Recombinant Candida glabrata Ubiquitin carboxyl-terminal hydrolase 4 (DOA4), partial

What is the biological function of DOA4 in Candida glabrata?

DOA4 in Candida glabrata functions as a deubiquitinating enzyme (DUB) that cleaves ubiquitin from protein substrates. Similar to its ortholog in Saccharomyces cerevisiae, it plays critical roles in recycling ubiquitin from proteins targeted for degradation by the proteasome, maintaining free ubiquitin pools, and ensuring proper protein turnover . DOA4 contributes to stress response mechanisms, which is particularly relevant given C. glabrata's adaptation to various stress conditions as a pathogen. The enzyme is likely involved in multiple cellular pathways including protein degradation, cell cycle regulation, and potentially virulence-related processes.

What role might DOA4 play in C. glabrata pathogenicity?

DOA4 likely contributes to C. glabrata pathogenicity through multiple mechanisms. As a deubiquitinating enzyme, it may regulate the stability and function of virulence factors through the ubiquitin-proteasome system. Research on other fungal pathogens suggests that proper ubiquitin recycling is essential for stress adaptation, biofilm formation, and host-pathogen interactions . Given that C. glabrata is known for its robust stress responses and adherence capabilities, DOA4 may regulate these critical virulence traits. Disruption of the ubiquitin homeostasis system in pathogens typically results in attenuated virulence, suggesting DOA4's importance in pathogenicity.

How does DOA4 interact with the SUMOylation machinery in C. glabrata?

DOA4 in C. glabrata likely has complex interactions with the SUMOylation machinery, potentially creating regulatory crosstalk between the ubiquitin and SUMO pathways. Research indicates that SUMOylation is essential for C. glabrata growth, division, and stress adaptation . DOA4 may play a role in regulating the stability or activity of SUMOylation machinery components. This interaction is particularly significant as SUMOylation has been shown to be crucial for virulence in C. glabrata, with loss of the deSUMOylating enzyme CgUlp2 leading to impaired growth, stress sensitivity, reduced adherence to epithelial cells, and poor tissue colonization in mouse models . The functional relationship between DOA4 and SUMOylation machinery represents an important area for investigation in understanding C. glabrata pathobiology.

What is the regulatory relationship between CgRpn4 and DOA4 in antifungal resistance?

The transcription factor CgRpn4 is a significant determinant of azole drug resistance in C. glabrata . While direct evidence of DOA4 regulation by CgRpn4 is not explicitly stated in the available research, there is a strong possibility of a regulatory relationship given that CgRpn4 regulates the expression of proteasome genes . Since DOA4 functions in the ubiquitin-proteasome system, it may be among the 212 genes regulated by CgRpn4, potentially in the set of 80 genes that are activated by this transcription factor during fluconazole exposure . This regulatory relationship could be crucial for understanding how C. glabrata modulates protein degradation pathways in response to azole antifungals, potentially contributing to drug resistance mechanisms.

How does recombinant DOA4 expression affect C. glabrata's response to environmental stressors?

Recombinant expression of DOA4 in C. glabrata likely modifies the organism's response to various environmental stressors through alteration of protein degradation dynamics. Overexpression or altered variants of DOA4 may enhance stress tolerance by ensuring efficient recycling of ubiquitin and proper proteasomal function. Research on related stress response mechanisms shows that C. glabrata has evolved robust adaptation strategies to oxidative and other stresses . DOA4 likely contributes to these mechanisms by regulating the turnover of stress-responsive proteins. The precise effects would depend on the specific recombinant construct and expression levels, potentially leading to enhanced survival under conditions such as oxidative stress, antifungal exposure, or nutrient limitation.

What is the optimal strategy for CRISPR-Cas9 mediated knockout of DOA4 in C. glabrata?

For efficient CRISPR-Cas9 mediated knockout of DOA4 in C. glabrata, researchers should employ a recombinant strain constitutively expressing the CRISPR-Cas9 system, such as the ∆HTL + CAS9 strain described in the literature . The optimal strategy includes:

• Guide RNA selection: Use specialized online tools to identify efficient guide RNAs targeting the DOA4 gene in C. glabrata, focusing on regions with minimal off-target effects .

• Homology-directed repair: Prepare a donor DNA with homology arms flanking the DOA4 target site. Research indicates that using 200 bp homology domains (HD) increases recombination frequency by up to 8-fold compared to using 20 bp HDs .

• Selection marker: Design the donor DNA to include a selection marker (such as XTAG or HIS3) to facilitate identification of successful transformants .

• Verification method: Employ the Surveyor technique and sequencing to confirm successful gene knockout .

This approach has been validated for gene disruption in C. glabrata with high efficiency and specificity, making it suitable for DOA4 knockout studies.

What expression systems are most effective for producing recombinant C. glabrata DOA4?

Based on established protocols for C. glabrata genetic manipulation, the following expression systems are most effective for producing recombinant DOA4:

What is the recommended protocol for purifying recombinant DOA4 while maintaining its enzymatic activity?

The following protocol is recommended for purifying recombinant C. glabrata DOA4 while preserving its deubiquitinating enzyme activity:

• Expression construct design: Include a dual affinity tag system (His6-tag and FLAG-tag) separated by a precision protease cleavage site to facilitate purification and tag removal.

• Cell lysis conditions: Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5% glycerol, 1 mM DTT, and protease inhibitor cocktail without serine protease inhibitors (which could affect DOA4 activity).

• Purification steps:

  • Initial capture: Ni-NTA affinity chromatography

  • Intermediate purification: Anti-FLAG affinity chromatography

  • Polishing: Size exclusion chromatography using Superdex 200

• Activity preservation measures:

  • Maintain temperature at 4°C throughout purification

  • Include 5% glycerol in all buffers to stabilize protein structure

  • Add 1 mM DTT to prevent oxidation of catalytic cysteine residues

  • Avoid freeze-thaw cycles; store purified protein at -80°C in single-use aliquots

• Activity verification: Test purified DOA4 using a fluorogenic ubiquitin substrate (Ub-AMC) assay to confirm retention of enzymatic activity.

This protocol balances high purity with preservation of enzymatic activity, which is essential for functional studies of recombinant DOA4.

How can researchers distinguish between direct and indirect effects of DOA4 deletion on C. glabrata phenotypes?

Distinguishing between direct and indirect effects of DOA4 deletion requires a multi-faceted experimental approach:

• Complementation studies: Reintroduce wild-type DOA4 or catalytically inactive mutants into the deletion strain. Phenotypes rescued only by the active enzyme are likely direct effects of DOA4 function .

• Temporal analysis: Monitor changes in phenotypes, gene expression, and protein levels at multiple time points after DOA4 deletion. Early changes are more likely to be direct effects, while later changes may represent secondary adaptations.

• Substrate identification: Use mass spectrometry-based proteomics to identify proteins with altered ubiquitination status in DOA4 deletion strains. These represent potential direct substrates of DOA4.

• Pathway analysis: Employ RNA-seq to identify differentially expressed genes in DOA4 mutants, similar to the approach used for CgRpn4 . This helps map the regulatory networks affected by DOA4 deletion.

• Epistasis analysis: Create double mutants of DOA4 with genes in related pathways to determine functional relationships and hierarchy of effects.

By integrating these approaches, researchers can build a comprehensive model distinguishing primary effects of DOA4 activity from downstream consequences of ubiquitin homeostasis disruption.

What statistical methods are most appropriate for analyzing DOA4-related virulence data from in vivo infection models?

For analyzing DOA4-related virulence data from in vivo infection models such as the Drosophila melanogaster system used in C. glabrata studies , the following statistical methods are most appropriate:

• Survival analysis: Kaplan-Meier survival curves with log-rank tests for comparing mortality rates between wild-type and DOA4 mutant infections. This approach accounts for time-to-event data and censored observations.

• Colony-forming unit (CFU) comparisons: Two-way ANOVA with Tukey's post-hoc test for comparing fungal burden across different organs and time points, incorporating both strain and time as variables .

• Multivariate analysis: Principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) to identify patterns in complex datasets involving multiple virulence parameters.

• Linear mixed-effects models: For longitudinal studies tracking infection progression over time, accounting for both fixed effects (strain, treatment) and random effects (individual host variation).

• Power analysis: A priori determination of sample sizes needed to detect meaningful differences in virulence with at least 80% power at α = 0.05.

The appropriate statistical method depends on the specific experimental design and data structure, but these approaches provide robust frameworks for analyzing DOA4's impact on virulence.

How should researchers interpret changes in global ubiquitination patterns following DOA4 manipulation?

Interpreting changes in global ubiquitination patterns following DOA4 manipulation requires careful consideration of several factors:

• Pattern classification: Categorize ubiquitination changes into:

  • Accumulation of K48-linked polyubiquitin chains (suggesting impaired proteasomal targeting)

  • Changes in K63-linked chains (indicating altered endocytosis or DNA repair pathways)

  • Alterations in monoubiquitination (affecting protein localization or activity)

• Functional context: Analyze affected proteins using Gene Ontology enrichment to identify biological processes disproportionately impacted by DOA4 manipulation.

• Temporal dynamics: Distinguish between immediate ubiquitination changes (direct DOA4 effects) and adaptive responses (secondary effects) by examining multiple time points.

• Stress response correlation: Correlate ubiquitination changes with stress response pathways known to be important in C. glabrata pathogenesis, similar to those regulated by the SUMOylation machinery or the CgRpn4 transcription factor .

• Pathway integration: Interpret ubiquitination changes in context of other post-translational modifications, particularly SUMOylation, which has established importance in C. glabrata stress response and virulence .

This multi-layered interpretation approach allows researchers to move beyond simply cataloging ubiquitination changes to understanding their functional significance in C. glabrata biology and pathogenicity.

What are common challenges in expressing functional recombinant DOA4 and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant DOA4 in C. glabrata. The following table outlines these issues and their solutions:

Additionally, when using CRISPR-Cas9 for gene manipulation, researchers should be aware that expressing Cas9 can lead to increased generation time, as observed in the ∆HTL + CAS9 strain . This growth defect should be considered when designing experiments and interpreting results.

How can researchers address antifungal resistance variations when studying DOA4 in clinical C. glabrata isolates?

When studying DOA4 in clinical C. glabrata isolates, addressing antifungal resistance variations requires a systematic approach:

• Baseline characterization: Determine minimum inhibitory concentrations (MICs) for multiple antifungals against each clinical isolate before any genetic manipulation. This establishes the inherent resistance profile of each strain.

• Genetic background analysis: Sequence key resistance genes, including those regulated by CgRpn4 (such as ergosterol biosynthesis genes ERG1, ERG2, ERG3, and ERG11) , to identify pre-existing mutations that might influence results.

• Standardized manipulation: Use identical genetic modification strategies across all isolates, preferably employing CRISPR-Cas9 with 200 bp homology domains for optimal recombination efficiency .

• Isogenic controls: Generate appropriate control strains for each clinical isolate by reintroducing the wild-type DOA4 gene at its native locus after deletion.

• Multifactorial testing: Evaluate resistance under various conditions (pH, temperature, nutrient availability) as C. glabrata's stress response pathways interact with drug resistance mechanisms .

• Transcriptional profiling: Perform RNA-seq to identify differential gene expression patterns between isolates, focusing on known resistance genes and pathways regulated by transcription factors like CgRpn4 .

This comprehensive approach accounts for the inherent variability in clinical isolates while ensuring that observed phenotypes can be reliably attributed to DOA4 manipulation.

What considerations are important when designing deubiquitinating activity assays for recombinant DOA4?

When designing deubiquitinating activity assays for recombinant C. glabrata DOA4, researchers should consider:

• Substrate specificity:

  • Use multiple ubiquitin chain types (K48, K63, linear) as DOA4 may have preferential activity

  • Include both artificial substrates (Ub-AMC) for quantitative kinetics and physiological substrates for biological relevance

  • Consider testing ubiquitin-like modifiers (SUMO, NEDD8) to assess cross-reactivity

• Assay conditions optimization:

  • Buffer composition: Test various pH conditions (typically pH 7.5-8.5)

  • Ionic strength: Optimize NaCl concentration (usually 50-150 mM)

  • Reducing agents: Include DTT (1-5 mM) to maintain catalytic cysteine activity

  • Temperature: Perform assays at both 30°C (optimal for yeast) and 37°C (host temperature)

• Controls and validation:

  • Catalytically inactive mutant (C→S mutation in active site) as negative control

  • Commercial DUBs of known activity as positive controls

  • Specific DUB inhibitors to confirm activity specificity

  • Assessment of contaminating DUB activity in preparations

• Detection methods:

  • Fluorescence-based assays for high sensitivity and real-time kinetics

  • Gel-based assays with immunoblotting for visualization of cleavage products

  • Mass spectrometry for precise identification of cleavage sites

These considerations ensure that the assay provides both scientifically valid and biologically relevant information about DOA4 activity, crucial for understanding its role in C. glabrata biology.

How does C. glabrata DOA4 function compare to homologs in other Candida species and pathogenic fungi?

A comparative analysis of DOA4 across various Candida species and pathogenic fungi reveals important functional similarities and differences:

What insights can be gained from studying the interaction between DOA4 and the SUMOylation machinery in C. glabrata compared to model organisms?

Studying the interaction between DOA4 and the SUMOylation machinery in C. glabrata provides several unique insights compared to model organisms:

• Pathogenicity-specific interactions: Unlike in S. cerevisiae, the DOA4-SUMOylation interplay in C. glabrata likely evolved to support pathogenesis. Research has shown that SUMOylation is essential for C. glabrata stress adaptation and virulence, with the deSUMOylating enzyme CgUlp2 being crucial for adherence to epithelial cells and tissue colonization .

• Stress response regulation: C. glabrata has a more robust stress response than S. cerevisiae, allowing it to survive in diverse host environments. The coordination between deubiquitination (via DOA4) and SUMOylation likely contributes to this enhanced stress tolerance, particularly for oxidative stress that C. glabrata encounters during host infection .

• Ubiquitin-SUMO crosstalk mechanisms: C. glabrata may employ unique crosstalk mechanisms between the ubiquitin and SUMO pathways to regulate virulence factors. This crosstalk could involve:

  • SUMO-targeted ubiquitin ligases (STUbLs) that recognize SUMOylated proteins

  • Competitive modification of the same lysine residues by ubiquitin or SUMO

  • Sequential modification cascades where one modification facilitates the other

• Therapeutic target potential: Understanding these interactions may reveal C. glabrata-specific vulnerabilities that could be exploited for antifungal development, particularly given the increasing resistance to conventional antifungals and the role of SUMOylation in azole resistance .

The comparison with model organisms highlights how core post-translational modification machineries have been adapted in C. glabrata to support its pathogenic lifestyle.

How do the functions of DOA4 in C. glabrata relate to azole resistance mechanisms compared to other resistance determinants?

The functions of DOA4 in C. glabrata likely contribute to azole resistance through several mechanisms that complement other known resistance determinants:

• Proteasomal regulation pathway: DOA4 functions within the ubiquitin-proteasome system, which intersects with the pathways regulated by the transcription factor CgRpn4. Research has shown that CgRpn4 regulates 212 genes during fluconazole exposure, including several proteasome and ergosterol biosynthesis genes (ERG1, ERG2, ERG3, and ERG11) . DOA4 may contribute to the stability and function of these resistance-related proteins.

• Comparative contribution to resistance:

• Stress response integration: DOA4's role in ubiquitin recycling likely supports the cellular stress response network that contributes to azole tolerance. Research on SUMOylation in C. glabrata has demonstrated that post-translational modification systems are essential for stress adaptation , suggesting DOA4 may similarly contribute to stress tolerance mechanisms that support azole resistance.

• Protein quality control: DOA4 contributes to protein quality control through the ubiquitin-proteasome system, which may help C. glabrata cope with proteotoxic stress induced by azoles. This function potentially complements the role of CgRpn4, which has been shown to be a determinant of azole drug resistance .

This multifaceted relationship positions DOA4 as a component of the broader cellular machinery that contributes to C. glabrata's notable ability to develop azole resistance.