Azole Susceptibility and Transcriptome Profiling in Candida albicans Mitochondrial Electron Transport Chain Complex I Mutants

Abstract

dMitochondrial dysfunction in pathogenic fungi or model yeast causes altered susceptibilities to antifungal drugs. Here we have characterized the role of mitochondrial complex I (CI) of Candida albicans in antifungal susceptibility. Inhibitors of CI to CV, except for CII, increased the susceptibility of both patient and lab isolates, even those with a resistance phenotype. In addition, in a C. albicans library of 12 CI null mutants, 10 displayed hypersusceptibility to fluconazole and were severely growth inhibited on glycerol, implying a role for each gene in cell respiration. We chose two other hypersusceptible null mutants of C. albicans, the goa1⌬ and ndh51⌬ mutants, for transcriptional profiling by RNA-Seq. Goa1p is required for CI activity, while Ndh51p is a CI subunit. RNA-Seq revealed that both the ndh51⌬ mutant and especially the goa1⌬ mutant had significant downregulation of transporter genes, including CDR1 and CDR2, which encode efflux proteins. In the goa1⌬ mutant, we noted the downregulation of genes required for the biogenesis and replication of peroxisomes, as well as metabolic pathways assigned to peroxisomes such as ␤-oxidation of fatty acids, glyoxylate bypass, and acetyl coenzyme A (acetyl-CoA) transferases that are known to shuttle acetyl-CoA between peroxisomes and mitochondria. The transcriptome profile of the ndh51⌬ mutant did not include downregulation of peroxisome genes but had, instead, extensive downregulation of the ergosterol synthesis gene family. Our data establish that cell energy is required for azole susceptibility and that downregulation of efflux genes may be an outcome of that dysfunction. However, there are mutant-specific changes that may also increase the susceptibility of both of these C. albicans mutants to azoles.

Multidrug-Resistant Transporter Mdr1p-Mediated Uptake of a Novel Antifungal Compound

“…We have also observed that fluorescent BQM coaccumulates in mitochondria with the mitochondrial stain MitoTracker (data not shown). We also observed that several mitochondrial mutants, such as the goa1 and ndh51 null mutants (22,42,43), showed increased resistance to BQM compared to the wild-type control (data not shown). The role of mitochondria in fluconazole susceptibility among Candida spp.…”

“…Drug susceptibility testing was carried out in flat-bottom, 96-well microtiter plates (Greiner Bio One) using the broth microdilution protocol according to the Clinical and Laboratory Standards Institute M-27A methods. Growth inhibition of all strains was evaluated in the presence of drugs and reported as percent inhibition of untreated cells as previously described (22). In brief, overnight cultures were prepared in YPD and washed, and ϳ10 3 cells/100 l were inoculated into microtiter wells.…”

Exploiting mitochondria as targets for the development of new antifungals

“…Our published data also correlate a loss of CI with increased susceptibility to fluconazole. 21,22 In this case, we have shown that 10 of 12 CI mutants were hypersusceptible to fluconazole. The two CI subunits that were not hypersusceptible were not associated with a respiration function.…”

“…The two CI subunits that were not hypersusceptible were not associated with a respiration function. 21 Thus, loss of mitochondrial ATP may be a benefit of an inhibitor that could specifically block CI respiration activity and, correspondingly, would ensure a reduction in resistance to antifungals such as the azoles.…”

Mechanisms of Antifungal Drug Resistance

“…C. albicans cannot undergo loss of mitochon-drial DNA and therefore is considered a "petitenegative" species. However, pharmacological inhibition of mitochondrial complexes involved in respiration and energy production results in higher susceptibility to azoles in this species (Sun et al 2013). The findings that mitochondrial defects are associated with azole resistance in C. glabrata, but susceptibility in C. albicans, suggests complex functional relationships between the azoles activity and mitochondrial function in fungal pathogens.…”