Relationships between Respiration and Susceptibility to Azole Antifungals in Candida glabrata

Abstract

Over the past two decades, the incidence of infections due to Candida glabrata, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly. Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species. In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of C. glabrata were evaluated. Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide. Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles. Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed. This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar. This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy. Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency. Finally, this mutant exhibited cross-resistance to all the azoles tested. In conclusion, blockage of respiration in C. glabrata induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA. This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain.

Roles of Cellular Respiration, Cg CDR1 , and Cg CDR2 in Candida glabrata Resistance to Histatin 5

“…Resistance to azoles has been associated with the upregulation of the genes CgCDR1 and CgCDR2 (also designated PDH1) which encode proteins belonging to the ATP binding cassette family of multidrug resistance membrane-associated efflux pumps (1,2,12,19,20,23). Other reported mechanisms of azole resistance in C. glabrata which may actually be related to CDR upregulation involve the generation or selection of respiratory-deficient petite mutants (3,4). In the quest to develop new antimycotics with possibly different cellular targets, histatins, a group of salivary cationic proteins, have received special attention (5,8,16,17,21).…”

“…Since C. glabrata resistance to fluconazole in some cases is explained by induction or selection of petite mutants (3,4) and petite mutation in C. albicans is known to abolish sensitivity to histatin 5 (7), the respiratory competence of C. glabrata cells grown in the presence of histatin 5 was evaluated. Two experimental approaches were followed.…”

Reduced Susceptibility to Polyenes Associated with a Missense Mutation in the ERG6 Gene in a Clinical Isolate of Candida glabrata with Pseudohyphal Growth

“…Moreover, like S. cerevisiae, C. glabrata cannot perform dimorphic transition from blastoconidia to hyphae or pseudohyphae under usual culture conditions (23). However, C. glabrata can produce pseudohyphae, as has been reported for both yeast species, in special culture conditions like nitrogen starvation (8) or in the presence of CuSO 4 (17). The recent recovery in our hospital laboratory of a clinical isolate of C. glabrata with a poor susceptibility to polyene drugs associated with a pseudohyphal growth led us to investigate the mechanisms responsible for its particular phenotype.…”

Candida glabrata PDR1 , a Transcriptional Regulator of a Pleiotropic Drug Resistance Network, Mediates Azole Resistance in Clinical Isolates and Petite Mutants

“…High-frequency acquired resistance (HFAR) to fluconazole has been reported in C. glabrata and S. cerevisiae in the laboratory setting (4,5,7,16,33,44), and overexpression of fluconazole-effluxing ABC transporters in the petite mutants has been documented in both S. cerevisiae and C. glabrata (33,44). Two transcriptional factors, Pdr1p and Pdr3p, are known to regulate the pleiotropic drug resistance (PDR) network in S. cerevisiae (1,9,15).…”