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

Abstract

Echinocandins target the fungal cell wall by inhibiting biosynthesis of the cell wall carbohydrate ␤-1,3-glucan. This antifungal drug class exhibits a paradoxical effect that is characterized by the resumption of growth of otherwise susceptible strains at higher drug concentrations (approximately 4 to 32 g/ml). The nature of this phenomenon is still unknown. In this study, we analyzed the paradoxical effect of the echinocandin caspofungin on the pathogenic mold Aspergillus fumigatus. Using a conditional fks1 mutant, we show that very high caspofungin concentrations exert an additional antifungal activity besides inhibition of the ␤-1,3-glucan synthase. This activity could explain the suppression of paradoxical growth at very high caspofungin concentrations. Additionally, we found that exposure to inhibitory caspofungin concentrations always causes initial growth deprivation independently of the capability of the drug concentration to induce the paradoxical effect. Paradoxically growing hyphae emerge from microcolonies essentially devoid of ␤-1,3-glucan. However, these hyphae expose ␤-1,3-glucan again, suggesting that ␤-1,3-glucan synthesis is restored. In agreement with this hypothesis, we found that expression of the ␤-1,3-glucan synthase Fks1 is an essential requirement for the paradoxical effect. Surprisingly, overexpression of fks1 renders A. fumigatus more susceptible, whereas reduced expression leads to hyphae that are more resistant to the growth-inhibitory and limited fungicidal activity of caspofungin. Upregulation of chitin synthesis appears to be of minor importance for the paradoxical effect, since paradoxically growing hyphae exhibit significantly less chitin than the growth-deprived parental microcolonies. Our results argue for a model where the paradoxical effect primarily relies on recovery of ␤-1,3-glucan synthase activity.

Caspofungin-Mediated Growth Inhibition and Paradoxical Growth in Aspergillus fumigatus Involve Fungicidal Hyphal Tip Lysis Coupled with Regenerative Intrahyphal Growth and Dynamic Changes in β-1,3-Glucan Synthase Localization

“…The switch to paradoxical growth is elicited in leading hyphae between 36 and 48 h after continuous exposure to a high caspofungin concentration. The paradoxical growth effect in liquid medium has been shown to occur within 3 days in the continuous presence of caspofungin in several strains of A. fumigatus (12). We confirmed the development of paradoxical growth in the CEA10, Af293 (20), and ΔKU80 strains and in several azole-resistant strains cultured in Aspergillus minimal medium, glucose minimal medium, and RPMI 1640 medium (data not shown).…”

“…We argue that the localization of Fks1 in vacuoles after caspofungin contact may be due to degradation of the enzyme, which correlates with the reduced production of ␤-1,3-glucan and impaired morphology (12,13,43). However, once paradoxical growth is triggered, this process is abolished due to the renewed localization of Fks1 at the tip followed by the recovery of the basal content of ␤-1,3-glucan and the normal hyphal morphology (12). Thus, this continuous accumulation of Fks1 in the vacuoles may be the reason for the absence of paradoxical growth with micafungin.…”

“…Caspofungin, the most commonly used echinocandin, disrupts the synthesis of the ␤-1,3-glucan in the cell wall, displaying what has been described as fungistatic activity against filamentous fungi (7), including A. fumigatus (8). However, caspofungin has also been reported to cause lysis of some hyphal cells and, thus, to exhibit partial fungicidal effects in A. fumigatus hyphae (9)(10)(11)(12). Treatment of A. fumigatus with caspofungin leads to swollen, short, and highly branched hyphae with thicker cell walls due to the reduction of ␤-1,3-glucan (13).…”

“…In Candida spp., the paradoxical effect has been shown to be independent of mutated or overexpressed ␤-1,3-glucan synthase but is mainly dependent on chitin upregulation (22). Using a conditional mutant, Loiko and Wagener (12) provided evidence that the paradoxical effect in A. fumigatus relies on the recovery of ␤-1,3-glucan synthase Fks1 activity.…”

“…Our first aim was to understand the fundamental process underlying caspofungin's limited fungicidal activity (9)(10)(11)(12) in order to elucidate the basic cellular mechanism of growth inhibition caused by the antifungal. We demonstrate that caspofunginmediated growth inhibition involves a highly dynamic fungicidal process at the colony periphery through repeated apical hyphal compartment lysis followed by regenerative, intrahyphal growth, which occurs in combination with increased septation and subapical hyperbranching.…”