Hypha-specific Genes Research Articles

Mitochondrial phosphate carrier plays an important role in virulence of Candida albicans

ABSTRACT Candida albicans is a common fungal pathogen that can cause life-threatening infections. MIR1 is considered to be a mitochondrial phosphate carrier of C. albicans, while its role in virulence has not been fully elucidated. In this study, we found that mir1Δ/Δ mutant exhibited severe virulence defect in both nematode and murine models. Further mechanism studies revealed that the mir1Δ/Δ mutant grew more slowly than the wild-type strain and showed severe filamentation defects on the hypha-inducing agar media, including YPD + serum, Lee, Spider + glucose, SLAD, SLD, and YPS. Furthermore, the loss of MIR1 resulted in unfermentable carbon utilisation defect, ATP decrease, and reactive oxygen species (ROS) accumulation in C. albicans. Antioxidant proanthocyanidins, vitamin E, and N-acetyl cysteine (NAC) could reduce intracellular ROS levels and partially rescue the filamentation defects of the mir1Δ/Δ mutant. Accordingly, hypha-specific genes, as well as CEK1 and RIM101 were down-regulated in the mir1Δ/Δ mutant, and this down-regulation could be partially rescued by the addition of the antioxidant NAC. Collectively, MIR1 plays an important role in C. albicans mitochondrial function, filamentation and virulence, and would be a promising antifungal target.

A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

The role of the C. albicans transcriptional repressor NRG1 during filamentation and disseminated candidiasis is strain dependent.

Candida albicans is one of the most common causes of superficial and invasive fungal diseases in humans. Its ability to cause disease is closely linked to its ability to undergo a morphological transition from budding yeast to filamentous forms (hyphae and pseudohyphae). The extent to which C. albicans strains isolated from patients undergo filamentation varies significantly. In addition, the filamentation phenotypes of mutants involving transcription factors that positively regulate hyphal morphogenesis can also vary from strain to strain. Here, we characterized the virulence, in vitro and in vivo filamentation, and in vitro and in vivo hypha-associated gene expression profiles for four poorly filamenting C. albicans isolates and their corresponding deletion mutants of the repressor of filamentation NRG1. The two most virulent strains, 57055 and 78048, show robust in vivo filamentation but are predominately yeast phase under in vitro hypha induction; the two low-virulence strains (94015 and 78042) do not undergo filamentation well under either condition. In vitro, deletion of NRG1 increases hyphae formation in the SC5314 derivative SN250, but only pseudohyphae are formed in the clinical isolates. Deletion of NRG1 modestly increased the virulence of 78042, which was accompanied by increased expression of hypha-associated genes without an increase in filamentation. Strikingly, deletion of NRG1 in 78048 reduced filamentation in vivo, expression of candidalysin (ECE1), and virulence without dramatically altering establishment of infection. Thus, the function of the conserved repressor NRG1 in C. albicans shows strain-based heterogeneity during infection.IMPORTANCEClinical isolates of the human fungal pathogen Candida albicans show significant variation in their ability to undergo in vitro filamentation and in the function of well-characterized transcriptional regulators of filamentation. Here, we show that Nrg1, a key repressor of filamentation and filament specific gene expression in standard reference strains, has strain-dependent functions, particularly during infection. Most strikingly, loss of NRG1 function can reduce filamentation, hypha-specific gene expression such as the toxin candidalysin, and virulence in some strains. Our data emphasize that the functions of seemingly fundamental and well-conserved transcriptional regulators such as Nrg1 are contextual with respect to both environment and genetic backgrounds.

Reprogramming in Candida albicans Gene Expression Network under Butanol Stress Abrogates Hyphal Development.

Candida albicans is the causative agent of invasive fungal infections. Its hyphae-forming ability is regarded as one of the important virulence factors. To unravel the impact of butanol on Candida albicans, it was placed in O+ve complete human serum with butanol (1% v/v). The Candida transcriptome under butanol stress was then identified by mRNA sequencing. Studies including electron microscopy demonstrated the inhibition of hyphae formation in Candida under the influence of butanol, without any significant alteration in growth rate. The numbers of genes upregulated in the butanol in comparison to the serum alone were 1061 (20 min), 804 (45 min), and 537 (120 min). Candida cells exhibited the downregulation of six hypha-specific transcription factors and the induction of four repressor/regulator genes. Many of the hypha-specific genes exhibited repression in the medium with butanol. The genes related to adhesion also exhibited repression, whereas, among the heat-shock genes, three showed inductions in the presence of butanol. The fungal-specific genes exhibited induction as well as repression in the butanol-treated Candida cells. Furthermore, ten upregulated genes formed the core stress gene set in the presence of butanol. In the gene ontology analysis, enrichment of the processes related to non-coding RNA, ribosome biosynthesis, and metabolism was observed in the induced gene set. On the other side, a few GO biological process terms, including biofilm formation and filamentous growth, were enriched in the repressed gene set. Taken together, under butanol stress, Candida albicans is unable to extend hyphae and shows growth by budding. Many of the genes with perturbed expression may have fitness or virulence attributes and may provide prospective sites of antifungal targets against C. albicans.

Inhibition of cell cycle-dependent hyphal and biofilm formation by a novel cytochalasin 19,20‑epoxycytochalasin Q in Candida albicans

Biofilm-mediated drug resistance is a key virulence factor of pathogenic microbes that cause a serious global health threat especially in immunocompromised individuals. Here, we investigated the antihyphal and antibiofilm activity of 19,20‑epoxycytochalasin Q (ECQ), a cytochalasin actin inhibitor isolated from medicinal mushroom Xylaria sp. BCC1067 against Candida albicans. Remarkably, 256 µg/ml of ECQ inhibited over 95% of C. albicans hyphal formation after 24 h-treatment. Combined ECQ and lipid-based biosurfactant effectively enhanced the antihyphal activity, lowering required ECQ concentrations. Hyphal fragmentation and reduction of biofilm biomass, shown by SEM and AFM visualization of ECQ-treated biofilms, were well corelated to the reduced metabolic activities of young and 24 h-preformed C. albicans biofilms. Induced intracellular accumulation of reactive oxygen species (ROS) also occurred in accompany with the leakage of shrunken cell membrane and defective cell wall at increasing ECQ concentrations. Transcriptomic analyses via RNA-sequencing revealed a massive change (> 1300 genes) in various biological pathways, following ECQ-treatment. Coordinated expression of genes, associated with cellular response to drugs, filamentous growth, cell adhesion, biofilm formation, cytoskeleton organization, cell division cycle, lipid and cell wall metabolisms was confirmed via qRT-PCR. Protein–protein association tool identified coupled expression between key regulators of cell division cyclin-dependent kinases (Cdc19/28) and a gamma-tubulin (Tub4). They coordinated ECQ-dependent hyphal specific gene targets of Ume6 and Tec1 during different phases of cell division. Thus, we first highlight the antihyphal and antibiofilm property of the novel antifungal agent ECQ against one of the most important life-threatening fungal pathogens by providing its key mechanistic detail in biofilm-related fungal infection.

Enniatins from a marine-derived fungus Fusarium sp. inhibit biofilm formation by the pathogenic fungus Candida albicans.

Candidemia is a life-threatening disease common in immunocompromised patients, and is generally caused by the pathogenic fungus Candida albicans. C. albicanscan change morphology from yeast to hyphae, forming biofilms on medical devices. Biofilm formation contributes to the virulence and drug tolerance of C. albicans, and thus compounds that suppress this morphological change and biofilm formation are effective for treating and preventing candidemia. Marine organisms produce biologically active and structurally diverse secondary metabolites that are promising lead compounds for treating numerous diseases. In this study, we explored marine-derived fungus metabolites that can inhibit morphological change and biofilm formation by C. albicans. Enniatin B (1), B1 (2), A1 (3), D (4), and E (5), visoltricin (6), ergosterol peroxide (7), 9,11-dehydroergosterol peroxide (8), and 3β,5α,9α-trihydroxyergosta-7,22-dien-6-one (9) were isolated from the marine-derived fungus Fusarium sp. Compounds 1-5 and 8 exhibited inhibitory activity against hyphal formation by C. albicans, and compounds 1-3 and 8 inhibited biofilm formation by C. albicans. Furthermore, compounds 1-3 decreased cell surface hydrophobicity and expression of the hypha-specific gene HWP1 in C. albicans. Compound 1 was obtained in the highest yield. An in vivo evaluation system using silkworms pierced with polyurethane fibers (a medical device substrate) showed that compound 1 inhibited biofilm formation by C. albicans in vivo. These results indicate that enniatins could be lead compounds for therapeutic agents for biofilm infections by C.albicans.

In vitro antifungal and antibiofilm activities of novel sulfonyl hydrazone derivatives against Candida spp.

The aim of this study was to investigate the antifungal and antibiofilm activity of the new sulfonyl hydrazones compound derived from sulphonamides. In this study, new sulfonyl hydrazone series were synthesized via a green chemistry method. The structures of the synthesized compounds were characterized by elemental analyses and spectroscopic methods. The antifungal activities of the Anaf compounds against Candida strains under planktonic conditions were tested. The biofilm-forming ability of Candida strains was determined and the inhibitory effects of Anaf compounds on Candida biofilms compared with fluconazole were measured by MTT assay. Expression analysis of biofilm-related genes was investigated with qRT-PCR. The statistical analysis was performed using a one-way ANOVA test. CANDIDA: strains was determined and the inhibitory effects of Anaf compounds on Candida biofilms compared with fluconazole were measured by MTT assay. Expression analysis of biofilm-related genes was investigated with qRT-PCR. The statistical analysis was performed using a one-way ANOVA test. A total of 16 (45.7%) out of 35 Candida isolates were determined as strong biofilm producers in this study. C. albicans was the most biofilm producer, followed by C. krusei and C. lusitaniae. The Anaf compounds had a broad spectrum of activity with MIC values ranging from 4μg/ml to 64μg/ml. Our data indicated that the Anaf compound had a significant effect on inhibiting biofilm formation in both fluconazole-susceptible and -resistant strains. The expression levels of hypha-specific genes als3, hwp1, ece1 and sap5 were downregulated by Anaf compounds. Our study revealed that the Anaf compounds had antifungal activity and inhibited fungal biofilms, which may be related to the suppression of C. albicans adherence and hyphal formation. These results suggest that Anaf compounds may have therapeutic potential for the treatment and prevention of biofilm-associated Candida infections.

Adhesion and biofilm formation by the opportunistic pathogen Candida tropicalis: what do we know?

Candida tropicalisis among the most importantCandidaspecies in terms of epidemiology, virulence and resistance. Considering the increase in C. tropicalis incidence and high rates of mortality associated with this species, knowledge of its adhesion and biofilm formation abilities is needed. These traits determine the persistence and survival of yeast on different indwelling medical devices and host sites. C. tropicalis is among the most adherent Candida species, and it has been described as a strong biofilm producer. Environmental factors, phenotypic switching and quorum sensing molecules can affect adhesion and biofilm growth. C. tropicalis can form sexual biofilms, which are promoted by mating pheromones. C. tropicalis biofilms are regulated by a wide and complex network of genes and signaling pathways that are currently poorly understood. Morphological studies showed improved biofilm architecture, which was related to the expression of several hypha-specific genes. Based on recent updates, research is still needed to increase our knowledge on the genetic network of adhesion and biofilm formation by C. tropicalis, as well as the protein diversity that mediates interactions with inert materials and biological surfaces. Here, we have reviewed the main aspects related to adhesion and biofilm formation in C. tropicalis and summarized current knowledge on the significance of these virulence factors in this opportunistic species.

A potential milk preservative----Phormicin C-NS, sorbic acid-modified housefly antimicrobial peptide, inhibits Candida albicans hypha and biofilm formation

Candida albicans causes mastitis in dairy cows, which reduces milk production, causes milk quality decline and increases the risk of foodborne infections. Antimicrobial peptides (AMPs) play important roles in insects due to their ability to inhibit the growth of bacteria, fungi and viruses. Moreover, sorbic acid and benzoic acid are widely used preservatives in the food industry. In this study, one of the housefly larval AMPs, Phormicin C-NS, modified with sorbic acid or benzoic acid, was synthesized, and the anti-C. albicans activity was evaluated. In addition, the potential mechanism of action of Phormicin C-NS against C. albicans was revealed by transcriptomic analysis. Phormicin C-NS inhibited the C. albicans bud-to-hyphal transition and biofilm formation by downregulating the hypha-specific genes HWP1, Ece1p and Als3. Moreover, it inhibited C. albicans propagation in pasteurized milk. The MIC of Phormicin C-NS for C. albicans was nearly 30 times lower than that of sorbic acid. Furthermore, Phormicin C-NS will significantly reduce sorbic acid usage in the milk industry as an alternative peptide. Eventually, the peptide is degraded into amino acids. In summary, Phormicin C-NS has good biosafety, enhances milk amino acid levels, and has great potential as a new preservative for use in the milk industry.

Promising Anti-Biofilm Agents and Phagocytes Enhancers for the Treatment of Candida albicans Biofilm-Associated Infections.

Little is known about the interactions among phagocytes and antifungal agents and the antifungal immunomodulatory activities on Candida species biofilms. Here, inhibition of C. albicans biofilms and the interactions among biofilms and phagocytes alone or in combination with essential oils, biological, and chemical agents, or fluconazole were investigated. Biofilm formation by a panel of 28 C. albicans clinical isolates from hospitalized patients, birds, and cattle was tested. The anti-biofilm activities of cinnamon and clove oils, sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and Enterococcus faecalis cell-free supernatant (CFS) in comparison with fluconazole were investigated using crystal violet and XTT reduction assays, expression of hypha-specific and hyphal regulator genes, and scanning electron microscopy (SEM) analysis. Of the tested C. albicans isolates, 15 of 28 (53.6%) were biofilm producers. Cinnamon followed by E. faecalis–CFS, SDS, and CTAB was the most effective inhibitors of planktonic C. albicans and biofilms. Fluconazole was an ineffective inhibitor of C. albicans biofilms. Sessile minimal inhibitory concentration (SMIC50) of cinnamon, SDS, CTAB, and E. faecalis–CFS downregulated the hypha-specific and regulator genes, albeit to various extents, when compared with untreated biofilms (P < 0.001). SEM analysis revealed disruption and deformity of three-dimensional structures in cinnamon oil–treated biofilms. C. albicans sessile cells within biofilm were less susceptible to phagocytosis than planktonic cells. The additive effects of phagocytes and the tested antifungals enabled phagocytes to engulf C. albicans cells rapidly in cinnamon, E. faecalis–CFS, or SDS-treated biofilms. No differences in anti-Candida or anti-biofilm eradication activities were detected among the tested isolates. Our findings reinforce the substantial anti-biofilm activity of cinnamon oil, SDS, and E. faecalis–CFS and provide new avenues for the development of novel anti-biofilm immunotherapies or antifungals that could be used prior to or during the management of cases with biofilm-associated infections.

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.

SAGA Complex Subunits in Candida albicans Differentially Regulate Filamentation, Invasiveness, and Biofilm Formation.

SAGA (Spt-Ada-Gcn5-acetyltransferase) is a highly conserved, multiprotein co-activator complex that consists of five distinct modules. It has two enzymatic functions, a histone acetyltransferase (HAT) and a deubiquitinase (DUB) and plays a central role in processes such as transcription initiation, elongation, protein stability, and telomere maintenance. We analyzed conditional and null mutants of the SAGA complex module components in the fungal pathogen Candida albicans; Ngg1, (the HAT module); Ubp8, (the DUB module); Tra1, (the recruitment module), Spt7, (the architecture module) and Spt8, (the TBP interaction unit), and assessed their roles in a variety of cellular processes. We observed that spt7Δ/Δ and spt8Δ/Δ strains have a filamentous phenotype, and both are highly invasive in yeast growing conditions as compared to the wild type, while ngg1Δ/Δ and ubp8Δ/Δ are in yeast-locked state and non-invasive in both YPD media and filamentous induced conditions compared to wild type. RNA-sequencing-based transcriptional profiling of SAGA mutants reveals upregulation of hyphal specific genes in spt7Δ/Δ and spt8Δ/Δ strains and downregulation of ergosterol metabolism pathway. As well, spt7Δ/Δ and spt8Δ/Δ confer susceptibility to antifungal drugs, to acidic and alkaline pH, to high temperature, and to osmotic, oxidative, cell wall, and DNA damage stresses, indicating that these proteins are important for genotoxic and cellular stress responses. Despite having similar morphological phenotypes (constitutively filamentous and invasive) spt7 and spt8 mutants displayed variation in nuclear distribution where spt7Δ/Δ cells were frequently binucleate and spt8Δ/Δ cells were consistently mononucleate. We also observed that spt7Δ/Δ and spt8Δ/Δ mutants were quickly engulfed by macrophages compared to ngg1Δ/Δ and ubp8Δ/Δ strains. All these findings suggest that the SAGA complex modules can have contrasting functions where loss of Spt7 or Spt8 enhances filamentation and invasiveness while loss of Ngg1 or Ubp8 blocks these processes.

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway.

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here we studied the roles of three sirtuin deacetylases, Sir2, Hst1, and Hst2, in hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hyphae formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hyphal-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to wild-type. This regulation of hyphae formation was dependent on the deacetylase activity of Sir2, as a point mutant lacking deacetylase activity had a similar defect in hyphae formation as the sir2Δ/Δ mutant. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and non-redundant role in hyphal growth of C. albicans.

The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans.

Candida albicans is an opportunistic human pathogen that exists as yeast, hyphal or pseudohyphal forms depending on pH, nutrients, and temperature. The morphological transition from yeast to hyphae, which is required for the complete virulence of C. albicans, is controlled by many transcription factors that activate or repress hypha-specific genes. The C. albicans transcriptional factor Cas5, a key regulator of genes involved in cell wall integrity, affects the susceptibility of C. albicans to fluconazole, an inhibitor of ergosterol synthesis. In this study, we found that deletion of CAS5 in C. albicans decreased the expression levels of a set of ergosterol biosynthesis genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24, resulting in the accumulation of lanosterol and zymosterol, which are intermediate metabolites in the ergosterol biosynthesis pathway. Interestingly, it was observed that the cas5Δ/Δ mutant could not maintain the yeast form under non-hypha-inducing conditions, while the CAS5-overexpressing cells could not form hyphae under hypha-inducing conditions. Consistent with these observations, the cas5Δ/Δ mutant highly expressed hypha-specific genes, ALS3, ECE1, and HWP1, under non-hypha-inducing conditions. In addition, CAS5 transcription was significantly downregulated immediately after hyphal initiation in the wild-type strain. Furthermore, the cas5Δ/Δ mutant reduced the transcription of NRG1, which encodes a major repressor of hyphal morphogenesis, while Cas5 overexpression increased the transcription of NRG1 under hypha-inducing conditions. Collectively, this study suggests the potential role of Cas5 as a repressor of hypha-specific genes during yeast-form growth of C. albicans.

Development of Carbazole Derivatives Compounds against Candida albicans: Candidates to Prevent Hyphal Formation via the Ras1-MAPK Pathway.

Morphogenesis contributes to the virulence of the opportunistic human fungal pathogen Candida albicans. Ras1-MAPK pathways play a critical role in the virulence of C. albicans by regulating cell growth, morphogenesis, and biofilm formation. Ume6 acts as a transcription factor, and Nrg1 is a transcriptional repressor for the expression of hyphal-specific genes in morphogenesis. Azoles or echinocandin drugs have been extensively prescribed for C. albicans infections, which has led to the development of drug-resistant strains. Therefore, it is necessary to develop new molecules to effectively treat fungal infections. Here, we showed that Molecule B and Molecule C, which contained a carbazole structure, attenuated the pathogenicity of C. albicans through inhibition of the Ras1/MAPK pathway. We found that Molecule B and Molecule C inhibit morphogenesis through repressing protein and RNA levels of Ras/MAPK-related genes, including UME6 and NRG1. Furthermore, we determined the antifungal effects of Molecule B and Molecule C in vivo using a candidiasis murine model. We anticipate our findings are that Molecule B and Molecule C, which inhibits the Ras1/MAPK pathway, are promising compounds for the development of new antifungal agents for the treatment of systemic candidiasis and possibly for other fungal diseases.

Candida albicans requires iron to sustain hyphal growth

Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. The ability to switch between yeast and hyphal growth forms is critical for its pathogenesis. Hyphal development in C. albicans requires two temporally linked regulations for initiation and maintenance. Here, we performed transcriptome sequencing (RNA-Seq) to analyze the transcriptional consequences for the two different phases of hyphal development. Genome-wide transcription profiling reveals that the sets associated with hyphal initiation were significantly enriched in genes for hyphal cell wall, biofilm matrix and actin polarization. In addition to hypha-specific genes, numerous genes involved in iron acquisition, such as FTR1 and SEF1, are highly induced specifically during sustained hyphal development even when additional free iron is supplied in the medium. Therefore, iron uptake genes are induced by signals that can support prolonged hyphal development in an iron-independent manner. The induction of iron acquisition genes during hyphal elongation was further confirmed by quantitative reverse transcription-PCR under various hypha-inducing conditions. Remarkably, preventing C. albicans from acquiring iron blocks BRG1 activation, leading to impaired hyphal maintenance, and ectopically expressed BRG1 can sustain hyphal development bypassing the requirement of iron. Our study elucidates an underlying mechanism of how multiple virulence factors are interconnected and are induced simultaneously during infection.

N-acetylglucosamine Signaling: Transcriptional Dynamics of a Novel Sugar Sensing Cascade in a Model Pathogenic Yeast, Candida albicans.

The amino sugar, N-acetylglucosamine (GlcNAc), has emerged as an attractive messenger of signaling in the pathogenic yeast Candida albicans, given its multifaceted role in cellular processes, including GlcNAc scavenging, import and metabolism, morphogenesis (yeast to hyphae and white to opaque switch), virulence, GlcNAc induced cell death (GICD), etc. During signaling, the exogenous GlcNAc appears to adopt a simple mechanism of gene regulation by directly activating Ngs1, a novel GlcNAc sensor and transducer, at the chromatin level, to activate transcriptional response through the promoter acetylation. Ngs1 acts as a master regulator in GlcNAc signaling by regulating GlcNAc catabolic gene expression and filamentation. Ndt80-family transcriptional factor Rep1 appears to be involved in the recruitment of Ngs1 to GlcNAc catabolic gene promoters. For promoting filamentation, GlcNAc adopts a little modified strategy by utilizing a recently evolved transcriptional loop. Here, Biofilm regulator Brg1 takes up the key role, getting up-regulated by Ngs1, and simultaneously induces Hyphal Specific Genes (HSGs) expression by down-regulating NRG1 expression. GlcNAc kinase Hxk1 appears to play a prominent role in signaling. Recent developments in GlcNAc signaling have made C. albicans a model system to understand its role in other eukaryotes as well. The knowledge thus gained would assist in designing therapeutic interventions for the control of candidiasis and other fungal diseases.

Effect of berberine hydrochloride on cell wall integrity of Candida albicans hypha

The aim of this paper was to investigate the effect of berberine hydrochloride on the cell wall integrity of Candida albicans hypha. The minimal inhibitory concentration(MIC) of berberine hydrochloride against clinical and standard C. albicans strains was detected by micro liquid-based dilution method; the effect of berberine hydrochloride on the colony formation of C. albicans SC5314 was investigated by spot assay; the effect of berberine hydrochloride on the metabolism of C. albicans SC5314 hypha was checked by XTT reduction assay, and the viability of C. albicans SC5314 hypha was tested by fluorescent staining assay. The effect of berberine hydrochloride on the morphology of C. albicans SC5314 hypha was examined by scanning electron microscope. The changes in the cell wall of C. albicans SC5314 hypha after berberine hydrochloride treatment were detected by transmission electron microscopy. The effect of berberine hydrochloride on β-glucan from C. albicans SC5314 was detected by flow cytometry. The effect of berberine hydrochloride on hypha-specific gene ECE1 and β-glucan synthase genes FKS1 and FKS2 in C. albicans was examined by qRT-PCR. The results showed that berberine hydrochloride showed a strong inhibitory effect on both clinical and standard strains of C. albicans, and the MIC was 64-128 μg·mL~(-1). Spot assay, XTT redunction assay and fluorescent staining assay showed that with the increase of berberine hydrochloride concentration, the viability of C. albicans SC5314 gradually decreased. The transmission electron microscopy scanning assay showed that this compound could cause cell wall damage of C. albicans. The flow cytometry analysis showed the exposure degree of C. albicans β-glucan. The qRT-PCR further showed that berberine hydrochloride could significantly down-regulate hypha-specific gene ECE1 and β-glucan synthase-related gene FKS1 and FKS2. In conclusion, this compound can down-regulate C. albicans and β-glucan synthase-related gene expressions, so as to destroy the cell wall structure of C. albicans, expose β-glucan and damage the integrity of the wall.

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.