Candida albicans causes superficial and life-threatening systemic infections. These are difficult to treat often due to drug resistance, particularly because C. albicans biofilms are inherently resistant to most antifungals. Sophorolipid (SL), a glycolipid biosurfactant, has been shown to have antimicrobial and anticancer properties. In this study, we investigated the effect of SL on C. albicans biofilm formation and preformed biofilms. SL was found to inhibit C. albicans biofilm formation as well as reduce the viability of preformed biofilms. Moreover, SL, when used along with amphotericin B (AmB) or fluconazole (FLZ), was found to act synergistically against biofilm formation and preformed biofilms. Effect of SL on C. albicans biofilm formation was further visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed absence of hyphae, typical biofilm architecture and alteration in the morphology of biofilm cells. We also found that SL downregulates the expression of hypha specific genes HWP1, ALS1, ALS3, ECE1 and SAP4, which possibly explains the inhibitory effect of SL on hyphae and biofilm formation.

Deletants of the sphingolipid biosynthetic pathway genes FEN1 and SUR4 of Saccharomyces cerevisiae, as well as deletants of their orthologs in Candida albicans, were found to be 2-to 5-fold-more sensitive to amphotericin B (AmB) than parent strains. The inhibition of sphingolipid biosynthesis in parent strains by myriocin sensitized them to AmB, which can be reversed by providing phytosphingosine, an intermediate in the sphingolipid pathway. These results indicate that sphingolipids modulate AmB resistance, with implications for mechanisms underlying AmB action and resistance. Candidiasis is the fourth most common hospital-acquired infection, and it is associated with high mortality rates in cases of invasive infections (1-5). Candida albicans is the main causative agent, but other Candida species such as C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. lusitaniae are becoming more prevalent (2, 6, 7). The rapid emergence of resistant strains against major antifungal drugs often renders therapy ineffective (8-10). Amphotericin B (AmB) is a commonly used fungicidal polyene drug, and resistance against it is reported mainly in some species of Candida (11). However, severe side effects are associated with high therapeutic doses of AmB (12). Thus, there is an urgent need for AmB analogs that have similar or higher potency than AmB but with minimal side effects. Another approach is to use AmB in combination with drugs that sensitize fungal cells to AmB or potentiate AmB action, such that less AmB is needed for effective therapy, thereby reducing AmB toxicity.The mechanisms underlying the fungicidal actions of AmB are becoming better understood; a recently reported study (13) showed that binding of AmB to ergosterol as such is sufficient to kill the cells, and leakage of ions due to pore formation is a secondary effect of AmB. Absence of ergosterol in ergosterol biosynthesis mutants results in AmB resistance (11,(14)(15)(16)(17)(18). Sterols (ergosterol in fungi and cholesterol in higher eukaryotes) and sphingolipids form distinct domains (lipid rafts) in plasma membrane and are required for several cellular processes, including maintenance of plasma membrane integrity, protein sorting, endocytosis, and proper functioning of certain membrane proteins (19-24). Altered composition or loss of these membrane constituents affects targeting of ATP-binding cassette transporter Cdr1p to lipid rafts and susceptibility of C. albicans and Saccharomyces cerevisiae to drugs that are substrates of drug efflux pumps (25, 26). Since ergosterol interacts physically as well as functionally with sphingolipids, and biosynthesis of sphingolipids is closely coordinated with that of sterols (27-29), we hypothesized that sphingolipids, like ergosterol, might be involved in modulation of AmB resistance. To test this, first we evaluated AmB resistance of S. cerevisiae deletants impaired in sphingolipid biosynthesis. We also tested the effect of myriocin (a sphingolipid biosynthesis inhibitor) and myriocin in combination with phyt...

Invasive opportunistic fungal infections of humans are common among those suffering from impaired immunity, and are difficult to treat resulting in high mortality. Amphotericin B (AmB) is one of the few antifungals available to treat such infections. The AmB resistance mechanisms reported so far mainly involve decrease in ergosterol content or alterations in cell wall. In contrast, depletion of sphingolipids sensitizes cells to AmB. Recently, overexpression of PMP3 gene, encoding plasma membrane proteolipid 3 protein, was shown to increase and its deletion to decrease, AmB resistance. Here we have explored the mechanistic basis of PMP3 effect on AmB resistance. It was found that ergosterol content and cell wall integrity are not related to modulation of AmB resistance by PMP3. A few prominent phenotypes of PMP3 delete strain, namely, defective actin polarity, impaired salt tolerance, and reduced rate of endocytosis are also not related to its AmB-sensitivity. However, PMP3 overexpression mediated increase in AmB resistance requires a functional sphingolipid pathway. Moreover, AmB sensitivity of strains deleted in PMP3 can be suppressed by the addition of phytosphingosine, a sphingolipid pathway intermediate, confirming the importance of this pathway in modulation of AmB resistance by PMP3.

Sphingolipids are involved in several cellular functions, including maintenance of cell wall integrity. To gain insight into the role of individual genes of sphingolipid biosynthetic pathway, we have screened Saccharomyces cerevisiae strains deleted in these genes for sensitivity to cell wall perturbing agents calcofluor white and congo red. Only deletants of FEN1 and SUR4 genes were found to be sensitive to both these agents. Candida albicans strains deleted in their orthologs, CaFEN1 and CaFEN12, respectively, also showed comparable phenotypes, and a strain deleted for both these genes was extremely sensitive to cell wall perturbing agents. Deletion of these genes was reported earlier to sensitise cells to amphotericin B (AmB), which is a polyene drug that kills the cells mainly by binding and sequestering ergosterol from the plasma membrane. Here we show that their AmB sensitivity is likely due to their cell wall defect. Further, we show that double deletant of C. albicans is defective in hyphae formation as well as biofilm development. Together this study reveals that deletion of FEN1 and SUR4 orthologs of C. albicans leads to impaired cell wall integrity and biofilm formation, which in turn sensitise cells to AmB.