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

Abstract

Caspofungin targets cell wall β-1,3-glucan synthesis and is the international consensus guideline-recommended salvage therapy for invasive aspergillosis. Although caspofungin is inhibitory at low concentrations, it exhibits a paradoxical effect (reversal of growth inhibition) at high concentrations by an undetermined mechanism. Treatment with caspofungin at either the growth-inhibitory concentration (0.5 μg/ml) or paradoxical growth-inducing concentration (4 μg/ml) for 24 h caused similar abnormalities, including wider, hyperbranched hyphae, increased septation, and repeated hyphal tip lysis, followed by regenerative intrahyphal growth. By 48 h, only hyphae at the colony periphery treated with the high caspofungin concentration displayed paradoxical growth. A similar high concentration of caspofungin also induced the paradoxical growth of during human A549 alveolar cell invasion. Localization of the β-1,3-glucan synthase complex (Fks1 and Rho1) revealed significant differences between cells exposed to the growth-inhibitory and paradoxical growth-inducing concentrations of caspofungin. At both concentrations, Fks1 initially mislocalized from the hyphal tips to vacuoles. However, only continuous exposure to 4 μg/ml of caspofungin for 48 h led to recovery of the normal hyphal morphology with renewed localization of Fks1 to hyphal tips. Rho1 remained at the hyphal tip after treatment with both caspofungin concentrations but was required for paradoxical growth. Farnesol blocked paradoxical growth and relocalized Fks1 and Rho1 to vacuoles. Our results highlight the importance of regenerative intrahyphal growth as a rapid adaptation to the fungicidal lytic effects of caspofungin on hyphal tips and the dynamic localization of Fks1 as part of the mechanism for the caspofungin-mediated paradoxical response in.

Chytrid rhizoid morphogenesis is adaptive and resembles hyphal development in ‘higher’ fungi

“…Inhibition of cell wall β-1,3-glucan synthesis and actin proliferation with caspofungin and cytochalasin B respectively induced a concentration-dependent decrease in the RGU and the development of atypical cells with hyperbranched rhizoids (Figures 2B-D; Supplementary Table 2; Supplementary Movies 6-7). These effects in R. globosum are similar to disruption of normal hyphal branching reported in Aspergillus fumigatus (Ascomycota) in the presence of caspofungin [24], and in Neurospora crassa (Ascomycota) in the presence of cytochalasins [25], suggesting that β-1,3-glucan-dependent cell wall synthesis and actin dynamics also govern branching in chytrid rhizoids by comparable processes.…”

The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of Aspergillus fumigatus

“…Interestingly, increased chitin content was also previously reported in A. fumigatus treated with caspofungin [29], an echinocandin which targets β-1,3-glucan synthase. Other similarities with caspofungin-treated hyphae include hyphal swelling and cell lysis (see [30]), although in the case of caspofungin, the cell lysis was hyphal tip specific, whereas olorofim-induced lysis can occur at any part of the cell [17]. Additionally, increased septation, observed in caspofungin-treated hyphae, was also observed in olorofim-treated hyphae, which again could be a potential stress response.…”

Echinocandins for the Treatment of Invasive Aspergillosis: from Laboratory to Bedside

“…Synthesis of ␤-1,3-glucan takes place at the cell membrane by the ␤-1,3-glucan synthase complex, consisting of a catalytic subunit, Fks1, and a regulatory subunit, RhoI. The crucial role of this complex in the PE was also supported by a recent study showing distinct localization patterns at low (non-PE) and high (PE) caspofungin concentrations (34). Under caspofungin exposure, Fks1 moves from the hyphal tips to vacuoles.…”

“…RhoI remains at the hyphal tips, where it is essential for Fks1 activation and the PE. Indeed, farnesol, which mislocalizes RhoI, abolishes the PE in A. fumigatus isolates (34).…”