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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.

[Characterization of Candida albicans ferric reductase genes in response to environmental stresses].

OBJECTIVE: Ferric reductases play a central role in iron acquisition and mobilization in C. albicans. This study focuses on stress response strategies exhibited by several ferric reductase genes through function and expression analyses. METHODS: Northern blot analysis was used to examine ferric reductase genes expression levels in different iron deficiency. We constructed ferric reductase-null mutants by a PCR-based homologous recombination, and examined the effects of gene deletion on cell-surface ferric reductase activity and growth ability under different conditions. Sub-cellular localization of Frpl-GFP fusion was imaged and analyzed by confocal laser scanning fluorescence microscopy. RESULTS: FRE10 was highly expressed at acidic pH, compared to that at alkaline pH, whereas the expression of FRE2 was just the opposite. Deletion of FRE10 resulted in a significant decreased surface reductase activity at acidic pH, with 75.5% down-regulation compared to wild-type levels. The fre2delta/delta mutant showed significantly attenuated growth ability and cell-surface ferric reductase activity at alkaline pH. Sub-cellular localization revealed that the green fluorescence was accumulated in the vacuoles. CONCLUSION: The expression of both FRE10 and FRE2 is induced in a pH-dependent manner. FRE2 encodes a major cell surface ferric reductase under alkaline pH condition. Frp1 localizes to the vacuole, and might support mobilization and transport of vacuolar ferric iron stores.

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.

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.

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.

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.

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.