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

Author

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

Subjects
Aspergillus fumigatus, host–fungal interaction, conidial surface, RodAp, hydrophobin, disulfide bonds, Biology (General), QH301-705.5
Abstract

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.

Published
2020
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2. The Role of RodA-Conserved Cysteine Residues in the Aspergillus fumigatus Conidial Surface Organization

Author

Vishukumar Aimanianda, Olaf Kniemeyer, Sarah Sze Wah Wong, Borja Rodríguez de Francisco, Isabel Valsecchi, J. Iñaki Guijarro, Margaret Sunde, Janet A. Willment, Jagadeesh Bayry, Emmanuel Stephen-Victor, Anupama Karnam, Gordon D. Brown, Thomas Krüger, Axel A. Brakhage, Jean-Paul Latgé, Aspergillus, Institut Pasteur [Paris], Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Mycologie moléculaire - Molecular Mycology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), The University of Sydney, Plateforme technologique de RMN biologique - Biological NMR Technological Platform, Leibniz Institute for Natural Product Research and Infection Biology (Hans Knoell Institute), University of Exeter, This work was supported by ANR-DFG AfuINF, ANR HYDROPHOBIN (ANR-10-BLAN-1309), ANR FUNHYDRO (ANR-16S-CE110020-01) and DFG CRC/TR FungiNet 124 (210879364, projects A1 and Z2) grants. SSWW was supported by a postdoctoral fellowship by ANR-DFG grant and Pasteur-Roux-Cantarini fellowship. MS was supported by an Australian Research Council Discovery Project (DP150104227). AK was supported by a fellowship from La Fondation pour la Recherche Médicale (FDT201805005552)., ANR-10-BLAN-1309,HYDROPHOBIN,HYDROPHOBINE DES CONIDIES d'Aspergillus fumigatus : ANALYSE STRUCTURALE ET REPONSE IMMUNE(2010), ANR-16-CE11-0020,FUNHYDRO,Amyloïdes fonctionnels formés par les hydrophobines du pathogène fongique Aspergillus fumigatus(2016), Institut Pasteur [Paris] (IP), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Microbiology (medical), hydrophobin, Hydrophobin, Aspergillus fumigatus, disulfide bonds, Plant Science, immunomodulation, Microbiology, Conidium, 03 medical and health sciences, Immunolabeling, Western blot, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, skin and connective tissue diseases, lcsh:QH301-705.5, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, 0303 health sciences, biology, medicine.diagnostic_test, 030306 microbiology, Chemistry, Point mutation, fungi, RodAp, biology.organism_classification, conidial surface, 3. Good health, host–fungal interaction, lcsh:Biology (General), Cytoplasm, Cysteine
Abstract

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 &alpha, (1,3)-glucan, ∆rodA conidia selectively bound to &beta, (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&ndash, fungal interaction.

Published
2020

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