Much progress has been made in the last decade in identifying genes responsible for antifungal resistance in Candida albicans Attention has focused on five major C albicans genes: ABC transporter genes CDR1 and CDR2, major facilitator efflux gene MDR1, and ergosterol biosynthesis genes ERG11 and ERG3 Resistance involves mutations in 14C-lanosterol demethylase, targeted by fluconazole (FLZ) and encoded by ERG11, and mutations that up-regulate efflux genes that probably efflux the antifungals Mutations that affect ERG3 mutations have been understudied as mechanism resistance among clinical isolates In vitro resistance in clinical isolates typically involves step-wise mutations affecting more than one of these genes, and often unidentified genes Different approaches are needed to identify these other genes Very little is understood about reversible adaptive resistance of C albicans despite its potential clinical significance; most clinical failures to control infections other than oropharyngeal candidiasis (OPC) occur with in vitro susceptible strains Tolerance of C albicans to azoles has been attributed to the calcineurin stress-response pathway, offering new potential targets for next generation antifungals Recent studies have identified genes that regulate CDR1 or ERG genes The focus of this review is C albicans, although information on Saccharomyces cerevisiae or Candida glabrata is provided in areas in where Candida research is underdeveloped With the completion of the C albicans genomic sequence, and new methods for high throughput gene overexpression and disruption, rapid progress towards understanding the regulation of resistance, novel resistance mechanisms, and adaptive resistance is expected in the near future

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An update on antifungal targets and mechanisms of resistance in Candida albicans

https://mmbr.asm.org/content/mmbr/42/1/161.full.pdf

Genome comparison in yeast systematics: delimitation of species within the genera Schwanniomyces, Saccharomyces, Debaryomyces, and Pichia.

The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy. The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2-7 genome units. A small proportion of mtDNA is present as supercoiled or relaxed circular molecules. Additional components, detected by electron microscopy, are circular molecules with either single- or double-stranded tails (lariats). The presence of lariats, together with the observation that the majority of mtDNA is linear and 2-7 genome units in length, suggests that replication occurs by a rolling circle mechanism. Replication of mtDNA in other yeasts is thought to occur by the same mechanism. For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length. Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of baker's yeast, although only circular DNA is detected when this plasmid occurs in the nucleus. The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.

Rolling circle replication of DNA in yeast mitochondria.

We previously showed that resistant colonies of Candida glabrata inside the azole inhibition zones had respiratory deficiency due to mutations in mitochondrial DNA. Here, we analyzed the mechanisms of azole resistance in petite mutants of C. glabrata obtained by exposure to fluconazole or induced by ethidium bromide. The respiratory deficiency of these mutants was confirmed by oxygraphy and flow cytometric analysis with rhodamine 123, and its mitochondrial origin was demonstrated by transmission electron microscopy and restriction endonuclease analysis of the mitochondrial DNA. Flow cytometry with rhodamine 6G suggested an increased drug efflux in mutant cells, which was further supported by Northern blot analysis of the expression of the C. glabrata CDR1 (CgCDR1) and CgCDR2 genes, encoding efflux pumps. Conversely, the expression of CgERG11, which encodes the azole target, was not affected by petite mutations, and no differences were seen in the sequence of this gene between parent isolates and mutants. Moreover, sterol analysis showed similar overall amount of sterols in parent and mutant cells, but quantitative modifications were observed in the mutants, with almost undetectable biosynthesis intermediates. Further analysis performed after separation of free sterols from steryl esters revealed a defect in sterol esterification in mutant cells, with free ergosterol representing 92% of the overall sterol content. Thus, resistance or decreased susceptibility to azoles in petite mutants of C. glabrata is associated with increased expression of CgCDR1 and, to a lesser extent, of CgCDR2. In addition, the marked increase in free ergosterol content would explain their increased susceptibility to polyenes.

Mechanisms of Azole Resistance in Petite Mutants of Candida glabrata

The yeast mitochondrial ATPase complex. Subunit composition and evidence for a latent protease contaminant.

Purified mitochondria from the petite positive yeast Torulopsis glabrata contain a circular deoxyribonucleic acid (DNA) with a length of 6 mum and a buoyant density of 1.686 g/cm3. This DNA is absent from ethidium bromide induced respiratory-deficient mutants. Images

G D Clark-Walker

Papers

Respiratory-deficient mutants of Torulopsis glabrata, a yeast with circular mitochondrial deoxyribonucleic acid of 6 mu m.