The rod cell, a small form of Candida albicans, possesses superior fitness to the host gut and adaptation to commensalism.

Candida albicans deploys various morphological forms through complex switching mechanisms, ensuring its survival and thriving as a commensal or pathogen in vastly different human niches. In this study, we demonstrate that a novel ''rod'' morphological form of C. albicans coexists and is interchangeable with previously reported white, gray, and opaque forms, constituting a tetra-stable phenotypic switching system. Rod cells arise from the efg1 mutant of SC5314 cells or from the clinical BJ1097 strain cultured under glucose-free conditions. They are characterized by a distinct gene expression profile and can be stably maintained through in vitro passaging or in vivo inhabitation of the gastrointestinal (GI) tract of mice. Remarkably, the majority of the efg1 mutant cells become rod cells in N-acetylglucosamine (GlcNAc)-containing medium, and the GlcNAc sensor Ngs1 is instrumental in converting the white or gray cells to the rod cells. Conversely, glucose inhibits rod cells through Cph1; consequently, the loss of Cph1 in the efg1 mutant cells permits their conversion to rod cells in glucose-replete media. Notably, rod cells of the efg1/ cph1 mutant display superior adaptation and longer persistence in the murine GI environment than wild-type white cells. Taken together, these findings establish rod cells as a previously unappreciated form that is not only morphologically and transcriptionally distinguishable but also defined by specific genetic and environmental determinants, shedding light on complex fungus-host interactions.

Ino80 is required for H2A.Z eviction from hypha-specific promoters and hyphal development of Candida albicans.

ATP-dependent chromatin remodeling complexes play important roles in many essential cellular processes, including transcription regulation, DNA replication, and repair. Evicting H2A.Z, a variant of histone H2A, from the promoter of hypha-specific genes is required for hyphal formation in Candida albicans. However, the mechanism that regulates H2A.Z removal during hyphal formation remains unknown. In this study, we demonstrated that Ino80, the core catalytic subunit of the INO80 complex, was recruited to hypha-specific promoters during hyphal induction in Arp8 dependent manner and facilitated the removal of H2A.Z. Deleting INO80 or mutating the ATPase site of Ino80 impairs the expression of hypha-specific genes (HSGs) and hyphal development. In addition, we showed that Ino80 was essential for the virulence of C. albicans during systemic infections in mice. Interestingly, Arp5, an INO80 complex-specific component, acts in concert with Ino80 during DNA damage responses but is dispensable for hyphal induction. Our findings clarified that Ino80 was critical for hyphal development, DNA damage response, and pathogenesis in C. albicans.

Fun30 nucleosome remodeller regulates white-to-opaque switching in Candida albicans.

Candida albicans ( C. albicans) is an opportunistic pathogen in humans and possesses a white-opaque heritable switching system. Wor1 is a master regulator of white-opaque switching and is essential for opaque cell formation in C. albicans. However, the regulatory network of Wor1 in white-opaque switching is still vague. In this study, we obtain a series of Wor1-interacting proteins using LexA-Wor1 as bait. Among these proteins, function unknown now 30 (Fun30) interacts with Wor1 in vitro and in vivo. Fun30 expression is upregulated in opaque cells at the transcriptional and protein levels. Loss of FUN30 attenuates white-to-opaque switching, while ectopic expression of FUN30 significantly increases white-to-opaque switching in an ATPase activity-dependent manner. Furthermore, FUN30 upregulation is dependent on CO 2; loss of FLO8, a key CO 2-sensing transcriptional regulator, abolishes FUN30 upregulation. Interestingly, deletion of FUN30 affects the WOR1 expression regulation feedback loop. Thus, our results indicate that the chromatin remodeller Fun30 interacts with Wor1 and is required for WOR1 expression and opaque cell formation.

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.

A ketogenic diet enhances fluconazole efficacy in murine models of systemic fungal infection.

Invasive fungal infections are a significant public health concern, with mortality rates ranging from 20% to 85% despite current treatments. Therefore, we examined whether a ketogenic diet could serve as a successful treatment intervention in murine models of Cryptococcus neoformans and Candida albicans infection in combination with fluconazole-a low-cost, readily available antifungal therapy. The ketogenic diet is a high-fat, low-carbohydrate diet that promotes fatty acid oxidation as an alternative to glycolysis through the production of ketone bodies. In this series of experiments, mice fed a ketogenic diet prior to infection with C. neoformans and treated with fluconazole had a significant decrease in fungal burden in both the brain (mean 2.66 ± 0.289 log10 reduction) and lung (mean 1.72 ± 0.399 log10 reduction) compared to fluconazole treatment on a conventional diet. During C. albicans infection, kidney fungal burden of mice in the keto-fluconazole combination group was significantly decreased compared to fluconazole alone (2.37 ± 0.770 log10-reduction). Along with higher concentrations of fluconazole in the plasma and brain tissue, fluconazole efficacy was maximized at a significantly lower concentration on a keto diet compared to a conventional diet, indicating a dramatic effect on fluconazole pharmacodynamics. Our findings indicate that a ketogenic diet potentiates the effect of fluconazole at multiple body sites during both C. neoformans and C. albicans infection and could have practical and promising treatment implications.IMPORTANCEInvasive fungal infections cause over 2.5 million deaths per year around the world. Treatments for fungal infections are limited, and there is a significant need to develop strategies to enhance antifungal efficacy, combat antifungal resistance, and mitigate treatment side effects. We determined that a high-fat, low-carbohydrate ketogenic diet significantly potentiated the therapeutic effect of fluconazole, which resulted in a substantial decrease in tissue fungal burden of both C. neoformans and C. albicans in experimental animal models. We believe this work is the first of its kind to demonstrate that diet can dramatically influence the treatment of fungal infections. These results highlight a novel strategy of antifungal drug enhancement and emphasize the need for future investigation into dietary effects on antifungal drug activity.

ECM17-dependent methionine/cysteine biosynthesis contributes to biofilm formation in Candida albicans.

Candida albicans has become the fourth leading pathogen of nosocomial bloodstream infections largely due to biofilm formation on implanted medical devices. Previous microarray data indicated that almost all genes in methionine (Met)/cysteine (Cys) biosynthesis pathway were up-regulated during biofilm formation, especially during the adherence period. In this work, we studied the role of Met/Cys biosynthesis pathway by disrupting ECM17, a gene encoding sulfite reductase in C. albicans. It was found that the ecm17Δ/Δ mutant failed to catalyze the biochemical reaction from sulfite to H(2)S and hardly grew in media lacking Met and Cys. NaSH, the donor of H(2)S, dose-dependently improved the growth of ecm17Δ/Δ in media lacking a sulfur source. Sufficient Met/Cys supply inhibited the expression of ECM17 in a dose-dependent manner. These results validated the important role of ECM17 in Met/Cys biosynthesis. Interestingly, the ecm17Δ/Δ mutant showed diminished ability to form biofilm, attenuated adhesion on abiotic substrate and decreased filamentation on solid SLD medium, especially under conditions lacking Met/Cys. Further results indicated that ECM17 affected the expressions of ALS3, CSH1, HWP1 and ECE1, and that the cAMP-protein kinase A (PKA) pathway was associated with ECM17 and Met/Cys biosynthesis pathway. These results provide new insights into the role of Met/Cys biosynthesis pathway in regulating cAMP-PKA pathway and benefiting biofilm formation.

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.

Transcriptional response of Candida albicans biofilms following exposure to 2-amino-nonyl-6-methoxyl-tetralin muriate.

AIM: To identify changes in the gene expression profile of Candida albicans (C albicans) biofilms following exposed to 2-amino-nonyl-6-methoxyl-tetralin muriate(10b) and clarify the mechanism of 10b against C albicans biofilms. METHODS: Anti-biofilm activity of 10b was assessed by tetrazolium (XTT) reduction assay and the action mechanism against biofilms was investigated by cDNA microarray analysis and real-time RT-PCR assay. RESULTS: Ten differentially expressed genes were directly linked to biofilm formation and filamentous or hyphal growth (eg, NRG1, ECE1 and CSA1). Decreased gene expression was involved in glycolysis (eg, HXK2 and PFK1) and antioxidant defense (eg, SOD5), while increased gene expression was associated with enzymes that specifically hydrolyzed beta-1,3 glucan (XOG1), and with lipid, fatty acid and sterol metabolism (eg, SLD1, ERG6 and ERG2). Functional analysis indicated that addition of anti-oxidant ascorbic acid reduced inhibitory efficiency of 10b on mature biofilm. CONCLUSION: Inhibition of 10b on biofilm formation possibly depends on impairing the ability of C albicans to change its morphology via altering the expression of biofilm formation genes. Mitochondrial aerobic respiration shift and endogenous ROS augmentation might be a major contribution to reduce mature biofilm metabolic activity. The data may be useful for the development of new strategies to reduce the incidence of device-associated infections.

Proteomic analysis reveals a synergistic mechanism of fluconazole and berberine against fluconazole-resistant Candida albicans: endogenous ROS augmentation.

Our previous study showed that concomitant use of berberine (BBR) and fluconazole (FLC) provided a synergistic action against FLC-resistant Candida albicans (C. albicans) clinical strains in vitro. To clarify the mechanism underlying this action, we performed a comparative proteomic study in untreated control cells and cells treated with FLC and/or BBR in 2 clinical strains of C. albicans resistant to FLC. Our analyses identified 16 differentially expressed proteins, most of which were related to energy metabolisms (e.g., Gap1, Adh1, and Aco1). Functional analyses revealed that FLC + BBR treatment increased mitochondrial membrane potential, decreased intracellular ATP level, inhibited ATP-synthase activity, and increased generation of endogenous reactive oxygen species (ROS) in FLC-resistant strains. In addition, checkerboard microdilution assay showed that addition of antioxidant ascorbic acid or reduced glutathione reduced the synergistic antifungal activity of FLC + BBR significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the synergistic action of FLC + BBR against FLC-resistant C. albicans.

Candida albicans cells lacking CaMCA1-encoded metacaspase show resistance to oxidative stress-induced death and change in energy metabolism.

Candida albicans, an opportunistic pathogen, can undergo programmed cell death upon various stimuli, including oxidative stress. In this study, we showed that deletion of CaMCA1, a homologue of Saccharomyces cerevisiae metacaspase YCA1, could both attenuated oxidative stress-induced cell death and caspase activation. Compared to wild-type strain, Camca1Delta mutant showed higher accumulation of trehalose and transcription of the genes related to trehalose biosynthesis (TPS2 and TPS3) under the condition of oxidative stress. Furthermore, lower intracellular ATP concentration and mitochondrial membrane potential, less endogenous reactive oxygen species (ROS) generation were observed in Camca1Delta mutant. Our results suggest that CaMCA1 might mediate the sensitiveness to oxidative stress by affecting energy metabolism in C. albicans.

2-Amino-nonyl-6-methoxyl-tetralin muriate inhibits sterol C-14 reductase in the ergosterol biosynthetic pathway.

AIM: To investigate the action mechanism of a novel chemical structural aminotetralin derivate, 2-Amino-Nonyl-6-Methoxyl-Tetralin Muriate (10b), against Candida albicans (C albicans) in the ergosterol biosynthetic pathway. METHODS: Antifungal susceptibility test of 10b was carried out using broth microdilution method, the action mechanism of 10b against C albicans was investigated by GC-MS spectrometry and real-time RT-PCR assay, and cytotoxicity of 10b in vitro was assessed by MTS/PMS reduction assay. RESULTS: 10b reduced the ergosterol content markedly, and the 50% ergosterol content inhibitory concentration (ECIC(50) value) was 0.08 microg/mL. Although the sterol composition of 10b-grown cells was completely identical with that of erg24 strain, the content of ergosta-8,14,22-trienol in 10b-grown cells was much higher than that in erg24 strain. Real-time RT-PCR assay revealed a global upregulation of sterol metabolism genes. In addition, the 50% inhibitory concentration (IC(50) value) of 10b was 11.30 microg/mL for murine embryonic fibroblasts and 35.70 microg/mL for human normal liver cells. CONCLUSION: 10b possessed a mode of action different from that of azoles and morpholines, whose targets were sterol C-14 reductase (encoded by ERG24 gene) and sterol C-5 desaturase (encoded by ERG3) related enzyme. Although 10b seemed to reduce MTS/PMS reduction in a dose dependent manner, IC(50) value for mammalian cells was much higher than 50% minimum inhibitory concentration (MIC(50)) value for C albicans. This indicates that the formulation is preliminarily safe and warrants further study for possible human applications.

Baicalein induces programmed cell death in Candida albicans.

Recent evidence has revealed the occurrence of an apoptotic phenotype in Candida albicans that is inducible with environmental stresses such as acetic acid, hydrogen peroxide, and amphotericin B. In the present study, we found that the Chinese herbal medicine Baicalein (BE), which was one of the skullcapflavones, can induce apoptosis in C. albicans. The apoptotic effects of BE were detected by flow cytometry using Annexin V-FITC and DAPI, and it was confirmed by transmission electron microscopy analysis. After exposure to 4 microg/ml BE for 12 h, about 10% of C. albicans cells were apoptotic. Both the increasing intracellular levels of reactive oxygen species (ROS) and upregulation of some redox-related genes (CAP1, SOD2, TRR1) were observed. Furthermore, we compared the survivals of CAP1 deleted, wild-type, and overexpressed strains and found that Cap1p attenuated BE-initiated cell death, which was coherent with a higher mRNA level of the CAP1 gene. In addition, the mitochondrial membrane potential of C. albicans cells changed significantly ( p<0.001) upon BE treatment compared with control. Taken together, our results indicate that BE treatment induces apoptosis in C.albicans cells, and the apoptosis was associated with the breakdown of mitochondrial membrane potential.

In vitro activity of baicalein against Candida albicans biofilms.

Candidiasis can be associated with the formation of biofilms on bioprosthetic surfaces. The intrinsic resistance of Candida albicans biofilms to the most commonly used antifungal agents has been demonstrated. Here we examined the effect of baicalein (BE) on C. albicans biofilm formation. Confocal laser scanning microscopy showed that C. albicans biofilm was inhibited and growth was predominantly composed of yeast cells and pseudohyphae. The effect of inhibition was dose-dependent. Over 70% inhibition of biofilms was observed at BE concentrations between 4 microg/mL and 32 microg/mL. Moreover, BE was active against different growth stages of biofilms, with 89% and 52% inhibition when BE was added at 0 h and 24 h of the incubation period, respectively. The water-hydrocarbon two-phase assay showed a decrease in cell surface hydrophobicity in the BE-treated groups compared with the control group. Consistent with this, real-time reverse transcriptase polymerase chain reaction (RT-PCR) results showed that BE-treated cells expressed lower levels of CSH1 mRNA than cells grown in the absence of BE. Our data provide useful information for the development of new strategies to reduce the incidence of device-associated infections.

Antifungal Activity of the Ethanol Extract from Flos Rosae Chinensis with Activity against Fluconazole-Resistant Clinical Candida.

This study was designed to investigate the antifungal activity of a hydroalcoholic extract from Flos Rosae Chinensis (FRC) combined with fluconazole (FCZ) against clinical isolates of Candida albicans resistant to FCZ. The minimum inhibitory concentration (MIC) of FRC was determined using a checkerboard microdilution assay. The synergistic effects of the combination of FRC and FCZ against clinical isolates of C. albicans resistant to FCZ were further confirmed by constructing time-growth curves and performing an agar diffusion test. FRC alone exerted efficient antifungal activities against C. albicans within a MIC80 ranging from 20 µg/ml to 40 µg/ml. FRC failed to enhance the effects of FCZ against sensitive C. albicans strains, although it rendered FCZ-resistant C. albicans more sensitive. These results were further confirmed by the result of in vivo study. Our study is the first to discover that FRC can inhibit the growth of C. albicans to a certain degree. An FRC antifungal mechanism study showed that FRC strengthens FCZ to inhibit the action of ergosterol biosynthesis by promoting the transformation of lanosterol to eburicol, suggesting that the antifungal mechanism of FRC involves the inhibition of ergosterol biosynthesis.

Cap1p attenuates the apoptosis of Candida albicans.

Candida albicans is the most common opportunistic fungal pathogen and its apoptosis is inducible by environmental stress. Based on our previous finding that transcription factor Cap1p was involved in baicalein-induced apoptosis, the present study aimed to further clarify the role of Cap1p in apoptosis by observing the impact of CAP1 deletion on cell fate. It was found that apoptotic stimulation with amphotericin B, acetic acid and hydrogen peroxide increased the number of apoptotic and necrotic cells, caspase activity and the accumulation of reactive oxygen species, whereas it decreased the mitochondrial membrane potential and intracellular ATP level in the cap1Δ/Δ mutant. The cell fate was, at least partly, caused by glutathione depletion and attenuation of the expression of the glutathione reductase gene in the cap1Δ/Δ mutant. Collectively, our data suggest that Cap1p participated in the apoptosis of C. albicans by regulating the expression of the glutathione reductase gene and glutathione content.

Hsp90 is involved in apoptosis of Candida albicans by regulating the calcineurin-caspase apoptotic pathway.

Candida albicans is the most common human fungal pathogen. Recent evidence has revealed the occurrence of apoptosis in C. albicans that is inducible by environmental stresses such as hydrogen peroxide, acetic acid, and amphotericin B. Apoptosis is regulated by the calcineurin-caspase pathway in C. albicans, and calcineurin is under the control of Hsp90 in echinocandin resistance. However, the role of Hsp90 in apoptosis of C. albicans remains unclear. In this study, we investigated the role of Hsp90 in apoptosis of C. albicans by using an Hsp90-compromised strain tetO-HSP90/hsp90 and found that upon apoptotic stimuli, including hydrogen peroxide, acetic acid or amphotericin B treatment, less apoptosis occurred, less ROS was produced, and more cells survived in the Hsp90-compromised strain compared with the Hsp90/Hsp90 wild-type strain. In addition, Hsp90-compromised cells were defective in up-regulating caspase-encoding gene CaMCA1 expression and activating caspase activity upon the apoptotic stimuli. Investigations on the relationship between Hsp90 and calcineurin revealed that activation of calcineurin could up-regulate apoptosis but could not further down-regulate apoptosis in Hsp90-compromised cells, indicating that calcineurin was downstream of Hsp90. Hsp90 inhibitor geldanamycin (GdA) could further decrease the apoptosis in calcineurin-pathway-defect strains, indicating that compromising Hsp90 function had a stronger effect than compromising calcineurin function on apoptosis. Collectively, this study demonstrated that compromised Hsp90 reduced apoptosis in C. albicans, partially through downregulating the calcineurin-caspase pathway.

Trehalose is an important mediator of Cap1p oxidative stress response in Candida albicans.

Trehalose, a nonreducing disaccharide which accumulates dramatically during stationary phase or under oxidative stress, is well known as a stress protectant in several organisms. Here we investigated the putative correlation of trehalose with Cap1p, which is a basic region-leucine zipper (bZip) transcription factor participating in oxidative stress tolerance in Candida albicans. HPLC-MS analysis showed that trehalose did not accumulate in the cap1/cap1 mutant during stationary phase. When the mutant was exposed to high concentration of H2O2, trehalose accumulation was still not induced. Under both of the conditions above, the cap1/cap1 mutant showed high sensitivity to H2O2, and the cell viability was rather low. Furthermore, when exogenous trehalose was added to the culture of the cap1/cap1 mutant, the tolerance of this strain to oxidative stress was increased. Real time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the transcript levels of TPS2 and TPS3 were increased in the wild type strain compared to that in cap1/cap1 mutant when exposed to H2O2. These results indicated that trehalose accumulation is important to the oxidative stress tolerance mediated by Cap1p in C. albicans.

CaIPF7817 is involved in the regulation of redox homeostasis in Candida albicans.

CaIPF7817, a functionally unknown gene in Candida albicans, was suggested to be involved in the redox system previously, but its exact role is unknown. In this study, ipf7817 null mutant was generated with the URA-blaster method. After the deletion of CaIPF7817, intracellular levels of reactive oxygen species were significantly increased; mitochondrial membrane potential, a direct indicator of mitochondrial function, was elevated; some important redox-related genes, including GLR1, SOD2, and TRR1, were up-regulated; and the GSH/GSSG ratio was raised. These changes indicated that CaIPF7817 played important roles in the regulation of redox homeostasis in C. albicans.