Abstract
Azoles are widely used antifungal drugs. This family of compounds includes triazoles, mostly used in the treatment of systemic infections, and imidazoles, such as clotrimazole, often used in the case of superficial infections. Candida glabrata is the second most common cause of candidemia worldwide and presents higher levels of intrinsic azole resistance when compared with Candida albicans, thus being an interesting subject for the study of azole resistance mechanisms in fungal pathogens.Since resistance often relies on the action of membrane transporters, including drug efflux pumps from the ATP-binding cassette family or from the Drug:H(+) antiporter (DHA)(1) family, an iTRAQ-based membrane proteomics analysis was performed to identify all the membrane-associated proteins whose abundance changes in C. glabrata cells exposed to the azole drug clotrimazole. Proteins found to have significant expression changes in this context were clustered into functional groups, namely: glucose metabolism, oxidative phosphorylation, mitochondrial import, ribosome components and translation machinery, lipid metabolism, multidrug resistance transporters, cell wall assembly, and stress response, comprising a total of 37 proteins. Among these, the DHA transporter CgTpo1_2 (ORF CAGL0E03674g) was identified as overexpressed in the C. glabrata membrane in response to clotrimazole. Functional characterization of this putative drug:H(+) antiporter, and of its homolog CgTpo1_1 (ORF CAGL0G03927g), allowed the identification of these proteins as localized to the plasma membrane and conferring azole drug resistance in this fungal pathogen by actively extruding the drug to the external medium. The cell wall protein CgGas1 was also shown to confer azole drug resistance through cell wall remodeling. Finally, the transcription factor CgPdr1 in the clotrimazole response was observed to control the expression of 20 of the identified proteins, thus highlighting the existence of additional unforeseen targets of this transcription factor, recognized as a major regulator of azole drug resistance in clinical isolates.
Highlights
Since resistance often relies on the action of membrane transporters, including drug efflux pumps from the ATPbinding cassette family or from the Drug:H؉ antiporter (DHA)1 family, an iTRAQ-based membrane proteomics analysis was performed to identify all the membraneassociated proteins whose abundance changes in C. glabrata cells exposed to the azole drug clotrimazole
The first iTRAQ-based membrane proteomics study focused on the fungal pathogen C. glabrata was undertaken, leading to functional characterization of the C. glabrata CgTpo1_1 and CgTpo1_2 drug:Hϩ antiporters, and of the cell wall assembly protein CgGas1 in the context of clotrimazole drug resistance
Using a membrane proteomics analysis, several proteins from distinct functional groups were found to be differentially expressed in C. glabrata clotrimazole response
Summary
Strains and Growth Media—S. cerevisiae parental strain BY4741 (MATa, ura3⌬0, leu2⌬0, his3⌬1, met15⌬0) and the derived single deletion mutant BY4741_⌬tpo were obtained from Euroscarf (http:// web.uni-frankfurt.de/fb15/mikro/euroscarf/). L5U1 C. glabrata cell suspensions used to inoculate the agar plates were midexponential cells grown in BM medium, supplemented with 50 M CuSO4 (Sigma), to induce protein overexpression, without uracil when using the L5U1 strain harboring the pGREG576-derived plasmids, until culture OD600 nm ϭ 0.4 Ϯ 0.02 was reached, and diluted in sterile water to obtain suspensions with OD600 nm ϭ 0.05 Ϯ 0.005. These cell suspensions and subsequent dilutions (1:5; 1:25) were applied as 4 l spots onto the surface of solid BM medium, without uracil for strains transformed with the pGREG576-derived plasmids, supplemented with 50 M CuSO4 and with adequate chemical stress concentrations. Statistical analysis of the results were performed using analysis of variance, and differences were considered significant for p values Ͻ .05
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