolved to recognize the conidial hydrophobin of its specialist fungal pathogen, whereas conidial hydrophobins from generalist fungi are able to evade recognition. Our results distinguish the immunogenic potential of conidial hydrophobins between specialist and generalist fungi.

The dynamic surface proteomes of allergenic fungal conidia

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static, but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

Conidial melanin of the human pathogenic fungus Aspergillus fumigatus disrupts cell autonomous defenses in amoebae

The human pathogenic fungus Aspergillus fumigatus is a ubiquitous saprophyte that causes fatal infections in immunocompromised individuals. Following inhalation, conidia are ingested by innate immune cells and can arrest phagolysosome maturation. How such general virulence traits could have been selected for in natural environments is unknown. Here, we used the model amoeba Dictyostelium discoideum to follow the antagonistic interaction of A. fumigatus conidia with environmental phagocytes in real time. We found that conidia covered with the green pigment 1,8-dihydroxynaphthalene-(DHN)-melanin were internalized at far lower rates when compared to those lacking the pigment, despite high rates of initial attachment. Immediately after uptake of the fungal conidia, nascent phagosomes were formed through sequential membrane fusion and fission events. Using single-cell assays supported by a computational model integrating the differential dynamics of internalization and phagolysosome maturation, we could show that acidification of phagolysosomes was transient and was followed by neutralization and, finally, exocytosis of the conidium. For unpigmented conidia, the cycle was completed in less than 1 h, while the process was delayed for conidia covered with DHN-melanin. At later stages of infection, damage to infected phagocytes triggered the ESCRT membrane repair machinery, whose recruitment was also attenuated by DHN-melanin, favoring prolonged persistence and the establishment of an intracellular germination niche in this environmental phagocyte. Increased exposure of DHN-melanin on the conidial surface also improved fungal survival when confronted with the fungivorous predator Protostelium aurantium, demonstrating its universal antiphagocytic properties.

Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic FungusAspergillus fumigatus

ABSTRACTAspergillus fumigatusis a common airborne fungal pathogen of humans and a significant source of mortality in immunocompromised individuals. Here, we provide the most extensive cell wall proteome profiling to date ofA. fumigatusresting conidia, the fungal morphotype pertinent to first contact with the host. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified proteins within the conidial cell wall by hydrogen-fluoride (HF)–pyridine extraction and proteins exposed on the surface using a trypsin-shaving approach. One protein, designatedconidialcell wallproteinA(CcpA), was identified by both methods and was found to be nearly as abundant as hydrophobic rodlet layer-forming protein RodA. CcpA, an amphiphilic protein, like RodA, peaks in expression during sporulation on resting conidia. Despite high cell wall abundance, the cell surface structure of ΔccpAresting conidia appeared normal. However, trypsin shaving of ΔccpAconidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen ΔccpAconidia led to higher activation of neutrophils and dendritic cells than was seen with wild-type conidia and caused significantly less damage to epithelial cellsin vitro. In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with ΔccpAconidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed toA. fumigatusconidia, suggesting a role for CcpA as a structural protein impacting conidial immunogenicity rather than possessing a protein-intrinsic immunosuppressive effect. Together, these data suggest that CcpA serves as a conidial stealth protein by altering the conidial surface structure to minimize innate immune recognition.IMPORTANCEThe mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case ofAspergillus fumigatus, the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis.A. fumigatuslacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.