Survival in the Presence of Antifungals

Abstract

How yeast cells respond to cell wall stress is relatively well understood; however, how filamentous fungi cope with cell wall damage is largely unexplored. Here we report the first transcriptome analysis of Aspergillus niger exposed to the antifungal compounds caspofungin, an inhibitor of ␤-1,3-glucan synthesis, and fenpropimorph, which inhibits ergosterol synthesis. The presence of sublethal drug concentrations allowed A. niger to adapt to the stress conditions and to continue growth by the establishment of new polarity axes and formation of new germ tubes. By comparing the expression profile between caspofungin-exposed and nonexposed A. niger germlings, we identified a total of 172 responsive genes out of 14,509 open reading frames present on the Affymetrix microarray chips. Among 165 up-regulated genes, mainly genes predicted to function in (i) cell wall assembly and remodeling, (ii) cytoskeletal organization, (iii) signaling, and (iv) oxidative stress response were affected. Fenpropimorph modulated expression of 43 genes, of which 41 showed enhanced expression. Here, genes predicted to function in (i) membrane reconstruction, (ii) lipid signaling, (iii) cell wall remodeling, and (iv) oxidative stress response were identified. Northern analyses of selected genes were used to confirm the microarray analyses. The results further show that expression of the agsA gene encoding an ␣-1,3-glucan synthase is up-regulated by both compounds. Using two PagsA-GFP reporter strains of A. niger and subjecting them to 16 different antifungal compounds, including caspofungin and fenpropimorph, we could show that agsA is specifically activated by compounds interfering directly or indirectly with cell wall biosynthesis.The fungal cell wall is a dynamic structure that is essential for sustaining cell morphology and for protection against lifethreatening environmental conditions. Morphological characteristics during developmental processes in fungi depend upon the temporal regulation and spatial localization of cell wall components and thereby ordered cell wall deposition (1-3). Moreover, cell wall rearrangements that guarantee the structural integrity of the cell wall are of vital importance to withstand environmental stress conditions such as osmotic stress or the presence of antifungal substances compromising cell wall and/or cell membrane integrity (4 -6). To prevent cell lysis and to ensure cell survival, fungi have developed mechanisms to sense cell surface stress and to respond to these stresses via a remodeling of the cell wall (see also reviews in Refs. 1, 7, 8).The composition of fungal cell walls and the mechanisms involved in ensuring cell surface integrity have been studied most intensively in the model yeast Saccharomyces cerevisiae. The cell wall of S. cerevisiae consists of a moderately branched, flexible ␤-1,3-glucan network to which to its external face ␤-1,6-glucan chains are bound which in turn are linked to GPI mannoproteins. At the inner side of the ␤-1,3-glucan network, chitin chains are attached (reviewed ...

Survival Strategies of Yeast and Filamentous Fungi against the Antifungal Protein AFP

“…We have decided to delete this chitin synthase for several reasons. Class V chitin synthases are specific for filamentous fungi (48), csmB expression levels are strongly affected during cell wall stress (19), and deletion of the class V chitin synthase chsV in F. oxysporum made the strain hyperresistant toward AFP (13). Indeed, for all yeast and fungal strains tested, we found a correlation between susceptibility and chitin synthesis (Table 2), i.e.…”

“…Using A. niger as model system, we could recently show that one defense mechanism to counteract AFP inhibitory effects is induction of the cell wall integrity (CWI) pathway, a highly conserved signaling cascade that secures cell surface protection in yeast and filamentous fungi (17)(18)(19)(20) and results in A. niger in increased expression of the agsA gene, encoding an ␣-1,3-glucan synthase (13). However, when induction of the CWI pathway is meant to protect A. niger, why then is the fungus killed by AFP?…”

A Transcriptome Meta-Analysis Proposes Novel Biological Roles for the Antifungal Protein AnAFP in Aspergillus niger

“…niger strain N402 in dormant conidia [36], during different stages of the germination process [36, 37], or in vegetative mycelium and aerial hyphae of a 7 day old culture grown on maltose [38]. The analysis of these microarray data did not reveal any anafp expression in dormant and germinating conidia but showed high intensity levels about 63-fold in the vegetative mycelium and about 227-fold in aerial hyphae compared to the baseline expression level (S1 Table).…”

“…In most of the studies, A . niger (N402 or derivatives thereof) was cultivated under controlled submerged conditions in bioreactors and stressed either during germination [37, 43, 44] or during the exponential growth phase [45]. Interestingly, none of the secretion stress agents (DTT, tunicamycin) and none of the cell membrane or cell-wall stressors applied in these studies (caspofungin, fenpropimorph, FK506, aureobasidin A, natamycin) provoked an increase in anafp expression.…”

8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae

“…Often, links between accumulation of 8-dehydrosterols, like fecosterol, and cell wall defects have been found in both plants and fungi [47,48]. We measured the sensitivity of erg2Δ and vph1Δ mutants to cell wall damage as the velocity of cell bursting under hypotonic conditions produced by lyticase treatment, a cell wall digesting commercial mixture of enzymes (Fig.…”