Recombinant Candida glabrata Enhancer of polycomb-like protein 1 (EPL1), partial

What is EPL1 and what is its functional significance in Candida glabrata?

Enhancer of polycomb-like protein 1 (EPL1) in Candida glabrata is a protein identified by UniProt accession number Q6FLZ0, belonging to the polycomb protein family . EPL1 plays a critical role in chromatin regulation and gene silencing mechanisms. Methodologically, researchers investigating EPL1 function should consider:

• Chromatin immunoprecipitation (ChIP) assays to identify genomic regions where EPL1 binds

• Gene expression profiling following EPL1 knockdown or overexpression

• Co-immunoprecipitation experiments to identify protein interaction partners

• Comparative genomic analyses with other fungal pathogens to establish evolutionary conservation patterns

EPL1's significance must be interpreted within the broader context of C. glabrata pathogenicity, as this organism represents the second most common cause of candidiasis behind C. albicans, accounting for 15-25% of invasive Candida infections .

What are the recommended storage and handling protocols for Recombinant EPL1?

For optimal experimental reproducibility when working with Recombinant C. glabrata EPL1, adhere to these evidence-based storage protocols:

• Long-term storage: The lyophilized form maintains stability for approximately 12 months at -20°C to -80°C

• Liquid preparations: Shelf life is typically limited to 6 months at -20°C to -80°C

• Working aliquots: Store at 4°C for a maximum of one week

• Avoid repeated freeze-thaw cycles, as this significantly reduces protein activity and integrity

Methodologically, researchers should:

• Document batch information and reconstitution dates

• Establish internal quality control procedures to verify activity before critical experiments

• Create multiple small-volume aliquots immediately after reconstitution

• Include appropriate controls in experimental design to account for potential batch-to-batch variation

What is the recommended reconstitution protocol for EPL1 partial protein?

The optimal reconstitution procedure for EPL1 involves:

• Centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Aliquot into appropriate volumes for experimental workflows

• Store aliquots at -20°C/-80°C for long-term use

For experimental validation, researchers should consider:

• Confirming protein stability post-reconstitution using SDS-PAGE or activity assays

• Optimizing buffer conditions based on downstream applications

• Testing protein functionality in preliminary experiments before conducting large-scale studies

How can researchers verify the purity and activity of EPL1 preparations?

Methodological approach to EPL1 quality assessment:

• Purity verification:

  • SDS-PAGE analysis: The commercial preparation typically shows >85% purity

  • Western blot using anti-EPL1 antibodies

  • Mass spectrometry for comprehensive protein identification

• Activity assessment:

  • Chromatin binding assays

  • Protein-protein interaction studies with known partners

  • Functional complementation in EPL1-deficient strains

How does EPL1 function relate to C. glabrata virulence mechanisms?

While the search results don't directly address EPL1's role in virulence, we can draw methodological parallels from research on other C. glabrata virulence factors:

• Generate EPL1 deletion mutants using CRISPR-Cas9 or traditional knockout methods

• Assess virulence using established infection models such as Galleria mellonella, which has proven effective for studying C. glabrata pathogenicity

• Monitor host survival rates and fungal proliferation in hemolymph

• Evaluate stress responses, particularly to oxidative and acidic environments common in phagocytes

Research on CgDtr1, another C. glabrata protein, demonstrated that expression levels directly correlated with virulence against G. mellonella, with deletion mutants showing a 30% reduction in larval mortality . EPL1 researchers might adopt similar experimental designs to evaluate this protein's contribution to pathogenicity.

What expression systems are most effective for producing functional EPL1?

For optimal EPL1 expression, researchers should consider:

• Yeast expression systems:

  • The commercial preparation utilizes yeast-based expression

  • Advantages include proper eukaryotic post-translational modifications

  • Consider Saccharomyces cerevisiae or Pichia pastoris for high-yield expression

• Methodological considerations:

  • Codon optimization for the expression host

  • Selection of appropriate promoters (constitutive vs. inducible)

  • Optimization of induction conditions (temperature, time, inducer concentration)

  • Purification strategy selection based on fusion tags

• Quality control metrics:

  • Yield assessment (typical yields range from 1-10 mg/L culture)

  • Activity verification through functional assays

  • Stability testing under various storage conditions

What techniques are recommended for studying EPL1's role in chromatin regulation?

As an enhancer of polycomb protein, EPL1 likely participates in chromatin modification and gene silencing. Advanced methodological approaches include:

• Genome-wide binding profile analysis:

  • ChIP-Seq to identify EPL1 binding sites across the C. glabrata genome

  • CUT&RUN or CUT&Tag for higher resolution binding profiles

  • Integration with RNA-Seq data to correlate binding with transcriptional outcomes

• Protein complex characterization:

  • Proximity labeling techniques (BioID, APEX)

  • Mass spectrometry-based interactome analysis

  • Co-immunoprecipitation coupled with Western blotting

• Functional genomics approaches:

  • CRISPR-Cas9 screens to identify genetic interactions

  • Conditional depletion systems to study essential functions

  • Fluorescence microscopy to track EPL1 localization during cell cycle and stress conditions

How might EPL1 contribute to antifungal resistance mechanisms in C. glabrata?

C. glabrata demonstrates significant resistance to azole antifungal agents like fluconazole , and as a chromatin regulator, EPL1 could potentially influence resistance mechanisms through epigenetic modulation. Research approaches should include:

• Comparative expression analysis:

  • Quantify EPL1 expression levels in susceptible versus resistant clinical isolates

  • Monitor expression changes following antifungal exposure

  • Perform RNA-Seq in EPL1 mutants to identify downstream targets

• Resistance phenotype testing:

  • Evaluate minimum inhibitory concentrations (MICs) in EPL1 overexpression and knockout strains

  • Assess multidrug resistance pump expression in EPL1 mutants

  • Test cross-resistance to different antifungal classes

• Mechanistic investigation:

  • Chromatin accessibility profiling (ATAC-Seq) at resistance gene loci

  • Histone modification mapping at key resistance genes

  • Drug efflux assays in relation to EPL1 expression levels

C. glabrata's increasing clinical significance (15-25% of invasive Candida infections) and its higher mortality rate, especially in bloodstream infections, underscores the importance of understanding potential resistance mechanisms mediated by chromatin regulators like EPL1.

What are the challenges in studying EPL1 interactions with host immune cells?

Investigating EPL1's potential role during host-pathogen interactions presents several methodological challenges:

• Experimental design considerations:

  • Selection of appropriate immune cell models (macrophages, neutrophils)

  • Establishing C. glabrata infection models that allow for protein-specific studies

  • Controlling for variables such as EPL1 expression levels and host cell activation states

• Technical approaches:

  • Live cell imaging to track EPL1-expressing cells during phagocytosis

  • Flow cytometry to quantify host cell responses to wild-type versus EPL1 mutants

  • Transcriptomics of both pathogen and host during interaction

• Relevant biological stressors:

  • Oxidative stress resistance assays, as phagocytes produce reactive oxygen species

  • Acidic stress tolerance testing, mimicking phagolysosomal conditions

  • Nutrient limitation models reflecting the intracellular environment

Drawing parallels from CgDtr1 research, which demonstrated roles in both oxidative and acetic acid stress resistance that influenced survival within hemocytes , EPL1 should be evaluated under similar stress conditions relevant to the host-pathogen interface.

How can EPL1 research contribute to understanding broader virulence mechanisms in Candida species?

EPL1 research can provide insights into conserved virulence mechanisms through:

• Comparative genomics approaches:

  • Sequence and functional comparison with homologs in C. albicans and other Candida species

  • Identification of conserved versus species-specific regulatory elements

  • Evolutionary analysis of chromatin regulation systems across pathogenic fungi

• Systems biology integration:

  • Network analysis incorporating EPL1 with known virulence factors

  • Pathway enrichment analysis of EPL1-regulated genes

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) in EPL1 mutants

• Translational research considerations:

  • Identification of EPL1-dependent virulence mechanisms as potential therapeutic targets

  • Evaluation of EPL1-mediated processes in the context of host-pathogen interactions

  • Assessment of EPL1's role in adaptation to host niches (bloodstream, mucosal surfaces)

C. glabrata's clinical significance in vulnerable populations, including older adults and immunocompromised patients , highlights the importance of understanding virulence mechanisms that might be influenced by chromatin regulators like EPL1.