The stress-protectant molecule trehalose mediates fluconazole tolerance in Candida glabrata

Abstract The incidence of non- albicans Candida infections has witnessed a substantial rise in recent decades. Candida glabrata (Nakaseomyces glabratus) , an opportunistic human fungal pathogen, is accountable for both superficial mucosal and life-threatening bloodstream infections, particularly in immunocompromised individuals. Distinguished by its remarkable resilience to environmental stressors, C. glabrata exhibits intrinsic tolerance to azoles and a high propensity to swiftly develop azole resistance during treatment. The molecular mechanism for the high tolerance is not fully understood. In this work we investigated the possible role of trehalose in this tolerance. We generated mutants in the C. glabrata TPS1 , TPS2 , and NTH1 genes, encoding trehalose 6-phosphate synthase (Tps1), trehalose 6-phosphate phosphatase (Tps2), and neutral trehalase (Nth1), respectively. As expected, the tps1Δ strain cannot grow on glucose. The tps2 Δ strain demonstrated diminished trehalose accumulation and very high levels of trehalose 6-phosphate (T6P), the biosynthetic intermediate, in comparison to the WT strain. Whereas these higher T6P levels did not affect growth, the lower trehalose levels clearly resulted in lower environmental stress tolerance and a lower susceptibility to fluconazole. More interestingly, the tps2Δ strain completely lost tolerance to fluconazole, characterized by the absence of slow growth at supra-MIC concentrations of this drug. All these phenotypes are reversed in the nth1 Δ strain, which accumulates high levels of trehalose. Our findings underscore the role of trehalose in enabling tolerance towards fluconazole in C. glabrata . We further show that the change in tolerance is a result of the effect that trehalose has on the sterol pattern in the cell, showing that accumulation of ‘toxic’ sterols correlate with absence of tolerance. Author summary C. glabrata is a yeast of significant medical importance, known for causing nosocomial outbreaks of invasive candidiasis. Its propensity to develop resistance to antifungal medications, notably azoles such as fluconazole, raises considerable concern. An underlying reason for the rapid development of resistance is its intrinsic tolerance to this drug. The underlying molecular mechanism of tolerance to fluconazole is heavily studied but not understood. This study sheds light on the involvement of trehalose in modulating tolerance to fluconazole. We have elucidated that trehalose serves not only as a protective agent against various stresses but also as a mediator of fluconazole resistance and tolerance. To start elucidating how this may work, we provide data that trehalose (or the enzymes affecting the amount of trehalose in the cell) alters the ergosterol type and level in the cells, thereby affecting tolerance.