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Protein Glycosylation in Aspergillus fumigatus Is Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development.

ABSTRACT: Glycosylation is a conserved posttranslational modification that is found in all eukaryotes, which helps generate proteins with multiple functions. Our knowledge of glycosylation mainly comes from the investigation of the yeast Saccharomyces cerevisiae and mammalian cells. However, during the last decade, glycosylation in the human pathogenic mold Aspergillus fumigatus has drawn significant attention. It has been revealed that glycosylation in A. fumigatus is crucial for its growth, cell wall synthesis, and development and that the process is more complicated than that found in the budding yeast S. cerevisiae. The present paper implies that the investigation of glycosylation in A. fumigatus is not only vital for elucidating the mechanism of fungal cell wall synthesis, which will benefit the design of new antifungal therapies, but also helps to understand the role of protein glycosylation in the development of multicellular eukaryotes. This paper describes the advances in functional analysis of protein glycosylation in A. fumigatus.

SUBMITTER: Jin C

PROVIDER: S-EPMC3184424 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Protein Glycosylation in Aspergillus fumigatus Is Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development.

Jin Cheng C

International journal of microbiology 20110928

Glycosylation is a conserved posttranslational modification that is found in all eukaryotes, which helps generate proteins with multiple functions. Our knowledge of glycosylation mainly comes from the investigation of the yeast Saccharomyces cerevisiae and mammalian cells. However, during the last decade, glycosylation in the human pathogenic mold Aspergillus fumigatus has drawn significant attention. It has been revealed that glycosylation in A. fumigatus is crucial for its growth, cell wall sy ...[more]

PMID: 21977037

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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:Aspergillosis associated with azole-resistant Aspergillus fumigatus has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the cyp51A gene that encodes the target of azole drugs, lanosterol ?-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of cyp51A and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of cyp51A as well as that of an important membrane transporter protein gene called abcG1 In this work, we characterize two different mutant alleles of atrR, either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of atrR for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the abcG1 and cyp51A promoters. The ATRE in cyp51A was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis.IMPORTANCE Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Infections associated with A. fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene cyp51A as an important contributor to resistance in A. fumigatus Here, we demonstrate that the transcription factor AtrR binds to a region in the cyp51A promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of A. fumigatus.

Dataset Information

Protein Glycosylation in Aspergillus fumigatus Is Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development.

Publications

Protein Glycosylation in Aspergillus fumigatus Is Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development.

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