Targeting calcineurin: a synergistic strategy with caspofungin to induce farnesol-mediated quorum sensing in <i>Nakaseomyces glabrata</i> —an inoculum-dependent tale

Using Nakaseomyces glabrata as a model organism, we demonstrate that targeting calcineurin can synergize with caspofungin to induce a quorum sensing (QS) effect mediated by farnesol. This QS effect requires calcineurin deficiency, sub-minimum inhibitory concentration (MIC) levels of caspofungin, and a high-density cell population. Cell growth and biofilm formation were significantly inhibited within a specific range of cell density and sub-MIC caspofungin treatment in the calcineurin mutant. The inhibition of biofilm formation follows the 'paradoxical growth,' showing a concentration-dependent response to caspofungin. We show that high cell density triggers two antagonistic effects: overcoming antibiotic inhibition, which promotes cell propagation, and QS-mediated growth inhibition, which negatively regulates cell proliferation. The QS molecule farnesol was detectable only in the calcineurin mutant, where the transcription of the farnesol synthase Dpp3 was significantly up-regulated, and deletion of DPP3 abolished the QS effect in both spot assay and biofilm formation of the calcineurin mutant. Besides this, we identified a Dpp3-dependent, ergosterol-farnesol metabolism-linked Crz1-independent regulatory mechanism that contributes to the calcineurin-mediated multi-stress resistance. We demonstrate that calcineurin, Dpp3, and caspofungin are all involved in regulating ergosterol metabolism and the transcription of ERG11 and FKS genes, leading to significant changes in membrane and cell wall stress tolerance. The cell wall composition undergoes substantial alterations upon deletion of calcineurin or treatment with caspofungin, while caspofungin also increases the levels of β-glucan and short peptides in the medium, tentatively pointing to the release of QS inducers from the cell wall.