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

Isabel Valsecchi,Gordon D Brown,Anupama Karnam,Margaret Sunde,Sarah Sze Wah Wong,Olaf Kniemeyer,Janet A Willment,Thomas Krüger,Emmanuel Stephen-Victor,Borja Rodríguez De Francisco,J Iñaki Guijarro,Vishukumar Aimanianda,Jean-Paul Latgé,Jagadeesh Bayry,Axel A Brakhage

doi:10.3390/jof6030151

Abstract

Immune inertness of Aspergillus fumigatus 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.

Highlights

Even though conidia produced by Aspergillus fumigatus, an opportunistic airborne fungal pathogen, are constantly inhaled by humans, the conidial surface rodlet layer masks immediate immune recognition of the conidia, resulting in a balanced immune response [1,2]
We showed that (i) the structure of RodAp monomers that are competent to assemble into rodlets is organized around four disulfide bonds formed by the eight conserved cysteine residues in the RodAp sequence [7] and that (ii) mutation of either one or four cysteine residues resulting in the disruption of one or all the four disulfide linkages generated mutant A. fumigatus conidia devoid of surface rodlets [9]
Our study indicates that compared to β-(1,3)-glucan, α-(1,3)-glucan is proportionately more exposed on the conidial surfaces during germination, suggesting a need to study the consequences of state-specific exposure of α-(1,3)-glucan during conidial germination during immune cells and conidial interactions

Summary

Introduction

Even though conidia (the asexual spores) produced by Aspergillus fumigatus, an opportunistic airborne fungal pathogen, are constantly inhaled by humans, the conidial surface rodlet layer masks immediate immune recognition of the conidia, resulting in a balanced immune response [1,2]. The outer coat of conidia consists of a rodlet layer composed of RodAp, a protein belonging to the Class-I hydrophobin family [6] that is characterized by the presence of eight conserved cysteine residues [7,8]. These cysteine residues are involved in the formation of four disulfide linkages that are critical in the structural organization of RodAp. Previously, we showed that the conserved cysteine residue point (ccrp) mutations in the RodA resulted in conidia devoid of a surface rodlet layer [9]. Ccrp-mutations of either single or of four cysteine residues that disrupt one or four disulfide linkages, respectively, resulted in mutant conidia with similar cell surface organization

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Conclusion