Caspofungin-Mediated Growth Inhibition and Paradoxical Growth in Aspergillus fumigatus involve Fungicidal Hyphal Tip Lysis Coupled with Regenerative Intrahyphal Growth and Dynamic Changes in {beta}-1,3-Glucan Synthase Localization [PublishAheadOfPrint]

Caspofungin targets cell wall β-1,3-glucan synthesis and is guideline-recommended to treat invasive aspergillosis as salvage therapy. 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 either growth inhibitory (0.5 μg/ml) or paradoxical growth-inducing (4 μg/ml) caspofungin concentrations 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. Similar high concentrations of caspofungin also induced paradoxical growth of Aspergillus fumigatus 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 growth inhibitory and paradoxical growth inducing concentrations of caspofungin. At both concentrations, Fks1 initially mislocalized from hyphal tips to vacuoles. However, only continuous exposure to 4 μg/ml of caspofungin for 48 h led to recovery of normal hyphal morphology with renewed localization of Fks1 to hyphal tips. Rho1 remained at the hyphal tip under 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 A. fumigatus.

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