| Candida albicans, the most common opportunistic human fungal pathogen, has both the benign and pathogenic association with its mammalian hosts. It typically exists as part of the mammalian commensal flora, occupying mucocutaneous surfaces such as skin, oral cavity, genitourinary tract and gastrointestinal tract. However, it also causes superficial mucosal infection as well as life-threatening systemic diseases in immuno-compromised individuals, including organ transplant recipients, cancer patients and people with AIDS. The rapid deveoplment of C. albicans comparative and functional genomics makes a significant contribution to our understanding of the pathogenicity and antifungal-drug resistance mechanisms of this important human pathogen. Several virulence determinants and activities that contribute to C. albicans infection have been extensively studied, including the yeast-hyphae transition, the white-opaque transition, adherence and invasion to host surfaces, secretion of hydrolases, biofilm formation, cell-wall integrity maintenance, pH regulation, contact sensing and environmental stress response. Among these virulence factors, multiple cellular processes were closely associated iron homeostasis.Iron is an essential nutrient with limited bioavailability that is required for the growth and metabolism in most organisms, including C. albicans. On the one hand, the strategies for iron exploitation within the host are important for microbial survival, colonization and infection in the hostile environments. On the other hand, excess iron is potentially toxic because of the formation of highly oxygen radicals by the Fenton reaction. Therefore, C. albicans has developed sophisticated strategies to control celluar iron homestasis, including iron acquisition, transport, storage and regulation, which is closely associated with the adaptation to the fluctuations of iron bioavailability. In recent years, our understanding of the mechanisms that C. albicans exploits iron from the host environment has significantly increased. Although many genes involved iron acquisition have been widely elaborated in C. albicans, the transporters involved in intracellular iron trafficking have not been characterized in detail. In addition, our previous study identified a C. albicans Aft-type functional homologue Aft2, but the underlying mechanisms remain unclear. In this study, we identified an intracellular iron transport system, explored the regulatory roles of Aft2transcription factor, and further investigated the effects on stress responses and morphogenesis in C. albicans. The main results were demonstrated as follows.(1) By bioinformatics and molecular cell biology techniques, we identified Mrs4as a mitochondrial transporter, Ccc1and Smf3as vacuolar transporters. C. albicans Mrs4was a new member of mitochondrial carrier family, and required for mitochondrial morphology and iron trafficking. C. albicans Cccl was an iron transporter that increased vacuolar iron stores at the expense of cytosolic iron, whereas C. albicans Smf3was responsible for the mobilization of vacuolar iron stores. In conclusion, these results suggest that C. albicans Mrs4, Cccl and Smf3form a pathway to modulate intracellular iron distribution, which is crucial for the maintenance of cellular iron homeostasis.(2) By PCR-mediated homologous recombination, we constructed mrs4Δ/Δ, ccc1Δ/Δ, smf3Δ/Δ single mutant strains, and mrs4Δ/Δ.smf3Δ/Δ, mrs4Δ/Δccc1Δ/Δ double mutant strains. Deletion of MRS4increased cellular iron content by altering the expression levels of genes involved in iron homoeostasis. Many iron regulon genes implicated in high-affinity iron uptake system were greatly induced in the mrs4Δ/Δ mutant. However, deletion of MRS4reduced the expression of some genes involved in mitochondrial iron utilization. Deletion of SMF3led to a further increase in cellular iron content of the mrs4Δ/Δ mutant, while deletion of CCC1obtained the capacity to prevent the accumulation of excess iron in the mrs4Δ/Δ mutant. In addition, the Mrs4-Ccc1-Smf3(MCS) pathway was essential for numerous cellular processes, including fitness, environmetal iron response, metal ion homeostasis, oxidative response, cell-wall stability, antifungal-drug susceptibility, mitochondrial respiration, iron-sulfur cluster formation, mitochondrial DNA (mtDNA) stability, mitochondrial membrane potential maintenance, filamentous development, adhesion and virulence. These results support a simple model for the MCS pathway:The MCS pathway is implicated in cellular iron homeostasis and distribution, in which Mrs4belongs to the mitochondrial carrier family and Cccl/Smf3are putative vacuolar iron transporters. Simultaneous deletion of MRS4and SMF3further exacerbates cellular iron accumulation in the absence of MRS4, whereas deletion of CCC1in the mrs4Δ/Δ mutant restores iron homeostasis to normal wild-type level. The disturbance of cellular iron levels results in mitochondrial dysfunction, and subsequently, has a profound influence on some vital cellular processes, including cell-wall stability, drug tolerance, stress response and morphogenesis. The model also suggests that cellular iron homeostasis, mitochondrial function and filamentous development are closely related, and all these processes contribute to fungal pathogenesis. Moreover, the MCS pathway might serve as an attractive target for future antifungal therapies.(3) C. albicans Aft2plays an important role in the regulation of iron metabolism. By atomic absorption spectroscopy, the results indicated that deletion of AFT2reduced cellular iron accumulation under iron-deficient conditions, suggesting a potential role of AFT2in iron metabolism. Real-time PCR data showed that C. albicans Aft2exhibited bi-directional regulation effects, acting as both a negative regulator to govern the expression of some iron-regulon genes (SIT1, MRS4, SMF3, HAP43), and a positive regulator to induce the expression of some other iron-related genes (FRP1, CFL1, FET3, FET34, FTR1). Through site-specific mutation and promoter-LacZ reporter system, the results showed that Aft2factor functioned as a negative regulator of MRS4expression through the conserved core CACCC Aft-type sequence in a gene dose-dependent fashion. Further research revelaed that iron deficiency induced nuclear import of Aft2, providing additional evidence for the role of Aft2in transcriptional regulation.(4) In addition to iron metabolism, C. albicans Aft2was also associated with multiple cellular processes, including environmental stress response and phenotypic switching. Phenotypic analyses revealed that deletion of AFT2resulted in sensitivity to H2O2treatment in an iron-independent fashion. Both reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity were remarkably increased in the aft2Δ/Δ mutant, implying that the growth defects under oxidative stress conditions was caused by increased accumulation of cellular ROS that could not be eliminated efficiently by antioxidant enzymes. Moreover, deletion of AFT2affected surface adhesion properties, leading to increased cell surface hydrophobicity, cell flocculation and adhesion to polystyrene surfaces. Hyphal inducing assays indicated that there might be two distinct mechanisms by which Aft2participated in the response to various morphogenetic signals. C. albicans Aft2functioned as an activator to regulate hypha-specific genes in solid inducing medium, whereas, it switched to a repressor by reducing the expression of hypha-specific genes in liquid inducing conditions. Furthermore, morphogenetic signals elevated Aft2mRNA and protein expression levels, and induced the accumulation of Aft2transcription factor in the nucleus, supporting the conclusion that Aft2plays an important role in filamentous development by regulating the expression of hypha-specific genes. In addition, we demonstrated that C. albicans Aft2was required for embedded filamentous growth and opaque cell-type formation. Under matrix-embedded conditions, Aft2functioned downstream of Czf1-mediated pathway and was required for invasive filamentation. Co-immunoprecipitation experiments revealed that Aft2physically interacted with Czfl under all tested conditions, whereas the interaction between Aft2and Efgl was barely detectable under embedded conditions, supporting the hypothesis that Aft2, together with Czf1, contributed to activate invasive filamentous growth by antagonizing Efgl-mediated repression. However, in the presence of GlcNAc inducer, Aft2and Czfl might operate through two distinct signaling and ectopic expression of Czfl partially bypass the requirement for Aft2in white-opaque switching.In this study, we characterized the MCS pathway and Aft2regulator as important virulence attributes, and investigated the potential roles of these two strategies in C. albicans physiology. Taken together, our results propose that both the MCS pathway and Aft2regulator might be potentially attractive targets for better antifungal therapies and drug development. |
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