Recombinant Candida albicans Dihydroorotate dehydrogenase (quinone), mitochondrial (URA9)

What is the role of URA9 in C. albicans pyrimidine biosynthesis?

URA9 encodes dihydroorotate dehydrogenase (DHOD), a critical enzyme in the de novo pyrimidine biosynthesis pathway in C. albicans. This Class-II DHOD catalyzes the conversion of dihydroorotate to orotate, which is a key step in the synthesis of UMP (uridine monophosphate). This reaction is particularly significant because it connects pyrimidine biosynthesis with the electron transport chain in many fungi.

In C. albicans, like other fungi, the de novo pyrimidine biosynthesis pathway is essential when external pyrimidine sources are unavailable. The pathway consists of six enzymatic steps, with DHOD catalyzing the fourth step. Unlike some other yeasts that possess both Class-I (soluble) and Class-II (membrane-bound) DHODs, C. albicans primarily relies on Class-II DHOD for this crucial reaction .

How does C. albicans URA9 differ from URA3 in function and regulation?

While both URA9 and URA3 participate in pyrimidine biosynthesis, they catalyze different reactions:

• URA9 (DHOD) catalyzes the oxidation of dihydroorotate to orotate

• URA3 encodes orotidine 5'-monophosphate (OMP) decarboxylase, which catalyzes the conversion of OMP to UMP in the final step of the de novo pathway

Regarding regulation, URA3 expression in C. albicans has been extensively studied as a selectable marker for gene manipulation techniques. The regulation of both genes appears to be linked to the TORC1 and Sch9 kinase pathways. Research has shown that Tor1-Sch9 kinase cascade stimulates the transcription of ribosomal protein genes, which may indirectly affect URA gene expression .

What is the relationship between URA9 and hyphal morphogenesis in C. albicans?

The connection between URA9 and morphogenesis in C. albicans appears to involve the Tor1-Sch9 signaling pathway. Research has revealed that during true hyphae formation, there is a reduction in the transcription of ribosomal protein genes, which is regulated through the TORC1 and Sch9 kinases . This suggests that metabolism and morphogenesis are coordinated processes in C. albicans.

The Tor1-Sch9 kinase cascade mediates both the stimulation of ribosomal protein gene transcription and the inhibition of adhesion gene transcription in a mutually exclusive manner. Since URA9 is involved in basic metabolism, its activity may be downregulated during hyphal formation when the cellular resources are redirected toward the expression of hyphae-specific genes and adhesins .

What molecular techniques are most effective for gene manipulation of URA9 in C. albicans?

Several molecular techniques can be applied for URA9 manipulation in C. albicans:

• URA Blaster Method: This classical approach uses the URA3 marker for gene disruption. While not directly targeting URA9, this method provides a foundation for gene manipulation in C. albicans. The technique involves a cassette carrying two direct repeats of hisG sequences flanking the URA3 gene. After transformation, the hisG sequences can spontaneously recombine, looping out URA3 and allowing for multiple rounds of gene disruption .

• CRISPR-Cas9 System: This method offers precise genome editing capabilities. For URA9 manipulation, design guide RNAs targeting the URA9 locus and include a repair template containing your desired modifications.

• Gateway Cloning: For recombinant expression, Gateway cloning technology allows efficient transfer of the URA9 gene between different expression vectors through site-specific recombination.

• Homologous Recombination: Design constructs with URA9 flanking sequences of 300-500 bp for optimal integration efficiency. When targeting URA9, consider using NAT1 or SAT1 as selectable markers instead of URA3 to avoid complications in pyrimidine metabolism studies .

How can I optimize heterologous expression of C. albicans URA9?

For optimal heterologous expression of C. albicans URA9, consider the following methodological approach:

• Expression System Selection:

  • For structural studies: Pichia pastoris or Saccharomyces cerevisiae

  • For functional studies: S. cerevisiae ura1Δ strain (as demonstrated with other fungal URA9 orthologs)

  • For high yield: E. coli BL21(DE3) with optimization for codon usage

• Vector Design:

  • Include a strong inducible promoter (GAL1 for yeast, T7 for E. coli)

  • Add appropriate targeting sequences if mitochondrial localization is desired

  • Incorporate a purification tag (His6 or GST) that doesn't interfere with enzyme activity

• Expression Conditions:

  • For yeast: Induce at OD600 0.8-1.0, grow at 30°C for 12-24 hours

  • For E. coli: Induce at OD600 0.6-0.8, lower temperature to 18-20°C, extend expression time to 16-20 hours

  • Supplement media with riboflavin (10 μg/ml) to support flavin cofactor incorporation

• Verification of Expression:

  • Western blot analysis using anti-His or anti-GST antibodies

  • Functional complementation in S. cerevisiae ura1Δ strain by testing growth on uracil-free media under both aerobic and anaerobic conditions

What are the optimal conditions for purifying recombinant C. albicans URA9?

Purification of recombinant C. albicans URA9 requires careful attention to maintain enzyme activity:

• Cell Lysis:

  • For mitochondrial URA9: Isolate mitochondria first using differential centrifugation

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM DTT, protease inhibitor cocktail

  • Gentle lysis using non-ionic detergents (0.5-1% Triton X-100) to preserve membrane association

• Chromatography Steps:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Ion exchange chromatography (IEX) as a secondary purification step

  • Size exclusion chromatography (SEC) as a final polishing step

• Quality Control:

  • SDS-PAGE with Coomassie staining to assess purity (>90%)

  • Western blot to confirm identity

  • Activity assay measuring dihydroorotate oxidation spectrophotometrically

  • Thermal shift assay to evaluate protein stability

How does URA9 activity differ under aerobic versus anaerobic conditions?

Class-II DHODs like URA9 show distinctive activity patterns under different oxygen conditions:

Under aerobic conditions, Class-II DHODs typically use ubiquinone in the respiratory chain as an electron acceptor. The enzyme is anchored to the mitochondrial membrane where it can efficiently interact with the electron transport chain. In the case of C. albicans URA9, this enables the organism to link pyrimidine biosynthesis with respiration in oxygen-rich environments.

Under anaerobic conditions, the situation is more complex. Some fungi have evolved Class-II DHODs that can function anaerobically using alternative electron acceptors. For example, research with URA9 orthologs from Anaeromyces robustus (ArUra9), Schizosaccharomyces japonicus (SjURA9), and Dekkera bruxellensis (DbURA9) has shown they can support anaerobic pyrimidine prototrophy when expressed in S. cerevisiae ura1Δ strains .

Experiments with cell extracts demonstrated that ArUra9 could use free FAD and FMN as electron acceptors instead of quinones, allowing for anaerobic function . While the specific electron acceptors for C. albicans URA9 under anaerobic conditions have not been fully characterized, the enzyme likely exhibits similar adaptations to function in the low-oxygen environments C. albicans encounters during infection.

How does the Tor1-Sch9 signaling pathway regulate URA9 expression?

The Tor1-Sch9 signaling cascade plays a critical role in regulating metabolism and morphogenesis in C. albicans, with implications for URA9 expression:

The Target of Rapamycin Complex 1 (TORC1), which includes the Tor1 kinase, acts as a central regulator of cell growth in response to nutrient availability. Sch9 is a downstream effector kinase of TORC1. Research has revealed that this pathway mediates the stimulation of ribosomal protein gene transcription and inhibition of adhesion gene transcription in a mutually exclusive manner in C. albicans .

During hyphal morphogenesis, there is a reduction in ribosomal protein gene transcription that is regulated through TORC1 and Sch9 kinases. Since pyrimidine biosynthesis is closely linked to ribosome biogenesis (as ribosomes require large amounts of nucleotides), the expression of URA9 likely falls under similar regulatory control.

The study by Li et al. demonstrated that polysome and monosome levels decreased during hyphal stimuli through TORC1 and Sch9 kinases, providing evidence for a novel link between ribosome biogenesis and morphogenesis mediated by Tor1 and Sch9 . This suggests that URA9 expression may be downregulated during hyphal growth as cellular resources are redirected toward the expression of virulence factors.

How does C. albicans URA9 compare with DHODs from other fungal species?

C. albicans URA9 belongs to the Class-II DHOD family, which demonstrates significant diversity across fungal species:

Key differences include:

• Subcellular Localization: While most Class-II DHODs are mitochondrial, some like ArUra9 lack a mitochondrial targeting sequence and localize to the cytosol . This localization affects the enzyme's access to electron acceptors and integration with cellular respiration.

• Anaerobic Functionality: Several phylogenetically distant fungi have independently evolved Class-II DHODs capable of anaerobic function. For example, DbURA9 from D. bruxellensis supports anaerobic pyrimidine prototrophy, though with initially slower growth that can be improved through adaptation involving mutations in other genes like FUM1 (fumarase) .

• Electron Acceptor Flexibility: ArUra9 can use free FAD and FMN as electron acceptors, while most Class-II DHODs prefer quinones. This flexibility allows for function under various oxygen conditions .

The comparative analysis of these enzymes provides insights into evolutionary adaptations that enable fungi to survive in diverse ecological niches with varying oxygen availability.

Can C. albicans URA9 complement URA gene deletions in other yeast species?

Complementation studies with DHODs across different fungal species reveal important functional similarities and differences:

While specific data on C. albicans URA9 complementation in other species is limited, research with related fungal DHODs provides valuable insights. Studies have shown that Class-II DHODs from diverse fungi can functionally complement the S. cerevisiae ura1Δ mutation under both aerobic and anaerobic conditions .

Experimentally, this can be tested by:

• Cloning the C. albicans URA9 gene into an appropriate expression vector for the host species

• Transforming the construct into a URA deletion strain of the target species

• Assessing growth on media without uracil under both aerobic and anaerobic conditions

• Measuring growth rates and comparing them to wild-type and uncomplemented mutant controls

When interpreting complementation results, researchers should consider factors such as expression levels, protein stability in the heterologous host, and potential interactions with other metabolic pathways. For instance, expression of SjURA9 in S. cerevisiae resulted in loss of respiration and slower growth even with uracil supplementation, indicating broader metabolic effects beyond simple complementation .

Is URA9 a viable target for novel antifungal development?

URA9 presents several characteristics that make it a potentially attractive target for antifungal development:

To evaluate URA9 as an antifungal target, researchers should consider:

• Target Validation:

  • Create conditional URA9 mutants in C. albicans and assess virulence in animal models

  • Develop URA9-specific inhibitors and test their efficacy in vitro and in vivo

  • Evaluate compensatory mechanisms that might circumvent URA9 inhibition

• Druggability Assessment:

  • Perform structural analysis of C. albicans URA9 to identify potential binding pockets

  • Conduct in silico screening of chemical libraries against the URA9 structure

  • Develop high-throughput assays to screen for URA9 inhibitors

• Resistance Potential:

  • Assess the likelihood of resistance development through directed evolution studies

  • Identify potential compensatory pathways that might be upregulated upon URA9 inhibition

How can recombinant URA9 be used in diagnostic applications for invasive candidiasis?

Recombinant fungal proteins have proven valuable for detecting antibodies in patients with invasive candidiasis, suggesting potential diagnostic applications for URA9:

• Antibody Detection ELISA:

  • Purified recombinant C. albicans URA9 can be used to coat ELISA plates

  • Patient serum samples are added and anti-URA9 antibodies detected using labeled secondary antibodies

  • This approach could complement existing diagnostic methods for invasive candidiasis

Studies with other recombinant C. albicans antigens have demonstrated the utility of this approach. For example, detection of antibodies against recombinant Hwp1 by ELISA showed sensitivity and specificity values of 88.9% and 90.2%, respectively, for diagnosing invasive candidiasis .

• Multi-Antigen Arrays:

  • Combine recombinant URA9 with other C. albicans antigens in protein microarrays

  • This approach could improve diagnostic sensitivity by detecting antibodies against multiple targets simultaneously

  • The arrays could differentiate between Candida species based on response patterns

• Species-Specific Diagnostics:

  • Exploit structural differences between URA9 orthologs to develop species-specific antibody detection

  • This could help distinguish between infections caused by different Candida species

  • Important for treatment decisions as different species have different antifungal susceptibility profiles

For diagnostic development, researchers should optimize:

• Protein expression conditions to ensure native folding

• Purification protocols to obtain highly pure protein without contaminating antigens

• Assay conditions including blocking agents, serum dilutions, and cutoff values for positivity

The diagnostic utility of recombinant URA9 would need to be validated in clinical studies with well-characterized patient cohorts, including those with confirmed invasive candidiasis, superficial Candida infections, and appropriate control groups .

What methodological approaches can identify URA9 inhibitors?

Several methodological approaches can be employed to identify potential inhibitors of C. albicans URA9:

• High-Throughput Enzyme Assays:

  • Spectrophotometric assays measuring the reduction of electron acceptors (e.g., 2,6-dichloroindophenol)

  • Coupling DHOD activity to other redox reactions that generate fluorescent or luminescent signals

  • Optimize assay conditions in 384-well format for screening compound libraries

• Structure-Based Drug Design:

  • Determine the crystal structure of C. albicans URA9 through X-ray crystallography

  • Perform in silico docking studies with virtual compound libraries

  • Focus on compounds that bind to catalytic sites or unique structural features

  • Use molecular dynamics simulations to assess binding stability

• Fragment-Based Screening:

  • Screen libraries of low-molecular-weight fragments using nuclear magnetic resonance (NMR) or thermal shift assays

  • Identify fragments that bind to different sites on URA9

  • Link or grow promising fragments to develop higher-affinity inhibitors

• Phenotypic Screening:

  • Test compounds for growth inhibition of C. albicans under conditions where URA9 function is essential

  • Confirm target engagement through resistance mutation mapping and direct binding assays

  • Evaluate activity under both aerobic and anaerobic conditions to identify inhibitors effective in different host environments

• Repurposing Known DHOD Inhibitors:

  • Test known inhibitors of human or bacterial DHODs for activity against C. albicans URA9

  • Modify these compounds to enhance selectivity for fungal DHOD

For all approaches, promising hits should be validated through:

• Dose-response studies to determine IC50 values

• Mechanism of action studies to confirm competitive, non-competitive, or uncompetitive inhibition

• Selectivity profiling against human DHOD and other related enzymes

• Assessment of antifungal activity against C. albicans and other pathogenic fungi

• Evaluation of cytotoxicity against mammalian cell lines

The genome-scale metabolic model iRV781 could be valuable for predicting the effects of URA9 inhibition on C. albicans metabolism and identifying potential escape mechanisms that might limit inhibitor efficacy .

What are the most promising research directions for C. albicans URA9 studies?

Several promising research directions emerge for future studies on C. albicans URA9:

• Structural Biology: Determining the high-resolution structure of C. albicans URA9 would provide invaluable insights for inhibitor design and understanding species-specific features. Cryo-EM and X-ray crystallography approaches should be pursued, particularly focusing on the enzyme in complex with substrates and potential inhibitors.

• Systems Biology Integration: Further integration of URA9 function into genome-scale metabolic models like iRV781 would enhance our understanding of how pyrimidine metabolism interfaces with other cellular processes, particularly under different environmental conditions relevant to infection .

• Host-Pathogen Interface: Investigating how URA9 function and regulation change during host-pathogen interactions could reveal new aspects of C. albicans pathogenicity and adaptation strategies.

• Regulatory Networks: Deeper exploration of the Tor1-Sch9 regulatory network and its impact on URA9 expression would illuminate how metabolism and morphogenesis are coordinated in this important pathogen .

• Translational Applications: Development of URA9-based diagnostics and targeted therapeutics represents an important avenue for translating basic research findings into clinical applications to address the challenges of invasive candidiasis .