Membrane Proteome-Wide Response to the Antifungal Drug Clotrimazole in Candida glabrata: Role of the Transcription Factor CgPdr1 and the Drug:H+ Antiporters CgTpo1_1 and CgTpo1_2

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 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. 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.

Membrane Proteomics Analysis of the Candida glabrata Response to 5-Flucytosine: Unveiling the Role and Regulation of the Drug Efflux Transporters CgFlr1 and CgFlr2

“…Details on the protein quantification can be assessed at the Mass spectrometry Interactive Virtual Environment (MassIVE) repository (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp; MassIVE ID: MSV000079209). Annotated spectra for single peptide identification for each protein in the membrane-associated proteome is provided in MS-viewer (http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer; Search keys: qrvm65goix; xffxn22szm; tm0y9x0hqd) (Pais et al, 2016). …”

“…The deletion of CgPdr1 was consistently found to decrease the activation of about 50% of the membrane proteins found to be up-regulated in response to 5-FC. Among the proteins whose expression was seen to be affect by CgPdr1 deletion are the multidrug efflux transporter encoding genes CgCDR1 and CgQDR2 , but also CgRIP1 , encoding a putative ubiquinol-cytochrome C reductase iron-sulfur protein, which were previously found to be controlled by this transcription factor (Tsai et al, 2006; Vermitsky et al, 2006; Ferrari et al, 2011; Costa et al, 2013b; Pais et al, 2016). New putative targets of CgPdr1 up-regulated in the context of 5-FC response include four proteins involved in RNA metabolism, CgDbp2, CgNop1, CgRpl20B, and CgRps16A.…”

“…These two transporters constitute, thus, two additional players in the antifungal drug resistance phenomenon. Our group had previously shown that the acquaglyceroporins CgFps1 and CgFps2 (Costa et al, 2015), as well as the DHA transporters CgAqr1 (Costa et al, 2013a) and CgTpo1_1 and CgTpo1_2 (Pais et al, 2016) are determinants of flucytosine resistance as well, suggesting that 5-FC extrusion is an important mechanism of resistance against this antifungal drug and showing this phenomenon to be the consequence of the additive contribution of several players.…”

“…Upon 1 h of cultivation, cells were harvested by centrifugation and the membrane protein fraction was obtained as described before (Pais et al, 2016). Expression proteomics analysis of the obtained membrane-enriched fraction was carried out using an iTRAQ-MS procedure, carried out as a paid service at the Keck Foundation Biotechnology Resource Laboratory, Yale University, USA (http://medicine.yale.edu/keck/proteomics/index.aspx), using the method followed in Pais et al (2016). Briefly, samples were sonicated and proteins reduced by adding 50 mM TCEP (tris(2-carboxyethyl)phosphine), followed by 200 mM MMTS (methyl methane thiosulfonate).…”

Quantitative Proteomic Profiling of Cryptococcus neoformans

“…In Cryptococcus neoformans , an opportunistic human fungal pathogen, defining changes in protein abundance provides novel insight into the production of virulence factors and the responses of both the host and pathogen to infection (Geddes et al., , ; Pandey et al., ). Moreover, the role of MS‐based proteomics in drug discovery also promotes its application in defining biological processes or targets relevant during the evolution of antifungal resistance (Geddes‐Mcalister & Shapiro, ; Moloney, Owens, & Doyle, ; Pais, Costa, et al., ; Pais, Pires, et al., ). To promote an in‐depth analysis of protein‐level changes in C. neoformans in response to a changing environment (e.g., laboratory associated, natural, nutrient limitation), we have devised a step‐by‐step protocol for quantitative profiling of the fungal cellular proteome and extracellular environment (secretome).…”