Recombinant Candida glabrata 21S rRNA pseudouridine (2819) synthase (PUS5)

How does PUS5 differ from other pseudouridine synthases in Candida species?

PUS5 differs from other pseudouridine synthases primarily in its substrate specificity and cellular localization. While pseudouridine synthases like Pus7 target multiple substrates including tRNA, rRNA, and possibly mRNA , PUS5 appears to be more specialized, targeting specific residues in mitochondrial rRNA. Unlike Cbf5, which uses snoRNAs to recognize its targets, PUS5 likely employs a combination of RNA primary and secondary structure for target recognition . This targeting mechanism is distinct from snoRNA-guided modifications and represents an independent evolutionary path for RNA modification enzymes. The substrate specificity of pseudouridine synthases is not always predictable by consensus motifs alone, as the presence of a consensus sequence doesn't ensure modification .

What is the evolutionary conservation of PUS5 across fungal species?

PUS5 appears to be conserved across fungal species, but with potential variations in substrate specificity. The CTG clade of fungi, which includes C. albicans, diverged from Saccharomyces approximately 170 million years ago , potentially leading to functional divergence of pseudouridine synthases. Research on C. albicans PUS7 demonstrates that despite conservation of pseudouridine synthases between budding yeast taxa, their substrates and in vivo roles can differ significantly . Similar evolutionary divergence likely exists for PUS5, potentially reflecting adaptations to different ecological niches and stress conditions. Comparative genomic analyses would be required to fully understand the evolution of PUS5 in pathogenic and non-pathogenic fungi.

How can recombinant C. glabrata PUS5 be expressed and purified for in vitro studies?

Recombinant C. glabrata PUS5 can be expressed using similar approaches to those demonstrated for other yeast proteins. Based on established protocols, the following methodology would be appropriate:

• Gene Cloning: Amplify the PUS5 gene from C. glabrata genomic DNA using PCR with specific primers containing appropriate restriction sites or homology regions for cloning.

• Vector Construction: For expression in S. cerevisiae, construct a plasmid similar to pGREG576 with either a GAL1 or copper-inducible MTI promoter . For example:

  • Design primers with homology to both the PUS5 gene and the cloning site of the vector

  • Co-transform the amplified gene and linearized vector into S. cerevisiae for homologous recombination

  • Verify the construct by DNA sequencing

• Protein Expression: Transform the expression vector into an appropriate yeast strain and induce protein expression:

  • For GAL1 promoter: Grow cells in medium containing 0.5% glucose and 0.1% galactose, then transfer to medium with 0.1% glucose and 1% galactose

  • For MTI promoter: Induce with 50 μM CuSO₄

• Protein Purification: Employ affinity chromatography if the construct includes a tag (e.g., His-tag or GFP-fusion), followed by size exclusion chromatography for further purification.

• Verification: Assess protein purity by SDS-PAGE and verify activity using appropriate RNA substrates.

This methodology can be adapted based on specific research requirements and available resources.

What techniques can be used to identify specific target sites of PUS5 in C. glabrata mitochondrial rRNA?

Several complementary approaches can be employed to identify specific target sites of PUS5 in C. glabrata mitochondrial rRNA:

• Comparative Analysis: Generate a PUS5 deletion strain using CRISPR-Cas9 genome editing, similar to methods used for CaPUS7 . Extract mitochondrial rRNA from wild-type and deletion strains for comparative analysis.

• Pseudouridine Detection Methods:

  • CMC-primer extension: Treat RNA with N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide (CMC), which specifically modifies pseudouridines, followed by primer extension to identify modification sites

  • Mass spectrometry: Analyze digested rRNA fragments to detect mass shifts characteristic of pseudouridylation

  • Next-generation sequencing methods: Employ Ψ-seq or Pseudo-seq techniques that use chemical modification and deep sequencing to map pseudouridines at single-nucleotide resolution

• In vitro Verification: Incubate recombinant PUS5 with in vitro transcribed potential target RNA segments and analyze pseudouridylation using the methods described above.

• Mutational Analysis: Create point mutations in potential target sites and assess the impact on pseudouridylation in vivo and in vitro.

A combination of these approaches would provide comprehensive identification of PUS5 target sites with high confidence.

How can the subcellular localization of PUS5 be determined in C. glabrata?

To determine the subcellular localization of PUS5 in C. glabrata, fluorescence microscopy using GFP fusion proteins represents an effective approach, as demonstrated for other C. glabrata proteins:

• Construction of GFP Fusion: Create a C-terminal GFP fusion of PUS5 using a similar approach to that described for CgDtr1 :

  • Clone the PUS5 gene into a vector like pGREG576, which enables fusion with GFP

  • Ensure the construct maintains the native or a controllable promoter (e.g., MTI promoter)

  • Transform the construct into C. glabrata cells

• Fluorescence Microscopy: Visualize the localization using fluorescence microscopy:

  • Grow transformed cells to mid-exponential phase (OD600 nm = 0.5 ± 0.05)

  • Induce protein expression if using an inducible promoter (e.g., with 50 μM CuSO₄ for MTI promoter)

  • After approximately 5 hours of induction, observe living cells under a fluorescence microscope

  • Use appropriate excitation (approximately 395 nm) and emission (approximately 509 nm) wavelengths for GFP

• Co-localization Studies: Perform co-localization with known mitochondrial markers:

  • Use MitoTracker dyes for mitochondria

  • Compare with other subcellular markers to confirm specificity

• Confirmation by Cellular Fractionation: Isolate mitochondrial, nuclear, and cytosolic fractions biochemically and perform Western blotting to detect the GFP-tagged protein.

This multi-faceted approach would provide robust evidence for the subcellular localization of PUS5 in C. glabrata.

How does deletion of PUS5 affect mitochondrial function and virulence in C. glabrata?

Based on studies of other pseudouridine synthases and virulence factors in Candida species, deletion of PUS5 might affect mitochondrial function and virulence through several mechanisms:

• Impact on Mitochondrial Translation: Since PUS5 likely modifies mitochondrial rRNA, its deletion could impair mitochondrial protein synthesis, affecting:

  • Respiratory chain function

  • ATP production

  • Cellular responses to oxidative stress

• Assessment of Mitochondrial Function in Δpus5 Mutants:

  • Oxygen consumption rates

  • Mitochondrial membrane potential measurements

  • ATP production assays

  • Expression of mitochondrial genes

• Virulence Assessment: Similar to studies with CgDTR1 , the virulence of Δpus5 mutants could be evaluated using:

  • Galleria mellonella infection model, measuring larval survival rates over 72 hours

  • Hemocyte interaction assays to assess phagocytosis resistance

  • Proliferation assays in hemolymph at 1, 24, and 48 hours post-injection

• Stress Response Analysis: Test resistance to various stresses, particularly:

  • Oxidative stress (H₂O₂)

  • Weak acid stress (acetic acid, benzoic acid)

  • Antifungal agents

The relationship between mitochondrial function and virulence is complex, as demonstrated by studies of virulence factors like CgDtr1, which affects both stress resistance and virulence in the G. mellonella model .

How do RNA modifications by PUS5 influence ribosome biogenesis and function in C. glabrata?

RNA modifications introduced by PUS5 likely influence ribosome biogenesis and function through structural and functional alterations:

• Structural Impact Analysis:

  • Nuclear magnetic resonance (NMR) or X-ray crystallography to determine the structural changes induced by specific pseudouridylation

  • Computational modeling to predict the impact of modifications on rRNA folding and stability

• Ribosome Biogenesis Assessment:

  • Northern blot analysis using probes targeting specific regions of rRNA to detect processing intermediates, similar to methods used for CaPUS7 studies

  • Polysome profiling to evaluate the impact on translational efficiency

  • Ribosome assembly analysis using sucrose gradient centrifugation

• Translation Fidelity and Rate:

  • In vitro translation assays using ribosomes from wild-type and Δpus5 strains

  • Measurement of misincorporation rates using reporter constructs

  • Ribosome profiling to assess translation efficiency genome-wide

• Differentially Expressed Genes:

  • RNA sequencing to identify gene expression changes in Δpus5 mutants, potentially revealing:

    • Upregulation of stress response genes

    • Alterations in virulence factor expression

    • Compensatory mechanisms for ribosome function

Studies on CaPUS7 have demonstrated that deletion of pseudouridine synthases can lead to defects in rRNA processing , and similar effects might be observed with PUS5 deletion, albeit potentially restricted to mitochondrial ribosomes.

What is the three-dimensional structure of C. glabrata PUS5 and how does it inform substrate recognition?

The three-dimensional structure of C. glabrata PUS5 has not been experimentally determined, but structural insights can be gained through computational and experimental approaches:

• Homology Modeling:

  • Generate a structural model based on known structures of related pseudouridine synthases

  • Refine the model using molecular dynamics simulations

  • Identify potential substrate binding sites and catalytic residues

• Experimental Structure Determination:

  • Express and purify recombinant PUS5 for X-ray crystallography or cryo-electron microscopy

  • Optimize conditions for protein crystallization

  • Solve the structure with and without bound substrate analogs

• Structure-Function Analysis:

  • Site-directed mutagenesis of predicted catalytic residues

  • Enzymatic assays to correlate structural features with function

  • Binding assays to measure substrate affinity and specificity

• Substrate Recognition Mechanisms:

  • In vitro selection experiments to identify preferred RNA sequences/structures

  • Footprinting assays to map RNA-protein interactions

  • Computational docking of target rRNA sequences to the protein structure

Understanding the structural basis of substrate recognition would provide insights into how pseudouridine synthases in different fungi have evolved distinct substrate specificities despite conserved catalytic mechanisms .

How do the substrate specificities of PUS5 differ between C. glabrata and other fungal species?

The substrate specificities of PUS5 likely differ between C. glabrata and other fungal species due to evolutionary divergence:

• Comparative Substrate Analysis:

  Species	Primary Substrate	Target Position(s)	Consensus Sequence	Reference
  S. cerevisiae	Mitochondrial rRNA	Single residue	Not fully defined
  C. glabrata	Predicted: Mitochondrial rRNA	Predicted: Limited sites	To be determined	-
  C. albicans	Not specifically studied	Not determined	Not determined	-
  Other CTG clade fungi	Variable	Variable	Variable	-

• Factors Influencing Substrate Divergence:

  • Evolutionary distance (e.g., C. albicans diverged from Saccharomyces ~170 million years ago)

  • Ecological niches and associated selection pressures

  • Co-evolution with other RNA modification systems

  • Species-specific RNA structural variations

• Methodological Approach for Comparative Studies:

  • Express recombinant PUS5 from multiple species

  • Test activity on standardized RNA substrates

  • Perform cross-species complementation studies

  • Map modification sites in different species using high-throughput methods

Studies of CaPUS7 have demonstrated that despite conservation of pseudouridine synthases between budding yeast taxa, their substrates and in vivo roles can differ significantly . Similar evolutionary divergence may exist for PUS5.

How can PUS5 function be studied in the context of C. glabrata pathogenesis?

To study PUS5 function in the context of C. glabrata pathogenesis, researchers can employ a multi-faceted approach:

• Infection Models:

  • Galleria mellonella larval model, similar to that used for CgDTR1 studies

  • Murine models of disseminated candidiasis

  • Macrophage and neutrophil interaction assays in vitro

• Virulence Assessment Protocol:

  • Generate Δpus5 deletion mutants and complemented strains

  • Inject standardized inocula (~5 × 10⁷ cells) into infection models

  • Monitor survival rates over time (e.g., 72 hours)

  • Assess fungal burden in infected tissues

• Host-Pathogen Interaction Studies:

  • Phagocytosis assays with hemocytes or mammalian macrophages

  • Measurement of viable intracellular yeast cells at various time points

  • Assessment of oxidative burst responses

  • Cytokine profiling in response to wild-type vs. Δpus5 strains

• Stress Response Correlations:

  • Test susceptibility to stresses encountered in the host:

    • Oxidative stress (H₂O₂)

    • Weak acid stress (acetic acid, benzoic acid)

    • Antimicrobial peptides

    • Nutrient limitation

Based on studies of other C. glabrata genes like CgDTR1, which affects both stress resistance and virulence , PUS5 may similarly contribute to pathogenesis through its effects on mitochondrial function and cellular stress responses.

How does PUS5 interact with other RNA modification systems in C. glabrata?

PUS5's interaction with other RNA modification systems in C. glabrata represents a complex regulatory network:

• Potential Interactions with Other Modification Systems:

  • Coordinated activity with other pseudouridine synthases

  • Interdependence with methyltransferases

  • Sequential or competitive modifications at neighboring sites

  • Integration with RNA quality control mechanisms

• Experimental Approaches:

  • Generate double deletion mutants (e.g., Δpus5Δpus7)

  • Perform RNA modification profiling in various mutant backgrounds

  • Conduct protein-protein interaction studies (co-immunoprecipitation, yeast two-hybrid)

  • Analyze genetic interactions through synthetic genetic array analysis

• Regulatory Network Analysis:

  • Transcriptome analysis under various stress conditions

  • Identification of common regulatory factors

  • Assessment of modification changes in response to environmental cues

  • Computational modeling of RNA modification networks

• Evolutionary Conservation of Interactions:

  • Comparative analysis across Candida species

  • Identification of species-specific interaction patterns

  • Correlation with ecological niches and pathogenicity

Understanding these interactions would provide insights into how RNA modification systems collectively contribute to C. glabrata adaptation and virulence, potentially revealing novel therapeutic targets.

What are the challenges in expressing active recombinant PUS5 and how can they be overcome?

Expressing active recombinant PUS5 presents several challenges that researchers should address:

• Common Expression Challenges:

  • Protein misfolding or aggregation

  • Low expression levels

  • Loss of enzymatic activity

  • Improper post-translational modifications

• Optimization Strategies:

  • Expression System Selection:

    System	Advantages	Disadvantages	Recommendations
    E. coli	Fast, high yield	May lack proper folding	Try fusion tags (MBP, SUMO)
    S. cerevisiae	Native-like environment	Lower yields	Use strong inducible promoters
    C. glabrata	Most authentic	Technical challenges	MTI copper-inducible system
    Insect cells	Good for eukaryotic proteins	More complex	Consider for structural studies

  • Solubility Enhancement:

    • Optimize induction conditions (temperature, inducer concentration)

    • Use solubility-enhancing tags (MBP, SUMO, GST)

    • Co-express with molecular chaperones

  • Activity Preservation:

    • Include cofactors during purification

    • Optimize buffer conditions (pH, salt, reducing agents)

    • Minimize freeze-thaw cycles

• Expression Vector Design:

  • For expression in yeast, consider vectors similar to pGREG576 with appropriate promoters

  • Include purification tags that can be removed if needed

  • Optimize codon usage for the expression host

• Quality Control:

  • Verify protein integrity by mass spectrometry

  • Assess secondary structure by circular dichroism

  • Confirm activity with appropriate RNA substrates

These approaches have been successful for expressing other challenging fungal proteins and can be adapted for C. glabrata PUS5.

How can researchers differentiate between direct and indirect effects of PUS5 deletion?

Distinguishing between direct and indirect effects of PUS5 deletion requires careful experimental design:

• Complementation Analysis:

  • Reintroduce wild-type PUS5 to confirm phenotype rescue

  • Use a catalytically inactive mutant to distinguish enzymatic from structural roles

  • Perform cross-species complementation to assess functional conservation

• Target Site Mutation Approach:

  • Mutate specific PUS5 target sites in rRNA

  • Compare phenotypes with Δpus5 deletion

  • Identify which phenotypes are replicated by target site mutations

• Temporal Analysis:

  • Use time-course experiments to identify primary (early) versus secondary (late) effects

  • Employ inducible promoters for controlled PUS5 depletion

  • Monitor changes in RNA modification, gene expression, and phenotypes over time

• Biochemical Validation:

  • Perform in vitro assays with purified components

  • Reconstitute minimal systems to confirm direct effects

  • Use structure-function analysis to correlate specific protein features with phenotypes

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Use computational approaches to distinguish direct targets from downstream effects

  • Validate key nodes in the resulting network model

This comprehensive approach would enable researchers to build a causal model of how PUS5-mediated RNA modifications directly and indirectly affect cellular processes.

What are the potential therapeutic applications targeting PUS5 in Candida infections?

Exploring PUS5 as a therapeutic target for Candida infections presents several avenues for investigation:

• Therapeutic Potential Assessment:

  • Determine essentiality of PUS5 under infection-relevant conditions

  • Evaluate the impact of PUS5 inhibition on virulence

  • Assess species specificity to enable targeting C. glabrata over human pseudouridine synthases

• Drug Discovery Approaches:

  • High-throughput screening for small molecule inhibitors

  • Structure-based drug design targeting the catalytic domain

  • RNA mimetics that compete for the enzyme's active site

  • Allosteric inhibitors affecting enzyme conformation

• Combination Therapy Strategies:

  • Test synergy between PUS5 inhibitors and existing antifungals

  • Explore stress sensitization by PUS5 inhibition

  • Investigate immune response modulation when PUS5 function is compromised

• Target Validation Criteria:

  • Demonstrate in vivo efficacy in animal models

  • Establish pharmacokinetic and pharmacodynamic profiles

  • Assess resistance development potential

  • Evaluate effects on commensal microbiota

Given that other pseudouridine synthases and RNA modification enzymes affect stress responses and virulence in Candida species , PUS5 could represent a novel therapeutic target, particularly if it proves essential for mitochondrial function under host conditions.

How might CRISPR-Cas9 technology be applied to study PUS5 function in C. glabrata?

CRISPR-Cas9 technology offers powerful approaches for studying PUS5 function in C. glabrata:

• Gene Editing Applications:

  • Complete gene deletion using homology-directed repair

  • Introduction of point mutations to create catalytically inactive variants

  • Tagging with fluorescent proteins or epitope tags for localization and interaction studies

  • Creation of conditional alleles (e.g., degron-tagged versions)

• Implementation Strategy:

  • Design guide RNAs targeting PUS5 using C. glabrata-specific tools

  • Introduce stop codons or frameshift mutations early in the coding sequence, similar to the approach used for CaPUS7

  • Confirm edits by PCR, restriction digestion, and sequencing

  • Validate protein loss by Western blotting

• Advanced CRISPR Applications:

  • CRISPRi for tunable gene repression

  • CRISPRa for overexpression studies

  • CRISPR base editing for precise nucleotide changes

  • CRISPR screening to identify genetic interactions

• Experimental Design Considerations:

  • Include multiple guide RNAs to minimize off-target effects

  • Generate independent mutant isolates to confirm phenotypes

  • Use appropriate controls, including complemented strains

  • Consider the impact of strain background on phenotypic outcomes

This technology would enable precise genetic manipulation to elucidate PUS5 function in ways previously challenging with traditional methods in C. glabrata.