Deletion of the α-(1,3)-Glucan Synthase Genes Induces a Restructuring of the Conidial Cell Wall Responsible for the Avirulence of Aspergillus fumigatus

Anne Beauvais,Christine Henry,Axel A Brakhage,Jean-Paul Latgé,Robert W Roberson,Olaf Kniemeyer,Silvia Bozza,Viviane Balloy,Luigina Romani,Etienne Dague,Céline Formosa,Michel Chignard

doi:10.1371/journal.ppat.1003716

Abstract

α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.

Highlights

MethodsStrains and culture conditions All strains were grown in 2% (w/v) malt agar slants and 1 weekold conidia were recovered from the slants by vortexing with 0.05% (v/v) Tween 20 aqueous solution
Analysis of the conidia of the triple mutants showed that the lack of virulence of the mutants in vivo was associated to major changes occurring on the cell wall, especially on the surface of the resting and swollen conidia, which resulted in an increased killing by phagocytes
This was confirmed by the broncho-alveolar lavage (BAL) analysis, which showed a higher PMN recruitment after infection with ku80 conidia compared with agsD (Fig. 1C, Fig. S1C)

Summary

Methods

Strains and culture conditions All strains were grown in 2% (w/v) malt agar slants and 1 weekold conidia were recovered from the slants by vortexing with 0.05% (v/v) Tween 20 aqueous solution. The A. fumigatus parental strain AkuBku80DpyrG (ku80, [43]) and three agsD mutant strains independently obtained: ags1Dags2Dags3D_5T (agsD_5T) obtained previously [8] and two new ones, ags1Dags2Dags3Dn8and ags1Dags2Dags3D_n6.2 (agsD_n8 and agsD_n6.2), were used in this study. Since it had been impossible to complement agsD mutant for reasons explained previously [8], two new triple agsD mutants were constructed independently using the strategy described previously to exclude the possibility that undesired mutations had occurred during the deletion process. The lack of a-(1,3)-glucan in the cell wall of mutant strains was confirmed by both chemical and immunolabeling assays (Fig. S8). Chemical analysis of the cell wall was performed as previously described [44]. 5–10 h germinated conidia were labeled using the MOPC 104E monoclonal antibody, which binds to a-(1,3)-glucan [45]

Results

Discussion

Conclusion