Tulika Prasad

Membrane Sphingolipid-Ergosterol Interactions Are Important Determinants of Multidrug Resistance in Candida albicans

ABSTRACT In this study, we examined the importance of membrane ergosterol and sphingolipids in the drug susceptibilities of Candida albicans. We used three independent methods to test the drug susceptibilities of erg mutant cells, which were defective in ergosterol biosynthesis. While spot and filter disk assays revealed that erg2 and erg16 mutant cells of C. albicans became hypersensitive to almost all of the drugs tested (i.e., 4-nitroquinoline oxide, terbinafine, o-phenanthroline, itraconazole, and ketoconazole), determination of the MIC at which 80% of the cells were inhibited revealed more than fourfold increase in susceptibility to ketoconazole and terbinafine. Treatment of wild-type C. albicans cells with fumonisin B1 resulted in 45% inhibition of sphingolipid biosynthesis and caused cells to become hypersensitive to the above drugs. Although erg mutants displayed enhanced membrane fluidity and passive diffusion, these changes alone were not sufficient to elicit the observed hypersusceptibility phenotype of erg mutants. For example, the induction in vitro of a 12% change in the membrane fluidity of C. albicans cells by a membrane fluidizer, benzyl alcohol, did not affect the drug susceptibilities of Candida cells. Additionally, the surface localization of green fluorescent protein-tagged Cdr1p, a major drug efflux pump protein of C. albicans, revealed that any disruption in ergosterol and sphingolipid interactions also interfered with its proper surface localization and functioning. A 50% reduction in the efflux of the Cdr1p substrate, rhodamine 6G, in erg mutant cells or in cells with a reduced sphingolipid content suggested a strong correlation between these membrane lipid components and this major efflux pump protein. Taken together, the results of our study demonstrate for the first time that there is an interaction between membrane ergosterol and sphingolipids, that a reduction in the content of either of these two components results in a disruption of this interaction, and that this disruption has deleterious effects on the drug susceptibilities of C. albicans cells.

Unexpected Link between Iron and Drug Resistance of Candida spp.: Iron Depletion Enhances Membrane Fluidity and Drug Diffusion, Leading to Drug-Susceptible Cells

ABSTRACT Inthis study, we show that iron depletion in Candidaalbicans with bathophenanthrolene disulfonic acid and ferrozine as chelators enhanced its sensitivity to several drugs, including the most common antifungal, fluconazole (FLC). Several other species of Candida also displayed increased sensitivity to FLC because of iron restriction. Iron uptake mutations, namely,Δ ftr1 and Δftr2, as well as the copper transporter mutation Δccc2, which affects high-affinity iron uptake in Candida, produced increased sensitivity to FLC compared to that of the wild type. The effect of iron depletion on drug sensitivity appeared to be independent of the efflux pump proteins Cdr1p and Cdr2p. We found that iron deprivation led to lowering of membrane ergosterol by 15 to 30%. Subsequently, fluorescence polarization measurements also revealed that iron-restricted Candida cells displayed a 29 to 40% increase in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Northern blot assays revealed that the ERG11 gene was considerably down regulated in iron-deprived cells, which might account for the lowered ergosterol content. Our results show a close relationship between cellular iron and drug susceptibilities of C. albicans. Considering that multidrug resistance is a manifestation of multifactorial phenomena, the influence of cellular iron on the drug susceptibilities of Candida suggests iron as yet another novel determinant of multidrug resistance.

Curcumin Modulates Efflux Mediated by Yeast ABC Multidrug Transporters and Is Synergistic with Antifungals

ABSTRACT Curcumin (CUR), a natural product of turmeric, from rhizomes of Curcuma longa, is a known agent of reversal of drug resistance phenotypes in cancer cells overexpressing ATP-binding cassette (ABC) transporters, viz., ABCB1, ABCG2, and ABCC1. In the present study, we evaluated whether CUR could also modulate multidrug transporters of yeasts that belong either to the ABC family or to the major facilitator superfamily (MFS). The effect of CUR on multidrug transporter proteins was demonstrated by examining rhodamine 6G (R6G) efflux in Saccharomyces cerevisiae cells overexpressing the Candida albicans ABC transporters Cdr1p and Cdr2p (CaCdr1p and CaCdr2p, respectively) and the MFS transporters CaMdr1p and S. cerevisiae Pdr5p. CUR decreased the extracellular concentration of R6G in ABC transporter-expressing cells but had no effect on methotrexate efflux mediated through the MFS transporter CaMdr1p. CUR competitively inhibited R6G efflux and the photolabeling of CaCdr1p by [125I]iodoarylazidoprazosin, a drug analogue of the substrate prazosin (50% inhibitory concentration, 14.2 μM). Notably, the mutant variants of CaCdr1p that displayed abrogated efflux of R6G also showed reduced modulation by CUR. Drug susceptibility testing of ABC protein-expressing cells by spot assays and checkerboard tests revealed that CUR was selectively synergistic with drug substrates such as R6G, ketoconazole, itraconazole, and miconazole but not with fluconazole, voriconazole, anisomycin, cycloheximide, or FK520. Taken together, our results provide the first evidence that CUR modulates only ABC multidrug transporters and could be exploited in combination with certain conventional antifungal drugs to reverse multidrug resistance in Candida cells.

Functional Analysis of CaIPT1, a Sphingolipid Biosynthetic Gene Involved in Multidrug Resistance and Morphogenesis of Candida albicans

ABSTRACT In the present study we describe the isolation and functional analysis of a sphingolipid biosynthetic gene, IPT1, of Candida albicans. The functional consequence of the disruption of both alleles of IPT1 was confirmed by mass analysis of its sphingolipid composition. The disruption of both alleles or a single allele of IPT1 did not lead to any change in growth phenotype or total sphingolipid, ergosterol, or phospholipid content of the mutant cells. The loss of mannosyl diinositol diphosphoceramide [M(IP)2C] in the ipt1 disruptant, however, resulted in increased sensitivity to drugs like 4-nitroquinoline oxide, terbinafine, o-phenanthroline, fluconazole, itraconazole, and ketoconazole. The increase in drug susceptibilities of ipt1 cells was linked to an altered sphingolipid composition, which appeared to be due to the impaired functionality of Cdr1p, a major drug efflux pump of C. albicans that belongs to the ATP binding cassette superfamily. Our confocal and Western blotting results demonstrated that surface localization of green fluorescent protein-tagged Cdr1p was affected in ipt1 disruptant cells. Poor surface localization of Cdr1p resulted in an impaired ability to efflux fluconazole and rhodamine 6G. The effect of mannosyl inositol phosphoceramide accumulation in the ipt1 mutant and the absence of M(IP)2C from the ipt1 mutant on the efflux of drug substrates was very selective. The efflux of methotrexate, a specific substrate of CaMdr1p, another major efflux pump of major facilitator superfamily, remained unaffected in ipt1 mutant cells. Interestingly, changes in sphingolipid composition affected the ability of mutant cells to form proper hyphae in various media. Taken together, our results demonstrate that an altered composition of sphingolipid, which is among the major constituents of membrane rafts, affects the drug susceptibilities and morphogenesis of C. albicans.

Membrane raft lipid constituents affect drug susceptibilities of Candida albicans

By exploiting the biosynthetic pathways of raft lipid constituents, in this study we demonstrate that fluctuations in either sphingolipid or ergosterol levels result in increased drug sensitivity and morphological defects in Candida albicans cells. We show that any change in either ergosterol composition by conditionally disrupting ERG1 or in sphingolipid composition by homozygously disrupting its biosynthetic gene IPT1 leads to improper surface localization of a major ABC (ATP-binding cassette) drug efflux protein, Cdr1p. Results suggest that sterol/sphingolipid-rich membrane microdomains play an important role in positioning and functional maintenance of the integral efflux protein. The impaired ability of erg1/ipt1 mutant cells to efflux drugs mediated through Cdr1p appears to be the main cause of increased drug sensitivity of Candida cells.

Iron deprivation induces EFG1-mediated hyphal development in Candida albicans without affecting biofilm formation

In this study, we investigated the role of cellular iron status in hyphae and biofilm formation in Candida albicans. Iron deprivation by a chelator, bathophenanthrolene disulfonic acid, promoted hyphal development even in nonhyphal-inducing media without affecting growth of C. albicans cells. Iron-acquisition defective mutants, Deltaftr1 and Deltaccc2, also showed hyphal formation, which was prevented by iron supplementation. Notably, most of the tested morphological mutants Deltacph1, Deltaefh1 and Deltatpk1 continued to form hyphae under iron-deprived conditions, except the Deltaefg1 null mutant, which showed a complete block in hyphae formation. The role of EFG1 in filamentation under iron-deprived conditions was further confirmed by Northern analysis, which showed a considerable upregulation of the EFG1 transcript. Of notable importance, all the morphological mutants including Deltaefg1 mutant possessed enhanced membrane fluidity under iron-deprived conditions; however, this did not appear to contribute to hyphal development. Interestingly, iron deprivation did not affect the ability of C. albicans to form biofilms on the catheter surface and led to no gross defects in azole resistance phenotype of these biofilms of C. albicans cells. Our study, for the first time, establishes a link between cellular iron, Efg1p and hyphal development of C. albicans cells that is independent of biofilm formation.

Phospholipidome of Candida: each species of Candida has distinctive phospholipid molecular species.

By employing electrospray ionization tandem mass spectrometry (ESI-MS/MS), the phospholipidomes of eight hemiascomycetous human pathogenic Candida species have been characterized. Over 200 phospholipid molecular species were identified and quantified. There were no large differences among Candida species in phosphoglyceride class composition; however, differences in phosphoglycerides components (i.e., fatty acyl chains) were identified. In contrast, differences in sphingolipid class composition as well as in molecular species were quite evident. The phospholipid compositions of C. albicans, C. glabrata, C. parapsilosis, C. kefyr, C. tropicalis, C. dubliniensis, C. krusei, and C. utilis could be further discriminated by principal component analysis. Notwithstanding that a single strain of each species was analyzed, our data do point to a typical molecular species imprint of Candida strains.

Morphogenic regulator EFG1 affects the drug susceptibilities of pathogenic Candida albicans

This study shows that the morphogenic regulator EFG1 level affects the drug susceptibilities of Candida albicans when grown on solid growth media. The Deltaefg1 mutant showed sensitivity particularly to those drugs that target ergosterol or its metabolism. Efg1p disruption showed a gene-dosage effect on drug susceptibilities and resulted in enhanced susceptibility to drugs in the homozygous mutant as compared with the wild type, heterozygous and revertant strains. The enhanced sensitivity to drugs was independent of the status of ATP-binding cassette and MFS multidrug efflux pumps of C. albicans. The Deltaefg1 mutant displayed increased membrane fluidity that coincided with the downregulation of ERG11 and upregulation of OLE1 and ERG3, leading to enhanced passive diffusion of drugs. Interestingly, Deltaefg1 mutant cells displayed enhanced levels of endogenous ROS levels. Notably, the higher levels of ROS in the Deltaefg1 mutant could be reversed by the addition of antioxidants. However, the restoration of ROS levels did not reverse the drug sensitivities of the Deltaefg1 mutant. Taken together, we, for the first time, establish a new role to EFG1 in affecting the drug susceptibilities of C. albicans cells, independent of ROS and known drug efflux mechanisms.

Variation in transpiration efficiency and carbon isotope discrimination in cowpea

Genotypic variation in transpiration efficiency (TE) was investigated in a set of cowpea (Vigna unguiculata (L.) Walp.) genotypes grown as isolated plants in pots and under canopy conditions in the field. In the field, plants were grown in mini-lysimeters embedded in the ground around which a crop was grown, to simulate crop canopy condition. Two moisture regimes (100 and 60% of field capacity) were imposed from 30 to 60 days after sowing in both pot and field experiments. TE was determined by measuring transpiration and dry matter (DM) produced by the genotypes during the treatment period. Genotypes differed significantly in DM although the variation in the amount of water transpired (T) was relatively small. The TE ranged from 2.2 to 3.7 g kg–1, representing a significant genotypic and environmental effect on the variation. There was a significant negative correlation (r = –0.77, P<0.01) between TE adjusted for prevailing vapour pressure deficit and carbon isotope discrimination ratio (Δ) across all the experiments and treatments. A significantly positive correlation of TE measured in pot and field experiments suggested a low G x E interaction. There was a negative correlation (r = –0.62, P < 0.01) between T and TE while the correlation of TE with net assimilation rate was non-significant, suggesting that the major cause for variation of TE in cowpea was in general associated with stomatal rather than mesophyll factors. Because of the positive relationship between T and DM, and negative relationship between TE and T, selection for high TE might therefore be associated with reduced T and hence lower DM. However, the present investigation showed a possibility of identifying specific genotypes with a combination of high TE and high net assimilation rate. The genotypes with high TE and high net assimilation rates were able to produce high DM under moisture deficit conditions.

Vitellogenin from the Silkworm, Bombyx mori: An Effective Anti-Bacterial Agent

Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host.