Recombinant Candida glabrata ATP-dependent rRNA helicase RRP3 (RRP3)

What is the basic structure and function of C. glabrata RRP3 helicase?

RRP3 is an ATP-dependent RNA helicase encoded by the RRP3 gene in Candida glabrata. It belongs to the DEAD-box helicase family and is primarily involved in ribosomal RNA processing. The protein has a UniProt accession number Q6FNK8 and includes characteristic ATP-binding motifs and RNA-binding domains .

The protein sequence begins with "MAGKVGKVSK KSDDVSSLAA KIRARALENQ KKMQAASRKD SESDSSDEEV ERPAKKQAKD EKVEEPEEEV TFESFAQLNL VPELIQACQN LNFTKPTPIQ ARAIPPALAG SDVIGLAQTG SGKTAAFAIP ILNKLWEDQQ PYYACVLAPT" and contains the conserved sequence motifs typical of DEAD-box helicases . These motifs are essential for its ATP-dependent unwinding activity of RNA secondary structures during ribosome biogenesis.

What are the optimal storage conditions for recombinant RRP3 protein?

For optimal stability and activity retention, storage conditions for recombinant RRP3 protein vary depending on the formulation:

The shelf life is influenced by multiple factors including buffer ingredients, storage temperature, and the intrinsic stability of the protein itself . For research applications requiring long-term storage, the lyophilized form is preferable due to its extended shelf life.

How is the purity of recombinant RRP3 typically assessed?

The purity of recombinant RRP3 is commonly assessed using SDS-PAGE, with commercial preparations typically achieving >85% purity . For researchers requiring higher purity levels for specialized applications, additional purification steps may be necessary:

• Size exclusion chromatography to separate based on molecular weight

• Ion exchange chromatography to separate based on charge differences

• Affinity chromatography using ATP-binding properties

• Western blot analysis with RRP3-specific antibodies to confirm identity

When interpreting experimental results, it's important to consider the purity level and potential effects of contaminants, particularly when studying enzymatic activities or protein-protein interactions.

How does RRP3 helicase activity contribute to C. glabrata drug resistance mechanisms?

The RRP3 helicase likely contributes to C. glabrata drug resistance through several interconnected mechanisms, though this relationship is still being fully elucidated:

• Ribosomal RNA processing: As an ATP-dependent rRNA helicase, RRP3 plays a critical role in ribosome biogenesis. Alterations in ribosome assembly can affect translation of key resistance genes .

• Stress response modulation: RNA helicases often function in stress response pathways. C. glabrata is known to generate strong stress responses against reactive oxygen species (ROS), enabling it to survive within phagosomes . RRP3 may contribute to this stress adaptation.

• Genetic plasticity: C. glabrata demonstrates remarkable genetic diversity and microevolution within patients, particularly affecting cell surface proteins and drug resistance genes like ERG4 and FKS1/2 . RNA helicases may facilitate RNA metabolism changes needed for this adaptability.

Experimental approaches to investigate these connections include:

• Gene knockout/knockdown studies comparing wild-type and RRP3-deficient strains

• Transcriptomic analysis under antifungal pressure

• Biochemical assays measuring helicase activity in the presence of antifungals

What experimental approaches are most effective for studying RRP3 interactions with other cellular components?

Several complementary approaches can effectively elucidate RRP3's interactions with other cellular components:

When designing these experiments, it's crucial to consider the native conditions in which RRP3 functions, including ATP concentration, RNA substrates, and potential regulatory partners. For instance, when performing Co-IP experiments, using mild detergents and physiological salt concentrations helps preserve native interactions .

How does RRP3 expression vary across different C. glabrata genetic backgrounds and under different stress conditions?

C. glabrata demonstrates significant genetic diversity with at least 19 distinct sequence types identified globally, plus newly discovered variants . This genetic heterogeneity likely affects RRP3 expression and function:

• Strain-dependent variation: Different clinical isolates show variations in gene expression patterns that may affect RRP3 levels and activity. Research approaches should include:

  • qRT-PCR analysis across multiple clinical isolates

  • Western blot quantification of protein levels

  • Functional assays for helicase activity

• Stress-induced regulation: C. glabrata adapts to various stresses including antifungal exposure, nutrient limitation, and host immune responses. RRP3 expression may be modulated under these conditions:

  • Oxidative stress (relevant to phagocyte survival)

  • Azole exposure (connects to drug resistance mechanisms)

  • Nutrient deprivation (common in biofilm environments)

• Host-pathogen interface: Expression may change during infection progression, particularly as C. glabrata adapts to different host niches and immune pressures .

Researchers should design experiments that capture these variables by using multiple strains and creating relevant in vitro stress conditions that mimic the host environment.

What are the key considerations when designing RRP3 helicase activity assays?

Designing robust helicase activity assays for RRP3 requires attention to several critical factors:

• Substrate selection:

  • RNA structure (single vs. double-stranded regions)

  • Length and sequence composition

  • Labeled vs. unlabeled substrates

  • Natural rRNA targets vs. synthetic substrates

• Reaction conditions optimization:

  • ATP concentration and regeneration system

  • Divalent cation requirements (typically Mg²⁺)

  • pH and buffer composition

  • Temperature (physiological relevance)

  • Presence of RNase inhibitors

• Detection methods:

  • Fluorescence-based unwinding assays

  • Gel-shift assays for direct visualization

  • FRET-based real-time monitoring

  • Radiolabeled substrate tracking

A standardized approach involves using dual-labeled RNA substrates with a fluorophore and quencher, where helicase activity separates the strands and generates a measurable fluorescence signal. This allows for continuous real-time monitoring of activity and is amenable to high-throughput screening applications.

How can researchers effectively study the impact of RRP3 mutations on C. glabrata pathogenicity?

Investigating the relationship between RRP3 mutations and pathogenicity requires a multi-faceted approach:

• Genetic engineering strategies:

  • CRISPR-Cas9 for precise genomic editing

  • Homologous recombination for allele replacement

  • Conditional expression systems for essential genes

• In vitro virulence assays:

  • Adhesion to epithelial cells (C. glabrata shows enrichment for epithelial adhesins)

  • Biofilm formation capacity (connected to antifungal resistance)

  • Survival within macrophages (C. glabrata can survive within phagosomes)

  • Growth rates under various stress conditions

• In vivo infection models:

  • Murine systemic infection models

  • Tissue-specific infection models

  • Host-pathogen interaction analysis

• Molecular phenotyping:

  • Transcriptomics to identify affected pathways

  • Proteomics to detect compensatory mechanisms

  • Metabolomics to assess physiological changes

When analyzing results, researchers should be mindful of C. glabrata's remarkable adaptability and microevolution capacity within hosts, which can complicate interpretation of pathogenicity data . Additionally, the connection between RRP3 function and known virulence factors (proteases, phospholipases, hemolysins, and biofilm formation) should be specifically examined .

What techniques are most appropriate for studying RRP3's role in ribosome biogenesis in C. glabrata?

To elucidate RRP3's specific functions in ribosome biogenesis:

• Ribosome profiling and assembly analysis:

  • Sucrose gradient fractionation of ribosomes

  • Northern blot analysis of rRNA processing intermediates

  • Pulse-chase labeling of rRNA

  • Mass spectrometry of ribosomal subunits

• RNA-protein interaction mapping:

  • CLIP-seq (Cross-linking immunoprecipitation) to identify binding sites

  • RNA footprinting to detect structural changes

  • Secondary structure mapping (SHAPE-seq, DMS-seq)

• Functional complementation:

  • Expression of mutant variants to identify essential domains

  • Heterologous expression in S. cerevisiae helicase mutants

  • Depletion/replenishment experiments

• High-resolution imaging:

  • Fluorescence microscopy for localization

  • Immuno-electron microscopy for ultrastructural analysis

  • Live-cell imaging to track dynamics

The interpretation of results should consider the highly conserved nature of ribosome biogenesis pathways while remaining alert to C. glabrata-specific adaptations that may have evolved to support its pathogenic lifestyle.

How should researchers address data inconsistencies when studying RRP3 in different C. glabrata clinical isolates?

When confronting data inconsistencies across clinical isolates, consider these methodological approaches:

• Genetic background characterization:

  • Whole-genome sequencing to identify strain-specific variations

  • Determination of sequence type (ST) using established markers

  • Analysis of key regulatory elements affecting RRP3 expression

• Statistical approaches:

  • Increased biological replicates to account for strain variability

  • Mixed-effects models that include strain as a random effect

  • Meta-analysis techniques for combining heterogeneous data

• Experimental design modifications:

  • Inclusion of reference strains (ATCC 2001/CBS 138/JCM 3761)

  • Standardization of growth conditions and experimental protocols

  • Creation of isogenic strains differing only in RRP3 alleles

• Data reporting standards:

  • Detailed documentation of strain provenance and characteristics

  • Comprehensive reporting of experimental conditions

  • Transparent sharing of raw data for community reanalysis

C. glabrata's established genetic diversity (with at least 19 sequence types globally) makes this challenge particularly relevant. Some isolates show evidence of ancestral recombination, suggesting genetic exchange between geographically distinct populations that may affect RRP3 function and regulation.

What are the best practices for validating RRP3's specific contributions to drug resistance phenotypes?

Establishing causality between RRP3 and drug resistance requires rigorous validation approaches:

• Genetic manipulation experiments:

  • Gene deletion/complementation (phenotype rescue)

  • Point mutations in functional domains

  • Overexpression studies

  • Promoter swapping to alter expression levels

• Pharmacological validation:

  • Specific inhibitors of helicase activity

  • Combination studies with antifungals

  • Dose-response relationship analysis

  • Time-kill curves with and without helicase inhibition

• Clinical correlation studies:

  • Analysis of RRP3 mutations/expression in resistant clinical isolates

  • Longitudinal studies during treatment and resistance development

  • Correlation with known resistance mutations (e.g., in ERG11, PDR1)

• Mechanistic validation:

  • Transcriptomic changes upon RRP3 modulation

  • Direct measurement of antifungal drug accumulation

  • Assessment of target enzyme activities (e.g., ergosterol biosynthesis)

When interpreting results, consider C. glabrata's multiple resistance mechanisms, including overexpression of drug transporters, biofilm formation, and modifications to cell wall proteins , which may interact with or mask RRP3-specific effects.

How can researchers differentiate between direct and indirect effects of RRP3 on C. glabrata virulence pathways?

Distinguishing direct from indirect effects requires sophisticated experimental designs:

• Temporal resolution approaches:

  • Time-course experiments with fine granularity

  • Inducible expression systems with rapid kinetics

  • Pulse-chase labeling of RNA/proteins to track synthesis and turnover

• Spatial resolution techniques:

  • Subcellular fractionation

  • Proximity labeling of interacting partners

  • Super-resolution microscopy for co-localization

• Biochemical validation:

  • In vitro reconstitution of minimal systems

  • Direct binding assays with purified components

  • Structure-function analyses with domain mutants

• Computational approaches:

  • Network analysis to identify direct vs. indirect connections

  • Predictive modeling based on multi-omics data

  • Causal inference statistical methods

When interpreting results, researchers should be aware that C. glabrata possesses multiple virulence factors, including proteases, phospholipases, hemolysins, and biofilm formation capabilities . RRP3's role as an RNA helicase may affect multiple pathways simultaneously through its influence on RNA metabolism and translation.

What are the emerging technologies that could advance our understanding of RRP3's function in C. glabrata?

Several cutting-edge technologies could significantly enhance RRP3 research:

• CRISPR interference (CRISPRi) and activation (CRISPRa):

  • Allows tunable gene expression modulation

  • Enables tissue-specific or temporal control

  • Permits screening approaches for genetic interactions

• Single-cell technologies:

  • Single-cell RNA-seq to capture population heterogeneity

  • Single-cell proteomics for protein-level analysis

  • Microfluidic approaches for isolation and manipulation

• Structural biology advances:

  • Cryo-electron microscopy for complex assemblies

  • Integrative structural biology combining multiple data types

  • Computational modeling and molecular dynamics simulations

• Synthetic biology approaches:

  • Engineered RRP3 variants with novel properties

  • Biosensors for helicase activity in living cells

  • Optogenetic control of RRP3 function

These technologies could help address key knowledge gaps, such as understanding how C. glabrata's genetic diversity affects RRP3 function across different clinical isolates and elucidating RRP3's potential role in the remarkable stress response capabilities that allow C. glabrata to survive within phagosomes .

How might insights from RRP3 research contribute to novel antifungal strategies?

Understanding RRP3's functions could inform several therapeutic strategies:

• Direct targeting approaches:

  • Small molecule inhibitors of helicase activity

  • Peptide-based inhibitors of protein-protein interactions

  • Antisense oligonucleotides to modulate expression

• Combination therapy strategies:

  • Helicase inhibitors as resistance-breaking adjuvants

  • Targeting synergistic pathways identified through RRP3 studies

  • Sequential therapy regimens based on adaptation mechanisms

• Biomarker development:

  • RRP3 expression/mutation as a predictor of treatment response

  • Monitoring changes during treatment for resistance emergence

  • Personalized therapy selection based on strain characteristics

• Immunomodulatory approaches:

  • Targeting RRP3-dependent immune evasion mechanisms

  • Boosting host responses that counteract RRP3 functions

  • Vaccine strategies incorporating RRP3-derived epitopes

These approaches align with emerging needs in antifungal development, as current drug resistance patterns in C. glabrata vary geographically and can reach concerning levels (up to 15% for azoles), with prior azole exposure increasing susceptibility . Novel therapeutics targeting multiple pathways simultaneously will be vital to circumventing the sophisticated resistance mechanisms of C. glabrata .

What are the key research questions that remain unresolved regarding RRP3's role in C. glabrata biology?

Despite advances in C. glabrata research, several critical questions about RRP3 remain unanswered:

• Evolutionary considerations:

  • How has RRP3 evolved in C. glabrata compared to other Candida species?

  • Does sequence variation in RRP3 contribute to the unique pathogenicity traits of C. glabrata?

  • Are there selective pressures on RRP3 during host adaptation and antifungal exposure?

• Regulatory networks:

  • What transcription factors and signaling pathways regulate RRP3 expression?

  • How does RRP3 activity integrate with known stress response pathways?

  • Does RRP3 participate in quorum sensing or other population-level phenomena?

• Host-pathogen interface:

  • Does RRP3 influence recognition by host immune receptors?

  • How does RRP3 activity change during different stages of infection?

  • Can host factors directly modulate RRP3 function?

• Translational aspects:

  • Could RRP3 serve as a biomarker for predicting treatment outcomes?

  • Are there patient-specific factors that influence RRP3-dependent pathogenicity?

  • How does RRP3 contribute to C. glabrata's ability to survive within phagosomes?

Addressing these questions will require interdisciplinary approaches and consideration of C. glabrata's remarkable genetic diversity and adaptability , as well as its sophisticated virulence mechanisms that allow for evasion of host immune responses .