Functional Analysis Of Ofil And High-Affinity Iron Permeases In Candida Albicans

Candida albicans is a harmless commensal and can be found in the oral cavity, digestive tract and genital region of healthy people. It is an opportunistic fungal pathogen and has the ability to live in harmony with the host or a lifetime. It can also cause candidiasis including superficial infections and life-threatening systemic disease when the host immunity is compromised or the host ecological environment is disordered. The frequency of Candida infections has grown steadily during the past decade. This is due, at least in part, to an increase of use of modern medical technology such as invasive surgical techniques, prosthetic devices, intravascular catheters, and the development of new drug therapies. The most striking biological feature of C. albicans is which makes it a successful opportunistic fungal pathogen is its phenotypic plasticity. C. albicans can grow in several morphological forms including unicellular yeast-form, elongated hyphae and pseudohyphae; yeast -formed white cell or bean-shaped opaque cell. Under certain condition, this organism can undergo morphological transitions in response to various environmental cues. There are two typical morphogenetic switch systems:yeast-hypal switching system and white-opaque transition system. The morphogenetic switch is reversible and has been widely thought to be associated with the ability of C. albicans switching from commensal to pathogenic phase. The process of morphogenetic switch is complex and it has significance to understand the molecular mechanism involved in the phenotype switching for providing theoretical basis for the candidiasis control in future.Transcription factors are central to the regulation of gene expression in eukaryotic organisms. In the human fungal pathogen Candida albicans, zinc-finger transcription factors are involved in a variety of biological processes including filamentation, white-opaque switching, biofilm formation, virulence, antifungal resistance, and sexual reproductionln this study, we identified the role of opaque and filamentation inducer 1 (Ofil), a previously uncharacterized zinc-finger-containing protein encoded by the gene orf19.4972, in the regulation of white-opaque switching and filamentous growth. Moreover, we determined the function of high-affinity iron permeases, Ftrl and Ftr2 in cell growth and filamentation in the Candida albicans. The main results are shown as following:1. Analysis of molecular characteristics of the OFI1 gene and its expression characteristics.Nucleotides sequence analysis shows that ORF of OFI1 gene encodes 648 amino acid residues. The predicted molecular weight is 71262 Da and isoelectric point (pI) is 9.41. The putative protein sequence of Ofi1 contains three C2H2 type zinc-finger DNA-binding motifs (ZnF_ C2H2, aa 97-119, aa 125-147, and aa 153-173), which indicates that Ofi1 can potentially bind to DNA sequences. We analysised several global gene expression profile data including Microarray assay and RNAseq assay.The results indicate that the relative expression level of OFI1 was much higher in opaque cells than in white cells. We further confirmed the expression difference between white cell and opaque cell by RT-qPCR. The expression level of OFI1 in opaque cell is 120 folds higher than in white cell.2. Role of Ofi1 in white-opaque switching in C. albicansWe firstly constructed an OFI1 overexpression plasmid, pACT1-OFI1, by inserting the ORF region of the OFI1 gene into plasmid pACT1, and then make OFI1 over-expression stain by transformed the plasmid into strain WUM5A, a white-to-opaque switching competent cell. In the overexpression strain OFI1 is constitutively expressed under the promoter of ACT1 theoretically. The expression levels of OFI1 in the overexpression strains were examined using quantitative RT-qPCR analysis.The expression level of OFI1 in white cells of the overexpression strain was about 15 folds higher than in white cells of the control. This result indicates that OFI1 was successfully expressed in the overexpression strain. Of note, the expression level of OFI1 in opaque cells of the control was much higher than in white cells. We further analyzed the effect of the overexpression of OFI1 on the switching frequency. The results of White-opaque switching assays showed that Over-expression of OFI1 induced white-to-opaque switching in C. albicans under all conditions tested. The white-to-opaque switching in OFI1 overexpression is higher over 10 folds than that of control strain on Lee’s Glucose plate. Notably, on Lee’s GlcNAc medium plates, the white-opaque switching frequency of the control was 61.1%±0.6%, while overexpression of OFI1 increased the switching frequency to 100.0%±0.0%. However, overexpression of OFIl had no notable effect on the opaque-to-white transition on Lee’s Glucose plate. Futhermore, we propose that Ofil functions downstream of Worl, the master regulator of white-opaque switching. We over-expressed OFI1 in the WORI mutant strain. However, over-expression of OF11 in the worl-/- mutant could not induce the opaque phenotype, suggesting that Ofil does not work alone and other transcription factors downstream of Worl are also involved in this regulation. These results also suggest that the master regulator Worl, but not Ofi1, is essential for the opaque phenotype.Since the expression of OFI1 is enriched in opaque cells, we then examined the effect of deletion of the OFI1 gene on white-opaque switching. Because MTL heterozygous derivatives of the laboratory strain SC5314 are incompetent for white-to-opaque switching, the MTLa locus was disrupted in the ofil/ofil mutant with plasmid L23.14. We further reconstituted OFI1 gene in the ofil/ofi1 mutant using the fusion PCR strategy. When cultured on Lee’s glucose medium plates, the white-to-opaque and opaque-to-white switching frequencies in the ofil/ofil mutant were comparable to those of the control and the OFI1 reconstituted strain. On Lee’s GlcNAc medium plates, the white-to-opaque switching frequencies of all these three strains were extremely low, while the ofil/ofil mutant exhibited a relatively lower white-to-opaque switching frequency than the control and the OFI1 reconstituted strain. These results are consistent with the overexpression results. As previously mentioned, Worl can bind to the promoter of OFI1. Therefore, Ofil could function as an inducer of the opaque phenotype downstream of the Worl master regulator.3. Role of Ofi1 in filamentation and invasive growth in C. albicansFilamentation is another striking feature of the pathogenic yeast C. albicans. Some of these transcription factors are involved in the regulation of both filamentation and white-opaque switching. We were interested in whether Ofil regulates filamentation in C. albicans. To study the effect of the deletion of OFI1 on filamentation, the ofil/ofil mutant, control, and OFI1 reconstituted strain underwent filamentous growth at similar levels on plates with three different media (Lee’s glucose medium, Lee’s G1cNAc medium, and Spider medium) respectively at 25℃,30℃ or 37℃. The deletion of OFI1 did not obviously affect filmentation under the conditions tested. However, the over-expression of OFIl promoted filamentous growth on Lee’s glucose medium, Lee’s G1cNAc medium, and Spider medium at 25℃. At 30℃, OFI1 overexpression promoted filamentous growth on Lee’s glucose medium and and Spider medium. But on Lee’s G1cNAc medium, all strains underwent strong filamentous growth. At 37℃, all of the strains underwent filamentation. Therefore, it was difficult to discriminate the robustness of filamentation at high temperatures (37℃).In worl-/- mutant of C. albicans, overexpression of OFI1 verexpression of OFI1 did not promote opaque cell formation, but induced filamentous growth. It indicated that unlike the role in regulating white-to-opaque switching, Ofil can promote filamentous growth in other different pathway which is independent of Worl. The capacity for invasive growth is required for C. albicans to penetrate host tissues to initiate infections. Because filamentation is strongly associated with this ability, we next tested whether Ofil regulated invasive growth in C. albicans. Cells were cultured on Spider medium for three days and washed with ddH2O. At 25℃, overexpression of OFI1 in a WT strain clearly enhanced the invasive growth capacity of the fungus. Consistently, spots of the overexpression strain were more wrinkled than those of the control at 25℃, which indicated that the overexpression strain had undergone more robust filamentation. At 30℃, both the overexpression strain and the WT control underwent strong invasive growth. None of the spots could be removed by washing.Given the importance of Ofil in the regulation of white-opaque switching and filamentation, the current study establishes a new link between these two processes.4. Role of high-Affinity Iron Permeases, Ftrl and Ftr2, in growth and filamentation in C.albicansIron is a critical micronutrient required by almost all the organisms. C.albicans can thrive in either iron-enriched biological niches such as mammalian gastrointestinal tract or iron-deficient environment such as bloodstream. FTR1 and FTR2 encode two high-affinity iron permeases consisting of 381 and 382 amino acids, respectively, sharing 83% identity with each other and contain multiple putative transmembrane domains and potential iron binding sites. Ftrl and Ftr2 have different biology characteristic.FTR1 expression was induced under iron-limited conditions and repressed when iron supply was sufficient, whereas the expression of FTR2 was regulated in a reversed manner. Mutants lacking FTR1 but not FTR2 exhibited a severe growth defect in iron-deficient medium and were unable to establish systemic infection in mice. Thus, FTR1-mediated iron-uptake mechanism constitutes a virulence factor of Candida albicans. But little is known about the role of the two high-affinity iron permeases in filamentation in Candida albicans. To determine the function of high-affinity iron permeases, Ftrl and Ftr2, we investigated cell growth and filamentation ability of the ftrl/ftr1,ftr2/ftr2, and ftrl1 ftrl ftr2/ftr2 mutants under different culture conditions. Cells of the wild type and mutants were cultured on different solid media at different temperatures. Cell growth and filamention were observed. The results showed that deletion of either one of the FTR genes had no effect on the growth under all conditions tested. Deletion of both FTR1 and FTR1 led to obvious growth defect on Spider media, although addition of FeCl3 restored their growth ability. The double mutant also grew much more slowly on nutrients-limited synthetic media such as Lee’s glucose and Lee’s GlcNAc. Moreover, deletion of FTR1 enhanced filamentation, while deletion of FTR2 weakened this ability. Deletion of both FTRl and FTR2 recovered the ability of filamentation. These results indicate that Ftrl plays a negative, while Ftr2 plays a positive role in the regulation of filamentation in C.albicans.Ftrl and Ftr2 are very important for C. albicans growth under iron-limited condition and may participate in the utilization of some carbon sources including GlcNAc, ethnol, and glycerol.