A New Endogenous Overexpression System of Multidrug Transporters of Candida albicans Suitable for Structural and Functional Studies.

Fungal pathogens have a robust array of multidrug transporters which aid in active expulsion of drugs and xenobiotics to help them evade toxic effects of drugs. Thus, these transporters impose a major impediment to effective chemotherapy. Although the Saccharomyces cerevisiae strain AD1-8u(-) has catered well to the need of an overexpression system to study drug transport by multidrug transporters of Candida albicans, artifacts associated with a heterologous system could not be excluded. To avoid the issue, we exploited a azole-resistant clinical isolate of C. albicans to develop a new system devoid of three major multidrug transporters (Cdr1p, Cdr2p, and Mdr1p) for the overexpression of multidrug transporters under native hyperactive CDR1 promoter due to gain of function (GOF) mutation in TAC1. The study deals with overexpression and functional characterization of representatives of two major classes of multidrug transporters, Cdr1p and Mdr1p, to prove the functionality of this newly developed endogenous expression system. Expression of native Cdr1 and Mdr1 protein in C. albicans cells was confirmed by confocal microscopy and immunodetection and resulted in increased resistance to the putative substrates as compared to control. The system was further validated by overexpressing a few key mutant variants of Cdr1p and Mdr1p. Together, our data confirms the utility of new endogenous overexpression system which is devoid of artifactual factors as most suited for functional characterization of multidrug transporter proteins of C. albicans

Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1

Abstract Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its transient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understanding of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unexpected hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition

Structure of Ycf1 in the dephosphorylated state

Structure of the Ycf1 transporter in the dephosphorylated state. </p

Molecular mechanisms of action of herbal antifungal alkaloid berberine, in Candida albicans.

Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida

pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport

International audienc

An Assessment of Growth Media Enrichment on Lipid Metabolome and the Concurrent Phenotypic Properties of <i>Candida albicans</i>

<div><p>A critical question among the researchers working on fungal lipid biology is whether the use of an enriched growth medium can affect the lipid composition of a cell and, therefore, contribute to the observed phenotypes. One presumption is that enriched medias, such as YPD (yeast extract, peptone and dextrose), are likely to contain lipids, which may homogenize with the yeast lipids and play a role in masking the actual differences in the observed phenotypes or lead to an altered phenotype altogether. To address this issue, we compared the lipids of <i>Candida albicans</i>, our fungus of interest, grown in YPD or in a defined media such as YNB (yeast nitrogen base). Mass spectrometry-based lipid analyses showed differences in the levels of phospholipids, including phosphatidylinositol, phosphatidylglycerol, lyso-phospholipids; sphingolipids, such as mannosyldiinositolphosphorylceramide; and sterols, such as ergostatetraenol. Significant differences were observed in 70 lipid species between the cells grown in the two media, but the two growth conditions did not affect the morphological characteristics of <i>C. albicans</i>. The lipid profiles of the YNB- and YPD-grown <i>C. albicans</i> cells did vary, but these differences did not influence their response to the majority of the tested agents. Rather, the observed differences could be attributed to the slow growth rate of the <i>Candida</i> cells in YNB compared to YPD. Notably, the altered lipid changes between the two media did impact the susceptibility to some drugs. This data provided evidence that changes in media can lead to certain lipid alterations, which may affect specific pathways but, in general, do not affect the majority of the phenotypic properties of <i>C. albicans</i>. It was determined that either YNB or YPD may be suitable for the growth and lipid analysis of <i>C. albicans</i>, depending upon the experimental requirements, but additional precautions are necessary when correlating the phenotypes with the lipids.</p></div

Effect of cell wall perturbing agents and protein biosynthesis inhibitors on <i>C. albicans</i> cells grown in YPD or YNB.

<p>A) CW. B) CR. C) TX-100. D) SDS. E) CYCLO. Values are means ± SEM (n = 4). YPD and YNB control datasets are same for the subfigures A–E.</p

PGL composition of <i>C. albicans</i> cells grown in different medium.

<p>A) Total PGLs (as normalized total PGL+SL+SE mass spectral signal). B) Relative abundance of PGL classes (as % of normalized total PGL+SL+SE mass spectral signal). Values are mean of 2 independent analyses (n = 2). Data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113664#pone.0113664.s001" target="_blank">Supplementary Sheet S1</a>. “*” indicates that <i>p</i>-value is <0.05.</p

Molecular lipid species composition of <i>C. albicans</i> cells grown in YPD or YNB.

<p>Data is represented as % of total PGL+SL+SE mass spectral signal normalized to the internal standards. Values are means ± SEM (n = 2). Data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113664#pone.0113664.s001" target="_blank">Supplementary Sheet S1</a>. Only significant changes where <i>p</i>-value is <0.05 are depicted in this figure.</p

Growth phenotypes of <i>C. albicans</i> cells grown in YPD or YNB.

<p>A) Growth curve of CAI-4 cells grown in YPD and YNB medium. B) Generation time of CAI-4 cells grown in YPD or YNB medium. C) Cell morphology of CAI-4 cells grown in YPD or YNB medium. D) Spot assays showing growth of CAI-4 cells grown in YPD or YNB medium at variable temperatures. E) Hyphae formation of CAI-4 cells grown in YPD or YNB. <i>Candida</i> strains were cultured in YPD or YNB medium at 30°C as described in previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113664#pone.0113664-Singh1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113664#pone.0113664-Shah1" target="_blank">[18]</a>. To test the hyphae formation YPD or YNB grown cells were re-grown on spider agar plates at 37°C for 5 days. Values are means ± SEM (n = 3). “*” indicates that p-value is <0.05.</p