Recombinant Candida glabrata RuvB-like helicase 1 (RVB1)

What is the basic structure of Candida glabrata RuvB-like helicase 1 (RVB1)?

RVB1 from Candida glabrata is a protein of 457 amino acids with structural characteristics typical of AAA+ ATPases. According to sequence data, it contains conserved Walker A and B motifs necessary for ATP binding and hydrolysis. The protein forms hexameric structures that are essential for its ATP hydrolysis activity, and it can form dodecamers with RUVBL2 protein. The amino acid sequence includes multiple functional domains including ATP-binding regions and DNA interaction sites . The crystallographic structure reveals a core domain organization similar to other RuvB-like helicases, with the protein displaying both N-terminal and C-terminal domains connected by a flexible linker region that facilitates conformational changes during ATP hydrolysis.

How does RVB1 function in Candida glabrata at the molecular level?

RVB1 functions as an ATP-dependent DNA helicase with 3' to 5' directionality. Its activity is stimulated by single-stranded DNA, and hexamerization is critical for ATP hydrolysis, with adjacent subunits in the ring-like structure contributing to ATPase activity . In C. glabrata, RVB1 participates in several chromatin remodeling complexes, including SWR1 and INO80 complexes . These complexes are involved in multiple cellular processes including transcription regulation, DNA damage repair, and telomere maintenance. The protein's function depends on its ability to use ATP hydrolysis to drive conformational changes that enable DNA unwinding and protein complex assembly or disassembly.

Is there evidence that RVB1 affects C. glabrata's intracellular survival in host cells?

Recent research has demonstrated that C. glabrata can manipulate host proteins involved in endocytic processes and intracellular trafficking . The yeast avoids fusion of endocytic vacuoles with lysosomes and downregulates host cell autophagy in the early stages of infection, which correlates with its intracellular replication. Although RVB1 has not been directly implicated in these processes within the search results, as a chromatin remodeling factor, it may regulate genes involved in these survival mechanisms. Investigating RVB1's role in the regulation of genes related to intracellular survival would provide valuable insights into this aspect of C. glabrata pathogenicity.

What are the optimal expression systems for producing recombinant C. glabrata RVB1?

Based on commercial availability information, recombinant C. glabrata RVB1 can be produced in multiple expression systems including yeast, E. coli, baculovirus, and mammalian cells . Each system offers distinct advantages:

For functional studies requiring native-like protein activity, yeast or higher eukaryotic systems are preferable. For structural studies requiring large quantities of protein, E. coli expression followed by proper refolding protocols may be more suitable.

What purification strategies yield the highest activity for recombinant RVB1?

Effective purification of RVB1 requires a multi-step approach to maintain its oligomeric structure and enzymatic activity. A recommended protocol includes:

• Affinity chromatography using His-tag or other fusion tags (maintaining low temperatures of 4°C throughout)

• Size-exclusion chromatography to isolate hexameric forms

• Ion-exchange chromatography for final polishing

Critical factors affecting activity include:

• Buffer composition: 25-50 mM Tris-HCl pH 7.5-8.0, 150-300 mM NaCl, 1-5 mM MgCl₂ (essential for ATPase activity)

• Presence of reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain cysteine residues

• Glycerol (10-15%) to enhance stability during storage

• ATP or non-hydrolyzable ATP analogs to stabilize oligomeric structure

Protein activity should be assessed through ATPase assays and DNA helicase assays to confirm functional integrity after purification.

How does C. glabrata RVB1 differ from human RUVBL1 in structure and function?

C. glabrata RVB1 and human RUVBL1 share significant structural similarities as they both belong to the AAA+ ATPase family, but they exhibit several key differences:

These differences may provide opportunities for selective targeting in therapeutic development. Functional differences likely extend to species-specific protein-protein interactions within chromatin remodeling complexes, which could affect how the protein influences gene expression patterns related to virulence and stress response.

How does RVB1 function compare across different Candida species?

Comparative analysis of RVB1 across Candida species reveals both conservation and divergence:

Unlike C. albicans, C. glabrata lacks typical virulence factors such as hyphal development , suggesting RVB1 may play different roles in virulence between species. In C. albicans, chromatin remodeling factors influence morphological transitions important for virulence, such as the yeast-to-filament transition regulated by factors like Yak1 . The differential roles of RVB1 across Candida species likely reflect their divergent evolutionary adaptations to various host niches and immune pressures.

How can researchers effectively study RVB1's role in regulating the C. glabrata adhesin repertoire?

To investigate RVB1's role in regulating C. glabrata adhesins, researchers should implement a multi-faceted approach:

• Gene knockout and conditional expression systems:

  • CRISPR-Cas9 deletion of RVB1

  • Tetracycline-regulated expression systems to control RVB1 levels

  • Point mutations in key functional domains (Walker A/B motifs)

• Genome-wide analysis techniques:

  • ChIP-seq to identify RVB1 binding sites near adhesin genes

  • RNA-seq comparing wild-type and RVB1-depleted strains

  • ATAC-seq to assess chromatin accessibility changes at adhesin gene loci

• Functional assays:

  • Adhesion assays to various cell types (e.g., HeLa, HaCaT cells)

  • Biofilm formation quantification

  • In vivo infection models such as Galleria mellonella

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify RVB1 partners in C. glabrata

  • Proximity labeling techniques (BioID, APEX) to map the RVB1 interactome

  • Yeast two-hybrid screening focused on transcriptional regulators

Such comprehensive analysis would reveal how RVB1-containing chromatin remodeling complexes influence the expression of the extensive adhesin repertoire, which includes at least 22 paralogs of Awp1 and 20 paralogs of Epa1-like adhesins .

What approaches can resolve contradictory findings about RVB1's impact on antifungal susceptibility?

Contradictory findings regarding RVB1's impact on antifungal susceptibility can be addressed through:

• Standardized susceptibility testing:

  • Implement CLSI or EUCAST standardized methodologies

  • Test multiple antifungal classes (azoles, echinocandins, polyenes)

  • Use both planktonic and biofilm growth conditions

• Context-specific analysis:

  • Evaluate susceptibility under different environmental conditions (pH, nutrient availability)

  • Assess impact of host factors (serum proteins, immune cells)

  • Examine strain-specific differences using clinical isolates with varying resistance profiles

• Mechanistic investigations:

  • Quantify expression of drug efflux pumps in RVB1-depleted strains

  • Measure ergosterol content and membrane composition

  • Assess cell wall architecture and β-glucan exposure

• Combined genetic approaches:

  • Create double mutants with known resistance factors (e.g., PDR1 )

  • Perform suppressor screens to identify genetic interactions

  • Use heterologous expression in S. cerevisiae to isolate specific functions

• Advanced microscopy techniques:

  • Track antifungal drug localization in RVB1-depleted cells

  • Monitor real-time membrane damage responses

This systematic approach would help reconcile contradictory findings by identifying condition-specific effects and elucidating the mechanistic basis of any observed phenotypes.

What structural features of RVB1 could be exploited for selective antifungal drug development?

Analysis of C. glabrata RVB1 structure reveals several potential target sites for selective inhibition:

• ATP-binding pocket uniqueness:

  • The ATP-binding domain contains fungal-specific residues that differ from human RUVBL1

  • These differences can be exploited to design nucleotide analogs with selective binding

• Interface regions for oligomerization:

  • Hexamer formation is essential for RVB1 function

  • Peptide mimetics targeting oligomerization interfaces could disrupt assembly

• DNA-binding domain:

  • Structural differences in the DNA-binding regions between fungal and human proteins

  • Small molecules that interfere with DNA-RVB1 interactions may show selectivity

• Protein-protein interaction surfaces:

  • Species-specific interaction sites with chromatin remodeling complex components

  • Targeting these interfaces could disrupt fungal-specific protein complexes

Computational analysis using molecular dynamics simulations and virtual screening against these sites could identify lead compounds with selective activity against fungal RVB1 while sparing human RUVBL1.

How can researchers address the challenge of targeting nuclear proteins like RVB1 in antifungal development?

Targeting nuclear proteins like RVB1 presents unique challenges that can be addressed through:

• Drug delivery strategies:

  • Conjugation to cell-penetrating peptides

  • Lipid-based nanoparticle formulations

  • Pro-drug approaches that leverage fungal-specific metabolic activation

• Target validation methodologies:

  • Chemical genetics using analog-sensitive RVB1 mutants

  • Inducible protein degradation systems (e.g., auxin-inducible degron)

  • PROTAC-like approaches adapted for fungal systems

• Phenotypic screening refinements:

  • High-content imaging assays focused on nuclear processes

  • Reporter systems linked to RVB1-dependent chromatin remodeling

  • Growth inhibition assays under conditions that increase dependency on RVB1

• Combination approaches:

  • Co-targeting cell wall/membrane components to enhance nuclear delivery

  • Synergistic targeting of multiple components in the same chromatin remodeling complex

  • Sequential treatment strategies that create synthetic dependencies on RVB1 function

These approaches would help overcome the challenges of targeting nuclear proteins while maintaining the selectivity necessary for antifungal therapeutics with minimal host toxicity.

How can researchers design experiments to distinguish between direct and indirect effects of RVB1 on virulence gene expression?

Distinguishing direct from indirect effects of RVB1 on virulence gene expression requires sophisticated experimental designs:

• Temporal control systems:

  • Auxin-inducible degron tagging of RVB1 for rapid protein depletion

  • Time-course analysis of transcriptional changes following RVB1 depletion

  • Pulse-chase experiments with labeled nucleotides to identify immediate transcriptional effects

• Genomic approaches:

  • ChIP-seq with spike-in normalization to quantify absolute RVB1 occupancy

  • CUT&RUN or CUT&Tag for higher resolution mapping of binding sites

  • ChEC-seq (chromatin endogenous cleavage) to identify direct DNA contacts

• Genetic engineering:

  • Domain-specific mutations that separate chromatin remodeling from other functions

  • Anchor-away systems to rapidly relocalize RVB1 from the nucleus

  • Tethering RVB1 to specific genomic loci using dCas9-RVB1 fusions

• Single-cell approaches:

  • scRNA-seq to capture heterogeneity in responses to RVB1 perturbation

  • Live-cell imaging with fluorescent reporters for virulence genes

  • smFISH (single-molecule fluorescence in situ hybridization) to detect nascent transcripts

These approaches would help establish causal relationships between RVB1 activity and virulence gene expression patterns, distinguishing direct regulatory roles from secondary effects due to broader cellular perturbations.

What are the most rigorous methods to evaluate RVB1's contribution to antifungal resistance mechanisms?

Rigorous evaluation of RVB1's contribution to antifungal resistance requires:

• Evolution experiments:

  • Laboratory evolution of resistance in wild-type vs. RVB1-depleted strains

  • Whole genome sequencing to identify compensatory mutations

  • Competition assays to assess fitness costs of resistance mechanisms

• Clinical isolate analysis:

  • Sequencing RVB1 and associated factors in resistant clinical isolates

  • Correlation of RVB1 expression levels with minimum inhibitory concentrations

  • Complementation studies with wild-type RVB1 in resistant isolates

• Mechanistic approaches:

  • Ribosome profiling to assess translational responses to antifungals

  • Metabolomics to identify altered metabolic pathways in resistant strains

  • Chromatin accessibility mapping at drug efflux pump loci

• Direct measurement techniques:

  • Intracellular drug concentration quantification

  • Real-time monitoring of efflux pump activity

  • Assessment of changes in membrane composition and permeability

• In vivo resistance development:

  • Mouse models of persistent infection during antifungal treatment

  • Ex vivo analysis of resistant populations

  • Host-pathogen transcriptomics during treatment failure