The malfunction of peroxisome has an impact on the oxidative stress sensitivity in Candida albicans.

The peroxisome plays an essential role in eukaryotic cellular metabolism, including ß-oxidation of fatty acids and detoxification of hydrogen peroxide. However, its functions in the important fungal pathogen, C. albicans, remain to be investigated. In this study, we identified a homologue of Saccharomyces cerevisiae peroxisomal protein Pex1 in this pathogen, and explored its functions in stress tolerance. Fluorescence observation revealed that C. albicans Pex1 was localized in the peroxisomes, and its loss led to the defect in peroxisome formation. Interestingly, the pex1Δ/Δ mutant had increased tolerance to oxidative stress, which was neither associated with the Cap1 pathway, nor related to the altered distribution of catalase. However, under oxidative stress, the pex1Δ/Δ mutant showed increased expression of autophagy-related genes, with enhanced cytoplasm-to-vacuole transport and degradation of the autophagy markers Atg8 and Lap41. Moreover, the double mutants pex1Δ/Δatg8Δ/Δ and pex1Δ/Δatg1Δ/Δ, both of which were defective in autophagy and peroxisome formation, showed remarkable attenuated tolerance to oxidative stress. These results indicated that autophagy is involved in resistance to oxidative stress in pex1Δ/Δ mutant. Taken together, this study provides evidence that the peroxisomal protein Pex1 regulates oxidative stress tolerance in an autophagy-dependent manner in C. albicans.

The Ccz1 mediates the autophagic clearance of damaged mitochondria in response to oxidative stress in Candida albicans.

Autophagy plays a critical role in response to numerous cellular stresses, such as nutrient deprivation, hypoxia, starvation and organelle damage. The disruption of autophagy pathway affects multiple aspects of cellular stress response. Here we for the first time identified Ccz1 as an essential component for autophagy in Candida albicans. Our experiments demonstrated that loss of CCZ1 gene led to vacuolar fragmentation and disruption of the autophagy pathway. Our results also suggested that Ccz1 functioned in oxidative stress. In the ccz1Δ/Δ mutant, the levels of reactive oxidative species (ROS) sharply increased under H2O2 treatment. Further studies demonstrated that breakdown of the autophagic clearance pathway led to the accumulation of oxidative stress-damaged mitochondria, and consequently elevated cellular ROS levels in the ccz1Δ/Δ mutant. Furthermore, deletion of CCZ1 led to a significant defect in filamentous development at both 30°C and 37°C. The disruption of CCZ1 gene led to decreased capacity of macrophage killing and increased sensitivity to the macrophages. In addition, the ccz1Δ/Δ mutant exhibited attenuated virulence and decreased fungal burdens in the mouse systemic infection model, indicating that CCZ1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of autophagy-related gene in C. albicans.

Tfp1 is required for ion homeostasis, fluconazole resistance and N-Acetylglucosamine utilization in Candida albicans.

The vacuolar-type H+-ATPase (V-ATPase) is crucial for the maintenance of ion homeostasis. Dysregulation of ion homeostasis affects various aspects of cellular processes. However, the importance of V-ATPase in Candida albicans is not totally clear. In this study, we demonstrated the essential roles of V-ATPase through Tfp1, a putative V-ATPase subunit. Deletion of TFP1 led to generation of an iron starvation signal and reduced total iron content, which was associated with mislocalization of Fet34p that was finally due to disorders in copper homeostasis. Furthermore, the tfp1∆/∆ mutant exhibited weaker growth and lower aconitase activity on nonfermentable carbon sources, and iron or copper addition partially rescued the growth defect. In addition, the tfp1∆/∆ mutant also showed elevated cytosolic calcium levels in normal or low calcium medium that were relevant to calcium release from vacuole. Kinetics of cytosolic calcium response to an alkaline pulse and VCX1 (VCX1 encodes a putative vacuolar Ca2+/H+ exchanger) overexpression assays indicated that the cytosolic calcium status was in relation to Vcx1 activity. Spot assay and concentration-kill curve demonstrated that the tfp1∆/∆ mutant was hypersensitive to fluconazole, which was attributed to reduced ergosterol biosynthesis and CDR1 efflux pump activity, and iron/calcium dysregulation. Interestingly, carbon source utilization tests found the tfp1∆/∆ mutant was defective for growth on N-Acetylglucosamine (GlcNAc) plate, which was associated with ATP depletion due to the decreased ability to catabolize GlcNAc. Taken together, our study gives new insights into functions of Tfp1, and provides the potential to better exploit V-ATPase as an antifungal target.

Deletion of genes encoding fatty acid desaturases leads to alterations in stress sensitivity in Pichia pastoris.

Unsaturated fatty acids (UFAs) are key compounds which have important roles in maintaining cell membrane physiological functions and the adaption to tough conditions. Defects of fatty acid desaturases will change cellular UFA constitution. Pichia pastoris GS115 has four fatty acid desaturase genes, namely FAD9A, FAD9B, FAD12 and FAD15. Their products catalyze the synthesis of three kinds of UFAs, oleic acid (catalyzed by Fad9A and Fad9B), linoleic acid (catalyzed by Fad12) and α-linolenic acid (catalyzed by Fad15), respectively. In this study, we found that deletion of FAD12 led to increased resistance to oxidative stress. Cellular lipid peroxidation levels declined in the fad12Δ mutant upon H2O2 treatment. Cellular fatty acids compositions were changed with the increased expression of FAD9A. On the other hand, deletion of FAD9A resulted in increased tolerance to the plasma membrane (PM) damage agent SDS, and PM deformation was not detected in the fad9AΔ mutant under this stress. Our results showed that UFAs are related to cell adaption to adverse environmental changes.

Convergent Regulation of Candida albicans Aft2 and Czf1 in Invasive and Opaque Filamentation.

Candida albicans is the most common fungal pathogen of mucosal infections and invasive diseases in immuno-compromised humans. The abilities of yeast-hyphal growth and white-opaque switching affect C. albicans physiology and virulence. Here, we showed that C. albicans Aft2 regulator was required for embedded filamentous growth and opaque cell-type formation. Under low-temperature matrix embedded conditions, Aft2 functioned downstream of Czf1-mediated pathway and was required for invasive filamentation. Moreover, deletion of AFT2 significantly reduced opaque cell-type formation under N-acetylglucosamine (GlcNAc) inducing conditions. Ectopic expression of CZF1 slightly increased the white-opaque switching frequency in the aft2Δ/Δ mutant, but did not completely restore to wild-type levels, suggesting that Czf1 at least partially bypassed the essential requirement for Aft2 in response to opaque-inducing cues. In addition, multiple environmental cues altered AFT2 mRNA and protein levels, such as low temperature, physical environment and GlcNAc. Although the absence of Czf1 or Efg1 also increased the expression level of AFT2 gene, deletion of CZF1 remarkably reduced the stability of Aft2 protein. Furthermore, C. albicans Aft2 physically interacted with Czf1 under all tested conditions, whereas the interaction between Aft2 and Efg1 was barely detectable under embedded conditions, supporting the hypothesis that Aft2, together with Czf1, contributed to activate filamentous growth by antagonizing Efg1-mediated repression under matrix-embedded conditions.

Novel mechanisms of surfactants against Candida albicans growth and morphogenesis.

Candida albicans is a common opportunistic fungal pathogen, causing not only superficial mucosal infections but also life-threatening systemic candidiasis in immune-compromised individuals. Surfactants are a kind of amphiphilic compounds implemented in a wide range of applications. Although their antimicrobial activity has been characterized, their effect on C. albicans physiology remains to be elucidated. In this study, we investigated the inhibitory effect of two representative surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), on C. albicans growth and morphogenesis. Both surfactants exhibited inhibitory effect on C. albicans growth. This effect was not attributed to plasma membrane (PM) damage, but was associated with mitochondrial dysfunction. Excitingly, the surfactants, especially CTAB, showed strong inhibitory effect on hyphal development (IC50=0.183 ppm for CTAB and 6.312 ppm for SDS) and biofilms (0.888 ppm for CTAB and 76.092 ppm for SDS). Actin staining and Hwp1-GFP localization further revealed that this inhibition is related to abnormal organization of actin skeleton and subsequent defect in polarized transport of hyphae-related factors. This study sheds a novel light on the antimicrobial mechanisms of surfactants, and suggests these agents as potential drugs against C. albicans hyphae-related infections in clinical practice.

Interaction among the vacuole, the mitochondria, and the oxidative stress response is governed by the transient receptor potential channel in Candida albicans.

Candida albicans is one of the most important opportunistic pathogens, causing both mucosal candidiasis and life-threatening systemic infections. To survive in the host immune defense system, this pathogen uses an elaborate signaling network to recognize and respond to oxidative stress, which is essential for its pathogenicity. However, the exact mechanisms that this fungus employs to integrate the oxidative stress response (OSR) with functions of various organelles remain uncharacterized. Our previous work implicated a connection between the calcium signaling system and the OSR. In this study, we find that the vacuolar transient receptor potential (TRP) channel Yvc1, one of the calcium signaling members, plays a critical role in cell tolerance to oxidative stress. We further provide evidence that this channel is required not only for activation of Cap1-related transcription of OSR genes but also for maintaining the stability of both the mitochondria and the vacuole in a potassium- and calcium-dependent manner. Element assays reveal that this TRP channel affects calcium influx and potassium transport from the vacuole to the mitochondria. Therefore, the TRP channel governs the novel interaction among the OSR, the vacuole, and the mitochondria by mediating ion transport in this pathogen under oxidative stress.

Role of TFP1 in vacuolar acidification, oxidative stress and filamentous development in Candida albicans.

The vacuolar-type H(+)-ATPase (V-ATPase) is a multiprotein complex consisting of the V0 and V1 sectors, and is required for vacuolar acidification and virulence in the opportunistic fungal pathogen Candida albicans. In this study, we identified C. albicans Tfp1 as a putative subunit of V-ATPase, and explored its importance in multiple cellular processes. Our results revealed that Tfp1 played an essential role in vacuolar acidification and endocytic trafficking. In addition, the tfp1Δ/Δ mutant was sensitive to alkaline pH and elevated calcium concentrations, which is characteristic of loss of V-ATPase activity. The mutant also showed hypersensitivity to metal ions which might be attributed to a defect in sequestration of toxic ions to the vacuole through proton gradient produced by V-ATPase. Interestingly, deletion of TFP1 triggered endogenous oxidative stress even without exogenous oxidants. Compared with the wild-type strain, the tfp1Δ/Δ mutant showed significantly higher ROS levels and lower expression levels of redox-related genes with the addition of hydrogen peroxide (H2O2). Western blotting analysis showed that deletion of TFP1 significantly reduced the expression of Cap1 under H2O2 treatment, which contributes to the regulation of genes involved in the oxidative stress response. Furthermore, the tfp1Δ/Δ mutant showed significantly impaired filamentous development in hyphal induction media, and was avirulent in a mouse model of systemic candidiasis. Taken together, our results suggested that the putative V1 subunit Tfp1 is essential for vacuolar function and C. albicans pathogenesis, and provided a promising candidate for antifungal drugs.

A novel role of the ferric reductase Cfl1 in cell wall integrity, mitochondrial function, and invasion to host cells in Candida albicans.

Candida albicans is an important opportunistic pathogen, causing both superficial mucosal infections and life-threatening systemic diseases. Iron acquisition is an important factor for pathogen-host interaction and also a significant element for the pathogenicity of this organism. Ferric reductases, which convert ferric iron into ferrous iron, are important components of the high-affinity iron uptake system. Sequence analyses have identified at least 17 putative ferric reductase genes in C. albicans genome. CFL1 was the first ferric reductase identified in C. albicans. However, little is known about its roles in C. albicans physiology and pathogenicity. In this study, we found that disruption of CFL1 led to hypersensitivity to chemical and physical cell wall stresses, activation of the cell wall integrity (CWI) pathway, abnormal cell wall composition, and enhanced secretion, indicating a defect in CWI in this mutant. Moreover, this mutant showed abnormal mitochondrial activity and morphology, suggesting a link between ferric reductases and mitochondrial function. In addition, this mutant displayed decreased ability of adhesion to both the polystyrene microplates and buccal epithelial cells and invasion of host epithelial cells. These findings revealed a novel role of C. albicans Cfl1 in maintenance of CWI, mitochondrial function, and interaction between this pathogen and the host.

Inhibitory effect of verapamil on Candida albicans hyphal development, adhesion and gastrointestinal colonization.

Candida albicans morphogenesis and gastrointestinal colonization are closely associated with the pathogenicity of this pathogen. This study investigated the in vitro and in vivo effect of verapamil, a calcium channel blocker, on these processes. Exposure to ≥ 10 µg mL(-1) verapamil led to a significant decrease of C. albicans hyphal cells. The ability to adhere to a polystyrene surface and buccal epithelial cells was inhibited by exposure to ≥ 20 µg mL(-1) verapamil. Detection of the Hwp1-green fluorescent protein fusion protein showed that verapamil inhibited expression and transport of Hwp1, indicating its activity against both the regulation network of morphogenesis-associated proteins and the secretory pathway in C. albicans. Moreover, treatment with verapamil at 10 mg (kg day)(-1) led to a remarkable decrease in gastrointestinal-colonizing fungal cells. This study revealed the inhibitory effect of verapamil on C. albicans hyphal development, adhesion and gastrointestinal colonization, which is relevant to decreased expression and abnormal transport of the proteins required for morphogenesis. Therefore, verapamil may be taken into account when choosing an antifungal therapy against C. albicans colonization and infection.

Novel role of the Candida albicans ferric reductase gene CFL1 in iron acquisition, oxidative stress tolerance, morphogenesis and virulence.

Ferric reductase catalyzes the reduction of ferric iron into ferrous iron and plays an essential role in high-affinity iron acquisition. In this study, we found that the cfl1Δ/Δ (orf19.1263) mutant was not defective in iron acquisition. However, deletion of CFL1 increased cellular iron accumulation by elevating surface ferric reductase activity in Candida albicans, revealing that there existed functional redundancy and/or a compensatory upregulation mechanism among ferric reductase genes. The absence of CFL1 resulted in increased expression levels of other alternative ferric reductase genes, including FRP1, CFL2 and FRE10. In addition, CFL1 played an important role in the response to different oxidative stresses. Further research revealed that the cfl1Δ/Δ mutant exhibited higher levels of both ROS production and SOD activity under oxidative conditions. Moreover, deletion of CFL1 led to a profound defect in filamentous development in an iron-independent manner at both 30 and 37 °C. The cfl1Δ/Δ mutant exhibited highly attenuated virulence and reduced fungal burdens in the mouse systemic infection model, indicating that CFL1 might be a potential target for antifungal drug development. In summary, our results provide new insights into the roles of ferric reductase gene in C. albicans.

The type II Ca2+/calmodulin-dependent protein kinases are involved in the regulation of cell wall integrity and oxidative stress response in Candida albicans.

The type II Ca(2+)/calmodulin-dependent protein kinases (CaMKs) are thought to play a vital role in cellular regulation in mammalian cells. Two genes CMK1 and CMK2 in the Candida albicans genome encode homologues of mammalian CaMKs. In this work, we constructed the cmk1Δ/Δ, the cmk2Δ/Δ and the cmk1Δ/Δcmk2Δ/Δ mutants and found that CaMKs function in cell wall integrity (CWI) and cellular redox regulation. Loss of either CMK1 or CMK2, or both resulted in increased expression of CWI-related genes under Calcofluor white (CFW) treatment. Besides, CaMKs are essential for the maintenance of cellular redox balance. Disruption of either CMK1 or CMK2, or both not only led to a significant increase of intracellular ROS levels, but also led to a decrease of the mitochondrial membrane potential (MMP), suggesting the important roles that CaMKs play in the maintenance of the mitochondrial function.

Cellular iron homeostasis mediated by the Mrs4-Ccc1-Smf3 pathway is essential for mitochondrial function, morphogenesis and virulence in Candida albicans.

Iron bioavailability is crucial for mitochondrial metabolism and biosynthesis. Dysregulation of cellular iron homeostasis affects multiple aspects of mitochondrial physiology and cellular processes. However, the intracellular iron trafficking pathway in Candida albicans remains unclear. In this study, we characterized the Mrs4-Ccc1-Smf3 pathway, and demonstrated its important role in maintaining cellular iron levels. Double deletion of vacuolar iron exporter SMF3 and mitochondrial iron transporter MRS4 further elevated cellular iron levels in comparison with the single MRS4 deletion. However, deletion of vacuolar iron importer CCC1 in the mrs4delta/delta mutant restored cellular iron homeostasis to normal wild-type levels, and also normalized most of the defective phenotypes in response to various environmental stresses. Our results also suggested that both Mrs4 and Cccl contributed to the maintenance of mitochondrial function. The mrs4delta/delta and mrs4delta/deltasmf3delta/delta mutants exhibited an obvious decrease in aconitase activities and mitochondrial membrane potential, whereas deletion of CCC1 in the mrs4delta/delta mutant effectively rescued these defects. Furthermore, we also found that the Mrs4-Ccc1-Smf3 pathway was indispensable for cell-wall stability, antifungal drug tolerance, filamentous growth and virulence, supporting the novel viewpoint that mitochondria might be the promising target for better antifungal therapies. Interestingly, the addition of exogenous iron failed to rescue the defects on non-fermentable carbon sources or hyphae-inducing medium, indicating that the defects in mitochondrial respiration and filamentous development might result from the disturbance of cellular iron homeostasis rather than environmental iron deprivation. Taken together, our results propose the Mrs4-Ccc1-Smf3 pathway as a potentially attractive target for antifungal drug development.

Ecm7, a regulator of HACS, functions in calcium homeostasis maintenance, oxidative stress response and hyphal development in Candida albicans.

Calcium is a universal messenger that translates diverse environmental stresses and developmental cues into specific cellular and developmental responses. In yeast, Cch1 and Mid1 function as part of a high affinity Ca²âº influx system (HACS) that becomes activated rapidly in response to sudden stimuli. Here, we report that Ecm7, a regulator of HACS, plays important roles in calcium homeostasis maintenance, oxidative stress response and hyphal development in Candida albicans. Disruption of ECM7 led to increased sensitivity to calcium-depleted conditions. Flow cytometry analysis revealed that Ecm7 mediated Ca²âº influx under high pH shock. Cycloheximide chase experiments further showed that MID1 deletion significantly decreased the stability of Ecm7. We also provided evidences that ecm7Δ/Δ cells were hypersensitive to oxidative stress. ECM7 deletion induced the degradation of Cap1 when exposed to H2O2 treatment. Besides, the ecm7Δ/Δ mutant showed a defect in hyphal development, which was accompanied with the decreased expression of hyphal related gene HWP1. Though subsequent experiments revealed that the ecm7Δ/Δ mutant showed similar virulence to the wild-type strain, the ability of invasion and diffusion of the mutant in mouse kidneys decreased. Taken together, Ecm7 plays important roles in the adaptation and pathogenicity of C. albicans.

The P-type ATPase Spf1 is required for endoplasmic reticulum functions and cell wall integrity in Candida albicans.

Endoplasmic reticulum (ER) is crucial for protein folding, glycosylation and secretion in eukaryotic organisms. These important functions are supported by high levels of Ca(2+) in the ER. We have recently identified a putative ER Ca(2+) pump in Candida albicans, called Spf1, which plays key roles in maintenance of cellular Ca(2+) homeostasis, morphogenesis and virulence. In this study, we purified Spf1 and confirmed that it is a P-type ATPase, suggesting its role in maintaining high levels of ER Ca(2+). Disruption of SPF1 caused severe defects in glycosylation of the ER-localized protein Cdc101 and secretory acid phosphatase, and a decrease in expression of SEC61 which encodes an important ER protein. Moreover, the spf1Δ/Δ mutant showed increased sensitivity to cell wall stresses, abnormal cell wall composition, delayed cell wall reconstruction and decreased flocculation and adherence, indicating its defect in cell wall integrity (CWI). We also revealed that disruption of SPF1 has an impact on gene expression related to CWI and morphogenesis. This study provides evidence that Spf1, as a P-type ATPase, is essential for ER functions and consequent CWI, implicating a role of ER Ca(2+) homeostasis in C. albicans physiology.

Aft2, a novel transcription regulator, is required for iron metabolism, oxidative stress, surface adhesion and hyphal development in Candida albicans.

Morphological transition and iron metabolism are closely relevant to Candida albicans pathogenicity and virulence. In our previous study, we demonstrated that C. albicans Aft2 plays an important role in ferric reductase activity and virulence. Here, we further explored the roles of C. albicans Aft2 in numerous cellular processes. We found that C. albicans Aft2 exhibited an important role in iron metabolism through bi-directional regulation effects on iron-regulon expression. Deletion of AFT2 reduced cellular iron accumulation under iron-deficient conditions. Furthermore, both reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity were remarkably increased in the aft2Δ/Δ mutant, which were thought to be responsible for the defective responses to oxidative stress. However, we found that over-expression of C. albicans AFT2 under the regulation of the strong PGK1 promoter could not effectively rescue Saccharomyces cerevisiae aft1Δ mutant defects in some cellular processes, such as cell-wall assembly, ion homeostasis and alkaline resistance, suggesting a possibility that C. albicans Aft2 weakened its functional role of regulating some cellular metabolism during the evolutionary process. Interestingly, deletion of AFT2 in C. albicans increased cell surface hydrophobicity, cell flocculation and the ability of adhesion to polystyrene surfaces. In addition, our results also revealed that C. albicans Aft2 played a dual role in regulating hypha-specific genes under solid and liquid hyphal inducing conditions. Deletion of AFT2 caused an impaired invasive growth in solid medium, but an increased filamentous aggregation and growth in liquid conditions. Moreover, iron deficiency and environmental cues induced nuclear import of Aft2, providing additional evidence for the roles of Aft2 in transcriptional regulation.

Novel insight into the expression and function of the multicopper oxidases in Candida albicans.

Iron is an essential element required for most organisms. The high-affinity iron-uptake systems in the opportunistic pathogen Candida albicans are activated under iron-limited conditions and are also required for virulence. Here one component of high-affinity iron-uptake systems, the multicopper oxidase (MCO) genes, was characterized. We examined the expression of five MCO genes and demonstrated that CaFET3 and CaFET34 were the major MCO genes in response to iron deficiency. Complementation of the Saccharomyces cerevisiae fet3Δ mutant showed that CaFET34 could effectively rescue the growth phenotype in iron-limited medium. Deletion of CaFET33 and CaFET34 in C. albicans decreased cellular iron content and iron acquisition during iron starvation. However, the fet33Δ/Δ and fet34Δ/Δ mutants exhibited no obvious growth defect in solid iron-limited medium while the fet34Δ/Δ mutant showed a slight growth defect in liquid medium. Further analysis shows that other members of the five MCO genes, especially CaFET3, would compensate for the absence of CaFET33 and CaFET34. Furthermore, for the first time, we provide evidence that CaFET34 is implicated in hyphal development in an iron-independent manner and is required for C. albicans virulence in a mouse model of systemic infection. Together, our results not only expand our understanding about the expression of the MCO genes in C. albicans, but also provide a novel insight into the role of CaFET34 in iron metabolism, hyphal development and virulence.

Identification and characterization of a novel C20-elongase gene from the marine microalgae, Pavlova viridis, and its use for the reconstitution of two pathways of long-chain polyunsatured fatty acids biosynthesis in Saccharomyces cerevisiae.

The marine microalga, Pavlova viridis, contains long-chain polyunsatured fatty acids including eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3). A full-length cDNA sequence, pvelo5, was isolated from P. viridis. From sequence alignment, the gene was homologous to fatty acyl elongases from other organisms. Heterologous expression of pvelo5 in Saccharomyces cerevisiae confirmed that it encoded a specific C20-elongase within the n-3 and n-6 pathways. Elongation activity was confined exclusively to EPA and arachidonic acid (20:4n-6). GC analysis indicated that pvelo5 could co-express with other genes for biosynthesis to reconstitute the Δ8 and Δ6 pathways. Real-time PCR results and fatty acid analysis demonstrated that long-chain polyunsatured fatty acids production by the Δ8 pathway might be more effective than that by the Δ6 pathway.

In vitro activity of verapamil alone and in combination with fluconazole or tunicamycin against Candida albicans biofilms.

Calcium channels and pumps play important roles in morphogenesis, stress response and virulence in Candida albicans. We hypothesised that verapamil, a potent calcium channel blocker, may display an inhibitory effect on C. albicans biofilms. To test this hypothesis, the in vitro activity of verapamil was evaluated alone and in combination with fluconazole or tunicamycin against C. albicans biofilms using a 96-well microtitre plate model. As expected, verapamil exerted inhibitory activity against C. albicans biofilms. The combinations of verapamil/fluconazole and verapamil/tunicamycin yielded synergistic effects on biofilm formation and on pre-formed biofilms. Furthermore, verapamil alone or in combination with fluconazole or tunicamycin led to a significant decrease in the transcription level of ALS3, essential for biofilm development. Therefore, verapamil may be a potential agent to enhance the effect of antifungal drugs against C. albicans biofilms.

Identification of a novel C22-∆4-producing docosahexaenoic acid (DHA) specific polyunsaturated fatty acid desaturase gene from Isochrysis galbana and its expression in Saccharomyces cerevisiae.

Isochrysis galbana, produces long chain polyunsaturated fatty acids including docosahexaenoic acid (DHA, 22:6n-3). A novel gene (IgFAD4-2), encoding a C22-∆4 polyunsaturated fatty acid specific desaturase, has been isolated and characterized from I. galbana. A full-length cDNA of 1,302 bp was cloned by LA-PCR technique. The IgFAD4-2 encoded a protein of 433 amino acids that shares 78 % identity with a previously reported ∆4-desaturase (IgFAD4-1) from I. galbana. The function of IgFAD4-2 was deduced by its heterologous expression in Saccharomyces cerevisiae, which then desaturated docosapentaenoic acid (DPA, 22:5n-3) to DHA. The conversion ratio of DPA to DHA was 34 %, which is higher than other ∆4-desaturases cloned from algae. However, IgFAD4-2 did not catalyze the desaturation or elongation reactions with other fatty acids. These results confirm that IgFAD4-2 has C22-∆4-PUFAs-specific desaturase activity.