Recombinant Candida glabrata NAD-dependent histone deacetylase SIR2 (SIR2), partial

What is the molecular function of SIR2 in Candida glabrata?

In C. glabrata, SIR2 functions as a NAD⁺-dependent histone deacetylase that catalyzes the deacetylation reaction of the amino-terminal tails of histones H3 and H4. This deacetylation activity is central to SIR2's role in establishing and maintaining heterochromatin formation, which affects transcriptional silencing and genome stability. The protein plays a crucial role in subtelomeric silencing, where it works in concert with other silencing factors including Sir3, Sir4, and Rap1 . While its primary biochemical function remains histone deacetylation, the regulatory pathways and genomic targets show specific adaptations in C. glabrata compared to related yeasts.

How does the structure of SIR2 relate to its function in chromatin regulation?

SIR2 contains a highly conserved catalytic domain responsible for NAD⁺-dependent deacetylase activity. The protein's structure includes regions that facilitate interactions with other silencing complex components (Sir3, Sir4) and with histones. The partial recombinant form typically includes the catalytic core while excluding regions that might hinder solubility or activity in experimental systems. Crystal structure and functional domain analyses have revealed that the enzymatic pocket where NAD⁺ and the acetylated histone substrate bind is critical for the deacetylation reaction. The protein's three-dimensional conformation enables it to access histone tails within nucleosomes, thereby regulating chromatin structure and gene accessibility.

What are the most effective methods for generating recombinant C. glabrata SIR2 protein?

Production of recombinant C. glabrata SIR2 can be accomplished through several approaches, with heterologous expression in E. coli being most common. For optimal expression, consider the following protocol:

• Gene optimization: Synthesize the SIR2 coding sequence with codon optimization for bacterial expression.

• Vector selection: Use pET-based expression vectors with an N-terminal His-tag for purification.

• Expression conditions: Transform into BL21(DE3) or Rosetta strains; induce with 0.5mM IPTG at 18°C overnight to minimize inclusion body formation.

• Purification strategy: Use nickel affinity chromatography followed by size exclusion chromatography.

• Activity preservation: Include NAD⁺ at 1mM in storage buffers to maintain stability.

For partial SIR2 constructs, focus on expressing residues encompassing the catalytic domain, typically achieving higher solubility than full-length protein.

What gene deletion approaches are most suitable for studying SIR2 function in C. glabrata?

Two principal approaches for SIR2 deletion in C. glabrata have proven effective:

• Traditional homologous recombination method:

  • Amplify 5' and 3' flanking regions of SIR2 (800bp+ each)

  • Clone these fragments into a plasmid flanking a selectable marker (e.g., hygromycin resistance cassette)

  • Transform linearized constructs into C. glabrata

  • Verify deletions by PCR targeting internal and external regions

• CRISPR-Cas9 system (offering higher efficiency):

  • Use a plasmid expressing Cas9 under control of TEF1 (S. cerevisiae) or CYC1 (C. glabrata) promoter

  • Design sgRNAs targeting SIR2 using specialized tools like CASTING

  • Express sgRNAs under control of SNR52 (S. cerevisiae) or RNAH1 (C. glabrata) promoter

  • Verify mutations using Surveyor assay and sequencing

  • This approach enables efficient gene disruption with only 20-200bp homology arms, compared to traditional methods requiring 500bp

The CRISPR-Cas9 approach offers higher efficiency but requires more specialized tools; traditional methods may be more accessible but with lower efficiency.

What assays can accurately measure SIR2 deacetylase activity from recombinant preparations?

Several validated assays can quantify SIR2 deacetylase activity:

For recombinant C. glabrata SIR2, the fluorogenic assay provides the best balance of throughput and sensitivity for initial characterization, while mass spectrometry offers the most detailed insights into substrate specificity.

How does SIR2 contribute to epigenetic silencing and heterochromatin formation in C. glabrata?

SIR2 establishes heterochromatin through a sequential recruitment mechanism in C. glabrata. The process begins when Rap1 binds to telomeric repeats, recruiting Sir proteins to these sites. Once localized, SIR2's deacetylase activity removes acetyl groups from histones H3 and H4, creating binding sites for Sir3 and Sir4 . This deacetylation initiates a positive feedback loop where Sir3 and Sir4 spread along the chromatin fiber, recruiting additional SIR2 molecules that deacetylate adjacent nucleosomes.

The spreading mechanism creates regions of heterochromatin that can extend several kilobases from nucleation sites. In C. glabrata, this silencing affects subtelomeric regions where it regulates the expression of telomere-proximal genes. The silencing complex requires all components (SIR2, SIR3, SIR4) and is influenced by yKu70/yKu80 proteins to varying degrees . Unlike in some related fungi, silencing in C. glabrata shows distinct targeting patterns that reflect this organism's genomic organization and evolutionary history.

What is the relationship between SIR2 and genome stability in C. glabrata compared to other Candida species?

SIR2's role in genome stability shows species-specific variations within the Candida genus:

In C. glabrata:

• SIR2 primarily regulates subtelomeric silencing and stability

• Works in concert with Sir3, Sir4, Rap1, and variably with yKu70/yKu80

• Plays critical roles in maintaining the stability of repetitive regions

In C. albicans:

• SIR2 has evolved novel functions in regulating genome stability

• Is largely dispensable for repressing recombination at rDNA loci (unlike in other fungi)

• Represses recombination at subtelomeric regions containing TLO Recombination Elements (TREs)

• Environmental stressors can mask SIR2-mediated recombination control

This comparative analysis reveals that the SIR2-dependent regulation of genome stability has been distinctly rewired during evolution of different Candida species. While core enzymatic functions remain conserved, the genomic targets and regulatory networks have diverged significantly, likely reflecting adaptation to different host environments and lifestyles.

How does SIR2 influence the virulence and pathogenicity of C. glabrata?

SIR2 contributes to C. glabrata virulence through several mechanisms:

• Regulation of adhesin genes: SIR2-mediated chromatin modifications influence the expression of epithelial adhesins, surface proteins that facilitate adherence to host cells. These adhesins show evidence of positive selection across clinical isolates, suggesting their importance in host colonization .

• Adaptive genome plasticity: By regulating recombination at specific genomic loci, SIR2 balances genome stability with the ability to generate genetic diversity, potentially allowing adaptation during infection.

• Stress response modulation: During antifungal exposure or oxidative stress, SIR2's regulatory functions may be altered, affecting the organism's ability to survive host defense mechanisms .

• Phenotypic switching: Evidence suggests SIR2 may influence phenotypic switching mechanisms that contribute to immune evasion and altered drug susceptibility.

Infection models using Drosophila melanogaster have demonstrated that disruption of epigenetic regulators can reduce virulence in vivo . While SIR2's direct role in pathogenicity requires further investigation, its fundamental functions in regulating chromatin structure and gene expression strongly suggest its involvement in multiple aspects of host-pathogen interaction.

How has the function of SIR2 evolved in C. glabrata compared to related yeast species?

The evolution of SIR2 function across yeast species reveals important distinctions:

The divergence of SIR2 function across these species highlights how core epigenetic machinery can be repurposed through evolution to serve species-specific requirements while maintaining fundamental enzymatic activities.

What structural and functional differences exist between partial and full-length SIR2 in C. glabrata?

The structural and functional differences between partial and full-length SIR2 in C. glabrata have important implications for research:

Full-length SIR2:

• Contains complete N-terminal and C-terminal domains

• Includes regions for protein-protein interactions with Sir3/Sir4

• Possesses additional regulatory elements that modulate activity

• May exhibit lower solubility in recombinant expression systems

• Represents the native form found in vivo

Partial SIR2:

• Typically contains the catalytic core domain

• Retains NAD⁺-dependent deacetylase activity

• May have altered substrate specificity or kinetics

• Shows improved solubility and expression yields

• Useful for biochemical and structural studies

How can SIR2 inhibition be exploited as a potential antifungal strategy against C. glabrata infections?

SIR2 inhibition represents a promising antifungal strategy against C. glabrata for several reasons:

• Disruption of epigenetic regulation: SIR2 inhibitors would alter the expression of numerous genes, potentially including those involved in virulence and stress response.

• Potential synergy with existing antifungals: Epigenetic destabilization could potentiate the effects of current antifungals by preventing adaptive responses.

• Novel mechanism of action: Unlike conventional antifungals targeting cell wall, membrane, or DNA synthesis, SIR2 inhibitors would affect chromatin-based regulation.

• Potential selectivity: Structural differences between fungal and human sirtuins could be exploited to develop selective inhibitors.

Development approaches should include:

• Structure-based design targeting the unique features of fungal SIR2

• Screening of natural product libraries, particularly other fungal metabolites

• Investigation of NAD⁺ competitive inhibitors adapted for enhanced fungal uptake

• Evaluation of synergistic combinations with established antifungals

Recent studies with infection models such as Drosophila melanogaster suggest that disruption of epigenetic regulators can indeed reduce virulence in vivo , supporting the potential of this approach.

What microevolutionary changes occur in SIR2 during prolonged C. glabrata infections, and how might they impact treatment outcomes?

Microevolutionary changes in SIR2 during prolonged infections may include:

• Point mutations affecting catalytic efficiency: Small modifications in the enzymatic pocket could alter deacetylase activity and target specificity.

• Regulatory region variations: Changes in promoter or enhancer regions could affect SIR2 expression levels in response to host conditions.

• Structural variants: Less commonly, larger insertions/deletions might modify functional domains or interaction surfaces.

These changes can impact treatment outcomes through:

• Altered regulation of adhesins and other virulence factors, potentially affecting tissue tropism

• Changes in heterochromatin formation affecting drug resistance gene expression

• Modified stress responses, potentially enhancing survival during antifungal therapy

Population genetics studies of clinical C. glabrata isolates indicate significant genetic diversity and evidence of recombination , suggesting that microevolutionary processes actively shape this pathogen's genome during infection. Tracking SIR2 variations in serial clinical isolates could provide valuable insights into adaptation mechanisms during persistent infections.

What methodological approaches can address the challenges of studying SIR2 in genetic backgrounds with different mitochondrial genome variations?

Studying SIR2 in C. glabrata strains with mitochondrial variations presents unique challenges due to the interplay between nuclear and mitochondrial functions. Population genetics research has identified hypervariable mitochondrial genomes in clinical isolates , requiring specialized approaches:

• Cybrid analysis technique:

  • Generate ρ⁰ strains (lacking mitochondrial DNA) from different nuclear backgrounds

  • Repopulate with mitochondria from donor strains through cytoduction

  • Compare SIR2 function in isogenic nuclear backgrounds with different mitochondrial genomes

• NAD⁺/NADH ratio normalization:

  • Measure and standardize NAD⁺/NADH ratios across strains

  • Use controlled media formulations to minimize metabolic variations

  • Supplement with specific carbon sources to equalize mitochondrial activity

• Multi-omics integration approach:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Account for mitochondrial effects on NAD⁺ metabolism

  • Develop computational models incorporating both nuclear and mitochondrial variables

• Single-cell analysis methods:

  • Employ microfluidic systems to track individual cells

  • Correlate SIR2 activity with mitochondrial membrane potential

  • Identify heterogeneous responses within populations

These approaches collectively address the confounding effects of mitochondrial variation on SIR2 function, particularly important when studying clinical isolates with diverse genetic backgrounds.