Dataset Information

EphA2-Dependent Internalization of A. fumigatus Conidia in A549 Lung Cells Is Modulated by DHN-Melanin.

ABSTRACT: Dectin-1 and ephrin type-A receptor 2 (EphA2) receptors recognize ?-glucan present in the fungal cell wall. Inhibition of Dectin-1 with the monoclonal 2a11 antibody was shown to reduce internalization of conidia of the human pathogen Aspergillus fumigatus into epithelial cells. In this study, we investigated the role of the EphA2 receptor present on A549 epithelial type II lung cells in the interaction with A. fumigatus conidia. We assessed whether EphA2 is involved in association and internalization of conidia by receptor inhibition by an antibody or by using the kinase inhibitor dasatinib. A 50% reduction of internalization of conidia was observed when this receptor was blocked with either the EphA2-specific monoclonal antibody or dasatinib, which was similar when Dectin-1 was inhibited with the 2a11 monoclonal antibody. Inhibition of both receptors reduced the internalization to 40%. EphA2 inhibition was also assessed in a hydrophobin deletion strain (?rodA) that exposes more ?-glucan and a dihydroxynaphthalene (DHN)-melanin deletion strain (?pksP) that exposes more glucosamine and glycoproteins. The ?rodA strain behaved similar to the wild-type strain with or without EphA2 inhibition. In contrast, the ?pksP mutant showed an increase in association to the A549 cells and a decrease in internalization. Internalization was not further decreased by EphA2 inhibition. Taken together, the presence of DHN-melanin in the spore cell wall results in an EphA2-dependent internalization of conidia of A. fumigatus into A549 cells.

SUBMITTER: Keizer EM

PROVIDER: S-EPMC7573251 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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EphA2-Dependent Internalization of A. fumigatus Conidia in A549 Lung Cells Is Modulated by DHN-Melanin.

Keizer Esther M EM Wösten Han A B HAB de Cock Hans H

Frontiers in microbiology 20201006

Dectin-1 and ephrin type-A receptor 2 (EphA2) receptors recognize β-glucan present in the fungal cell wall. Inhibition of Dectin-1 with the monoclonal 2a11 antibody was shown to reduce internalization of conidia of the human pathogen Aspergillus fumigatus into epithelial cells. In this study, we investigated the role of the EphA2 receptor present on A549 epithelial type II lung cells in the interaction with A. fumigatus conidia. We assessed whether EphA2 is involved in association ...[more]

PMID: 33123097

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Project description:BACKGROUND: Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia. RESULTS: We used three A. fumigatus isolates which grew as white or brown powdery colonies, to demonstrate the role of melanin. Firstly, sequencing the genes responsible for biosynthesis of melanin (ALB1, AYG1, ARP1, ARP2, ABR1 and ABR2) showed point mutations (missense mutation, deletion or insertion) in the ALB1 gene for pigmentless isolates or in ARP2 for the brownish isolate. The isolates were then shown by scanning electron microscopy to produce numerous, typical conidial heads, except that the conidia were smooth-walled, as previously observed for laboratory mutants with mutations in the PKSP/ALB1 gene. Flow cytometry showed an increase in the fibronectin binding capacity of conidia from mutant isolates, together with a marked decrease in the binding of laminin to the conidial surface. A marked decrease in the electronegative charge of the conidia and cell surface hydrophobicity was also seen by microelectrophoresis and two-phase partitioning, respectively. Ultrastructural studies of mutant isolates detected considerable changes in the organisation of the conidial wall, with the loss of the outermost electron dense layer responsible for the ornamentations seen on the conidial surface in wild-type strains. Finally, analysis of the conidial surface of mutant isolates by atomic force microscopy demonstrated the absence of the outer cell wall rodlet layer which is composed of hydrophobins. CONCLUSION: These results suggest that, in addition to a protective role against the host's immune defences, melanin is also a structural component of the conidial wall that is required for correct assembly of the cell wall layers and the expression at the conidial surface of adhesins and other virulence factors.

Project description:Previously, 1,8-dihydroxynaphthalene (DHN)-melanin was described to protect Aspergillus fumigatus against hydrogen peroxide (H2O2), thereby protecting this opportunistic human pathogen from reactive oxygen species generated by the immune system. This was based on the finding that the ATCC 46645 mutant with mutations in the pksP gene of the DHN-melanin synthesis pathway showed increased sensitivity to reactive oxygen species compared to the wild type. Here, it is shown that deletion of the pksP gene in A. fumigatus strain CEA10 did not affect sensitivity for H2O2 and superoxide in a plate stress assay. In addition, direct exposure of the dormant white conidia of the pksP deletion strains to H2O2 did not result in increased sensitivity. Moreover, complementation of the ATCC 46645 pksP mutant strain with the wild-type pksP gene did result in pigmented conidia but did not rescue the H2O2-sensitive phenotype observed in the plate stress assay. Genome sequencing of the ATCC 46645 pksP mutant strain and its complemented strain revealed a mutation in the cat1 gene, likely due to the UV mutagenesis procedure used previously, which could explain the increased sensitivity toward H2O2. In summary, DHN-melanin is not involved in protection against H2O2 or superoxide and, thus, has no role in survival of conidia when attacked by these reactive oxygen species. IMPORTANCE Opportunistic pathogens like Aspergillus fumigatus have strategies to protect themselves against reactive oxygen species like hydrogen peroxides and superoxides that are produced by immune cells. DHN-melanin is the green pigment on conidia of Aspergillus fumigatus and more than 2 decades ago was reported to protect conidia against hydrogen peroxide. Here, we correct this misinterpretation by showing that DHN-melanin actually is not involved in protection of conidia against hydrogen peroxide. We show that UV mutagenesis that was previously used to select a pksP mutant generated many more genome-wide mutations. We discovered that a mutation in the mycelial catalase gene cat1 could explain the observed phenotype of increased hydrogen peroxide sensitivity. Our work shows that UV mutagenesis is not the preferred methodology to be used for generating mutants. It requires genome sequencing with single-nucleotide polymorphism analysis as well as additional validations to discard unwanted and confirm correct phenotypes.

Dataset Information

EphA2-Dependent Internalization of A. fumigatus Conidia in A549 Lung Cells Is Modulated by DHN-Melanin.

Publications

EphA2-Dependent Internalization of <i>A. fumigatus</i> Conidia in A549 Lung Cells Is Modulated by DHN-Melanin.

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