Mitochondrial dysfunctions trigger the calcium signaling-dependent fungal multidrug resistance

Abstract

Drug resistance in fungal pathogens has risen steadily over the past decades due to long-term azole therapy or triazole usage in agriculture. Modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance. However, mechanisms for nondrug target-induced resistance remain only loosely defined. Here, we explore the molecular mechanisms of multidrug resistance resulted from an efficient adaptation strategy for survival in drug environments in the human pathogen Aspergillus fumigatus. We show that mutants conferring multidrug resistance are linked with mitochondrial dysfunction induced by defects in heme A biosynthesis. Comparison of the gene expression profiles between the drug-resistant mutants and the parental wild-type strain shows that multidrug-resistant transporters, chitin synthases, and calcium-signaling-related genes are significantly up-regulated, while scavenging mitochondrial reactive oxygen species (ROS)-related genes are significantly down-regulated. The up-regulated-expression genes share consensus calcium-dependent serine threonine phosphatase-dependent response elements (the binding sites of calcium-signaling transcription factor CrzA). Accordingly, drug-resistant mutants show enhanced cytosolic Ca2+ transients and persistent nuclear localization of CrzA. In comparison, calcium chelators significantly restore drug susceptibility and increase azole efficacy either in laboratory-derived or in clinic-isolated A. fumigatus strains. Thus, the mitochondrial dysfunction as a fitness cost can trigger calcium signaling and, therefore, globally up-regulate a series of embedding calcineurin-dependent–response-element genes, leading to antifungal resistance. These findings illuminate how fitness cost affects drug resistance and suggest that disruption of calcium signaling might be a promising therapeutic strategy to fight against nondrug target-induced drug resistance.

Mitochondria-Mediated Azole Drug Resistance and Fungal Pathogenicity: Opportunities for Therapeutic Development

“…Mitochondrial dysfunction can initiate a mitochondrial–nuclear cross-talk that contributes to azole resistance in various pathogenic fungi [ 24 , 26 ]. For example, mitochondrial dysfunction caused by mitochondrial mutation can mediate azole resistance via activation of the pleiotropic drug resistance (Pdr) pathway that is involved in directly regulating the expression of drug efflux pumps ( CDR1 , CDR2 , and MDR1 ) ( Figure 1 ) [ 16 ].…”

“…Although the relationship between the calcineurin–Crz1 pathway and azole resistance has been determined, it is still unclear whether there is a direct relationship between the calcium signaling pathway and mitochondrial function. Recently, however, a study has established a close connection between mitochondrial function and calcium signaling-dependent azole resistance in Aspergillus fumigatus ( Figure 1 ) [ 24 ]. Abnormal cytoplasmic Ca 2+ transients induced by mitochondrial dysfunction cause a persistent nuclear localization of zinc finger transcription factor CrzA ( C. albicans Crz1 homolog in A. fumigatus ) and, therefore, triggers the global overexpression of multidrug transport genes ( mdr1 , atrB , atrF , abcE , atrA , and abcC ) ( Figure 1 ), leading to azole resistance [ 24 ].…”

“…Importantly, downregulation of CrzA markedly decreased the expression of the abovementioned multidrug transport genes [ 24 ], which suggested that it is indeed persistent nuclear localization of CrzA that determines the survival of strains with dysfunctional mitochondria in an azole-stress environment. Interestingly, in contrast to the persistent nuclear localization of CrzA induced by mitochondrial dysfunction in normal medium, supplementation of culture medium with calcium chelators strikingly prompted CrzA–GFP to localize in the cytoplasm, leading to significant restoration of azole drug susceptibility for A. fumigatus strains with dysfunctional mitochondria [ 24 ]; this indicates that it is indeed enhanced cytosolic Ca 2+ transients caused by mitochondrial dysfunction that determine the localization of CrzA and hence azole susceptibility. Taken together, these data strongly suggest that defects in mitochondrial function might be a signal leading to activation of calcium signaling in response to azole stress.…”

“…Generally, the regulation of drug efflux pumps includes gene expression regulation and activity regulation. As mentioned above, dysfunctional mitochondria exert translational regulation on efflux pump levels ( Figure 1 ) [ 24 ]. Recently, a series of studies has also confirmed that dysfunctional mitochondria are involved in the regulation of efflux pump activity [ 14 , 34 ].…”

“…Mitochondria house and integrate multiple metabolic functions relating to lipids, iron metabolism, energy production, and cell wall biosynthesis [ 14 , 15 , 16 , 17 , 18 , 19 ], which are associated with fungal virulence and resistance to azoles. Mutations affecting mitochondrial functions including functioning of the electron transport chain (ETC), protein import, calcium homeostasis, mitochondrial genome maintenance, and mitochondrial transcription can result in avirulence and azole resistance or susceptibility [ 17 , 20 , 21 , 22 , 23 , 24 , 25 ]. The functions of mitochondria in these pathways are complex, and research findings open avenues for new, mitochondria-targeted therapeutic approaches.…”