Rim101-upregulated Fets contribute to dark pigment formation in gray cells of Candida albicans.

Candida albicans has long been known to switch between white and opaque phases; however, a third cell type, referred to as the 'gray' phenotype, was recently characterized. The three phenotypes have different colonial morphologies, with white cells forming white-colored colonies and opaque and gray cells forming dark-colored colonies. We previously showed that Wor1-upregulated ferroxidases (Fets) function as pigment multicopper oxidases that regulate the production of dark-pigmented melanin in opaque cells. In this study, we demonstrated that Fets also contributed to dark pigment formation in gray colonies but in a Wor1-independent manner. Deletion of both WOR1 and EFG1 locked cells in the gray phenotype in some rich media. However, the efg1/efg1 wor1/wor1 mutant could switch between white and gray in minimal media depending on the ambient pH. Specifically, mutant cells exhibited the white phenotype at pH 4.5 but switched to gray at pH 7.5. Consistent with phenotype switching, Fets expressions and melanin production were also regulated by ambient pH. Ectopic expression of the Rim101-405 allele in the mutant enabled the pH restriction to be bypassed and promoted gray cell formation in acidic media. Our data suggest that Rim101-upregulated Fets contribute to dark pigment formation in the gray cells.

Wor1-regulated ferroxidases contribute to pigment formation in opaque cells of Candida albicans.

Candida albicans is a harmless commensal resident in the human gut and a prevalent opportunistic pathogen. A key part of its commensalism and pathogenesis is its ability to switch between different morphological forms, including white-to-opaque switching. The Wor1 protein was previously identified as a master regulator of white-to-opaque switching in mating type locus (MTL) homozygous cells. The mechanisms by which the dark color of the opaque colonies is controlled and the pimpled surface of opaque cells is formed remain unknown. Candida albicans produces melanin pigment in vitro and during infection. However, the molecular mechanism underlying the regulation of melanin production is unclear. In this study, we demonstrated that ferroxidases (Fets) function as pigment multicopper oxidases and regulate the production of dark-pigmented melanin in opaque cells. The FET genes presented distinct regulation patterns in response to different extracellular stimuli. In YPD (1% yeast extract, 2% peptone and 2% dextrose)-rich medium, four of the five FET genes were up-regulated by Wor1, especially at the human body temperature of 37 °C. In minimal medium with low ammonium concentrations, all five FET genes were up-regulated by Wor1. However, at high ammonium concentrations, some FET genes were down-regulated by Wor1. Wor1-up-regulated Fets contributed to dark pigment formation in opaque colonies, but not to the elongated shape of these opaque cells. Increased melanin externalization was associated with the pimpled surface of the opaque cells. Melanized C. albicans cells were more resistant to fungal clearance. Deletion of the five FET genes completely blocked melanin production in opaque cells and resulted in the generation of white elongated 'opaque' cells. In addition, the up-regulated Fets are important for defense against oxidant attacks. The functional diversity of Fets may reflect the multiple strategies of C. albicans to rapidly adapt to diverse host niches.

Bre1 and Ubp8 regulate H2B mono-ubiquitination and the reversible yeast-hyphae transition in Candida albicans.

The reversible yeast-hyphae transition of the human fungal pathogen Candida albicans is tightly linked to its pathogenicity. In this study, we show that histone H2B mono-ubiquitination (H2Bub) at lysine 123 was maintained at a low level in the yeast state, whereas it increased significantly during yeast-to-hyphae transition and decreased when hyphae converted to yeast. The increased H2Bub level is correlated with activation of the hyphal program. H2B ubiquitination and deubiquitination are dynamically regulated by the E3 ligase Bre1 and the deubiquitinase Ubp8 during the reversible yeast-hyphae transition. The functions of Bre1 and Ubp8 in hypha-specific gene (HSG) regulation appears to be direct because both are recruited to the coding regions of HSGs during hyphal induction. The sequential recruitment of Bre1 and Ubp8 to HSGs coding regions is important for the initiation and maintenance of HSG expression. Additionally, Ubp8 contributes to the pathogenicity of C. albicans during early infection in a mouse model. Our study is the first to link H2B ubiquitination to the morphological plasticity and pathogenicity of the human fungal pathogen C. albicans and shed light on potential antifungal treatments.

Fabrication and data harvesting of casting sample of rat liver blood vessels.

BACKGROUND: The configuration and course of liver blood vessels (LBVs) are involved in the study of pathogenesis of hepatic diseases including liver cirrhosis, tissue engineering of the liver and surgical treatment of diseases of the liver and gallbladder. In the study of vascularization in tissue engineering of the liver in particular, the work we should do is to get the anatomy data of LBVs for computer-aided reconstruction of digital model of LBVs. In doing so, the casting sample of rat liver blood vessels (RLBVs) is fabricated and the data of each section of the sample is harvested. METHODS: Liquid polymer preparation (8%-10%), which was made of chlorinated poly vinyl chloride (CPVC) as a solute, acetone as solvent and pigment, was injected into the RLBVs of 40 rats. Once acetone evaporated, the preparation solidified. When the cells and connective tissue were dissolved by hydrochloric acid, a casting sample of RLBV was left. The sample was embedded in paraffin and cut into sections. The data of each section of RLBVs was collected by digital camera. RESULTS: In 36 rats, the casting sample of RLBVs was made successfully by this method. The diameter of the hepatic arteries varied from 0.8 to 0.2 mm, the portal veins from 2.0 to 0.1 mm, and the hepatic veins from 2.2 to 0.2 mm. In each rat, about 150 photographs of the sections of RLBVs were taken. CONCLUSION: The method described above is feasible for getting experimental data for computer-aided reconstruction of the digital model of RLBVs.