Aspergillus fumigatus devoid of cell wall β‐1,3‐glucan is viable, massively sheds galactomannan and is killed by septum formation inhibitors

Abstract

SummaryEchinocandins inhibit β-1,3-glucan synthesis and are one of the few antimycotic drug classes effective against Aspergillus spp. In this study, we characterized the β-1,3-glucan synthase Fks1 of Aspergillus fumigatus, the putative target of echinocandins. Data obtained with a conditional mutant suggest that fks1 is not essential. In agreement, we successfully constructed a viable Δfks1 deletion mutant. Lack of Fks1 results in characteristic growth phenotypes similar to wild type treated with echinocandins and an increased susceptibility to calcofluor white and sodium dodecyl sulfate. In agreement with Fks1 being the only β-1,3-glucan synthase in A. fumigatus, the cell wall is devoid of β-1,3-glucan. This is accompanied by a compensatory increase of chitin and galactosaminogalactan and a significant decrease in cell wall galactomannan due to a massively enhanced galactomannan shedding. Our data furthermore suggest that inhibition of hyphal septation can overcome the limitations of echinocandin therapy. Compounds inhibiting septum formation boosted the antifungal activity of caspofungin. Thus, development of clinically applicable inhibitors of septum formation is a promising strategy to improve existing antifungal therapy.

The Paradoxical Effect of Echinocandins in Aspergillus fumigatus Relies on Recovery of the β-1,3-Glucan Synthase Fks1

“…To further investigate this observation, we analyzed and compared the growth of the wild type and the conditional fks1 tetOn strain in the presence of caspofungin. In agreement with our previous results (14), growth of the fks1 tetOn strain under repressive conditions was not affected at any time, even in the presence of caspofungin concentrations as high as 32 g ml Ϫ1 (Fig. 3).…”

“…Under induced conditions, the fks1 tetOn strain showed caspofungin susceptibility that was similar to that of the wild type (14) (Fig. 3) and the ability for paradoxical growth (Fig.…”

“…This fks1 tetOn mutant was constructed in the same genetic background by replacing the endogenous promoter with a doxycycline-inducible Tet-On promoter. Under repressive conditions, the mutant mimics the phenotype of a Δfks1 deletion mutant and yields slowly growing hyperbranching hyphae very similar to those of the caspofungin-treated wild type (14). To allow discrimination of the wild type and the conditional fks1 tetOn control strain, experiments were performed with a wild-type strain that expresses a cytosolic variant of green fluorescent protein (GFP).…”

“…First, a compensatory increase of cell wall chitin was repeatedly observed after exposure of Aspergillus fumigatus and Candida albicans to echinocandins (11)(12)(13)(14). Second, disruption of calcineurin signaling or cell wall integrity signaling in C. albicans or A. fumigatus, either by pharmacologic inhibition or genetic modification, suppresses the paradoxical effect (15,16).…”

“…In this work, we exploited the ability of A. fumigatus to grow in the absence of the ␤-1,3-glucan synthase (Fks1) and cell wall ␤-1,3-glucan (14) to study the paradoxical effect. We show that expression of the ␤-1,3-glucan synthase is required for the paradoxical effect and that increased expression of the ␤-1,3-glucan synthase renders A. fumigatus more susceptible to caspofungin.…”