Link between Heat Shock Protein 90 and the Mitochondrial Respiratory Chain in the Caspofungin Stress Response of Aspergillus fumigatus

Abstract

Aspergillus fumigatus is an opportunistic mold responsible for invasive aspergillosis. Triazoles (e.g., voriconazole) represent the first-line treatment, but emerging resistance is of concern. The echinocandin drug caspofungin is used as second-line treatment but has limited efficacy. The heat shock protein 90 (Hsp90) orchestrates the caspofungin stress response and is the trigger of an adaptive phenomenon called the paradoxical effect (growth recovery at increasing caspofungin concentrations). The aim of this study was to elucidate the Hsp90-dependent mechanisms of the caspofungin stress response. Transcriptomic profiles of the wild-type A. fumigatus strain (KU80) were compared to those of a mutant strain with substitution of the native hsp90 promoter by the thiA promoter (pthiA-hsp90), which lacks the caspofungin paradoxical effect. Caspofungin induced expression of the genes of the mitochondrial respiratory chain (MRC), in particular, NADH-ubiquinone oxidoreductases (complex I), in KU80 but not in the pthiA-hsp90 mutant. The caspofungin paradoxical effect could be abolished by rotenone (MRC complex I inhibitor) in KU80, supporting the role of MRC in the caspofungin stress response. Fluorescent staining of active mitochondria and measurement of oxygen consumption and ATP production confirmed the activation of the MRC in KU80 in response to caspofungin, but this activity was impaired in the pthiA-hsp90 mutant. Using a bioluminescent reporter for the measurement of intracellular calcium, we demonstrated that inhibition of Hsp90 by geldanamycin or MRC complex I by rotenone prevented the increase in intracellular calcium shown to be essential for the caspofungin paradoxical effect. In conclusion, our data support a role of the MRC in the caspofungin stress response which is dependent on Hsp90.

Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation

“…Subsequently, we investigated the metabolic activity of the mature A. fumigatus biofilm consisting of hyphal cells exposed to HS. The 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) assay was employed to test the biofilm response to HS as previously described (Dhingra, Buckey, & Cramer, 2018). Consistent with the observed decrease in conidia viability, biofilms of pkcA G579R and ΔmpkA strains also exhibited a significant decrease in metabolic activity when the biofilm was incubated for 60 or 120 min at 48 C (Figure 1b).…”

“…Cooperating mechanisms between the CWIP and Hsp90 function have been observed in A. fumigatus since mutants of these genes are concomitantly more susceptible to CW stress and elevated temperatures (Lamoth, Juvvadi, Fortwendel, & Steinbach, 2012; Rocha et al, 2015; Valiante et al, 2009). Also, hsp90 mediates the caspofungin paradoxical effect (Aruanno, Bachmann, Sanglard, & Lamoth, 2019; Lamoth, Juvvadi, Gehrke, et al, 2014; Lamoth, Juvvadi, Soderblom, et al, 2014). At the protein level, the MpkA homologues in yeast and C. albicans are known Hsp90 clients (Diezmann, Michaut, Shapiro, Bader, & Cowen, 2012; LaFayette et al, 2010).…”

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect

“…Addition of respiratory inhibitors results in significant decreases of the caspofungin MICs for C. parapsilosis (39). Caspofungin exposure induces expression of A. fumigatus mitochondrial respiratory chain genes, specifically NADH-ubiquinone oxidoreductases (complex I), while CPE is abolished by addition of rotenone, a complex I inhibitor (58). Interestingly, those authors also described a connection between rotenone inhibition and increases in intracellular calcium concentrations.…”

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

“…However, there may be other downstream effectors in the complex network of Hsp90. Very recently, the mitochondrial respiratory chain was shown to play a role in the PE, and activation of mitochondria in response to caspofungin was dependent on Hsp90 (27).…”

“…Analyses of the intracellular trafficking of the different actors of the Hsp90calcineurin pathway showed that calcineurin and calmodulin localized at the hyphal tips and septa and that Hsp90 moves from the cytosol to the cell wall and sites of septa formation in the presence of caspofungin (27)(28)(29)(30). Physical interaction between Hsp90 and calcineurin has been demonstrated in yeasts (31).…”