Recombinant Candida glabrata Cell division cycle protein 123 (CDC123)

What is the basic function of CDC123 in Candida glabrata?

CDC123 is a highly conserved protein that plays crucial roles in cell cycle regulation and nutrient sensing in C. glabrata. Similar to its homolog in Saccharomyces cerevisiae, C. glabrata CDC123 is regulated by nutrient availability and participates in cellular proliferation control . Functionally, CDC123 is involved in the assembly of translation initiation factor 2 (eIF2), which is critical for protein synthesis initiation. The protein appears to interact specifically with domain III of Gcd11 (eIF2γ), independent of the G-domain that mediates interactions with other eIF2 subunits . This mechanism allows CDC123 to serve as a checkpoint connecting nutritional status to cell division decisions.

How does CDC123 structure in C. glabrata compare to its homologs in related species?

While complete structural characterization of C. glabrata CDC123 is still emerging, comparative genomic analyses suggest significant conservation within the Candida clade. Unlike Saccharomyces cerevisiae which has a single HAP1 homolog, C. glabrata maintains two Hap1 homologs (Zcf27 and Zcf4) that function in different oxygen conditions . This duplication pattern may extend to other regulatory proteins including CDC123, potentially providing functional redundancy or specialization. Molecular analyses indicate that C. glabrata CDC123 contains conserved regions for protein-protein interactions and post-translational modifications that regulate its stability and function.

What experimental approaches are recommended for expressing recombinant C. glabrata CDC123?

To express recombinant C. glabrata CDC123, researchers should consider:

Recommended expression systems:

• E. coli BL21(DE3) for high-yield bacterial expression

• Pichia pastoris for eukaryotic post-translational modifications

• Baculovirus-insect cell system for complex eukaryotic protein structures

Optimization protocol:

• Clone CDC123 with appropriate affinity tags (6xHis or GST) at either N or C-terminus

• Express at lower temperatures (16-20°C) to improve protein folding

• Include protease inhibitors during purification to prevent degradation

• Consider codon optimization, as C. glabrata uses a different codon bias than standard expression hosts

• Use detergent screens to determine optimal solubilization conditions if membrane-associated

When purifying CDC123, researchers should be aware that the protein may form oligomeric complexes under certain conditions, which can be stabilized with appropriate buffer components .

What are validated methods for detecting CDC123 in C. glabrata samples?

Multiple validated methods exist for detecting CDC123 in C. glabrata experimental samples:

For Western blot detection specifically, anti-CDC123 antibodies have been validated in multiple applications including ELISA, flow cytometry, immunofluorescence, immunohistochemistry, and Western blot analysis . When detecting CDC123 in clinical isolates, protocols may need optimization due to variable protein expression levels across different strains .

How can researchers assess CDC123 function in C. glabrata through genetic manipulation?

To assess CDC123 function through genetic manipulation:

• Gene deletion approach:

  • Use CRISPR-Cas9 system adapted for C. glabrata

  • Apply homologous recombination with selection markers (NAT1, HYG, URA3)

  • Construct conditional mutants if CDC123 is essential

• Complementation testing:

  • Re-introduce wild-type or mutant CDC123 variants

  • Use promoters with variable strength for expression level control

  • Apply episomal or integrative plasmids depending on experimental goals

• Protein interaction studies:

  • Use yeast two-hybrid or pull-down assays to identify interaction partners

  • Confirm interactions with co-immunoprecipitation

  • Map interaction domains with truncated protein variants

Recent genomic tools developed for C. glabrata, including deletion strain collections and optimized gene disruption protocols, facilitate these approaches . When assessing phenotypes, researchers should examine effects on growth rate, cell cycle progression, and stress responses under various nutrient conditions to capture CDC123's regulatory functions.

What is the role of CDC123 in C. glabrata virulence and host adaptation?

CDC123 may contribute to C. glabrata virulence through several mechanisms:

• Nutrient sensing and metabolic adaptation:
  CDC123 functions in nutrient detection pathways, potentially allowing C. glabrata to adapt to the nutrient-limited environment of the host. This metabolic flexibility enables survival within macrophage phagosomes where alternative carbon source utilization becomes essential .

• Cell cycle regulation during infection:
  As a cell cycle regulator, CDC123 likely facilitates appropriate proliferation in response to host environmental cues. The ability to control growth rate in different host niches is critical for establishing persistent infections.

• Stress response coordination:
  CDC123 may integrate with stress response pathways required for surviving host immune defenses. C. glabrata's ability to persist within phagocytes suggests sophisticated coordination between cell cycle and stress response mechanisms .

Researchers investigating CDC123's role in virulence should examine its expression patterns during different stages of infection and in various host niches. Comparative analysis between clinical isolates with different virulence profiles may reveal correlations between CDC123 sequence variations and pathogenicity .

How does CDC123 expression change under antifungal treatment conditions?

CDC123 expression patterns under antifungal stress remain incompletely characterized, but several observations are relevant:

• CDC123 may participate in stress response pathways activated during azole treatment. When C. glabrata is exposed to azole antifungals that target ergosterol biosynthesis, significant transcriptional reprogramming occurs, potentially involving cell cycle regulators like CDC123 .

• Serial clinical isolates collected before and after antifungal treatment show genomic changes including non-synonymous mutations in cell wall proteins and genes involved in stress responses . While CDC123 specifically hasn't been highlighted in these analyses, proteins in related pathways show adaptations.

• The metabolic flexibility that allows C. glabrata to survive in nutrient-limited conditions may also contribute to antifungal resistance, particularly in biofilms where decreased metabolic activity correlates with increased resistance . CDC123's role in metabolic regulation may indirectly influence susceptibility.

To properly assess CDC123 expression changes under antifungal treatment, researchers should use time-course experiments with subinhibitory antifungal concentrations and examine both transcriptional and post-translational regulation of CDC123.

How variable is the CDC123 gene sequence across clinical C. glabrata isolates?

Genomic analyses of C. glabrata clinical isolates reveal:

A comprehensive analysis of CDC123 sequence variation would require comparing sequences from diverse clinical isolates representing different geographic regions, infection sites, and patient populations. Researchers could contribute to this understanding by sequencing CDC123 from their clinical isolates and depositing the data in public databases.

What evidence exists for CDC123 gene duplication or divergence in pathogenic Candida species?

Evidence for gene duplication and functional divergence in C. glabrata is exemplified by the case of Hap1 homologs:

• Unlike S. cerevisiae which has a single HAP1 gene, C. glabrata maintains two homologs (Zcf27 and Zcf4) that have evolved distinct roles in adaptation to specific host and environmental conditions :

  • Zcf27 regulates ergosterol biosynthesis genes under aerobic conditions

  • Zcf4 expression is specifically induced under hypoxic conditions, where it represses ergosterol biosynthesis genes

• This functional specialization demonstrates how gene duplication events in C. glabrata can lead to adaptation to the diverse microenvironments encountered during infection.

While specific evidence for CDC123 duplication has not been reported in the provided sources, researchers should investigate whether similar patterns of duplication and specialization might apply to CDC123 or related cell cycle regulators. Comparative genomic approaches across Candida species could reveal evolutionary patterns in CDC123 structure and function related to pathogenicity.

How can researchers leverage CDC123 to understand translational regulation during stress in C. glabrata?

CDC123's role in eIF2 assembly positions it as a valuable tool for studying translational regulation during stress:

• Research approach:

  • Create reporter systems with CDC123 fusion proteins to monitor localization and activity

  • Develop CDC123 activity assays based on eIF2 assembly efficiency

  • Use ribosome profiling to assess translational responses in CDC123 mutants

• Experimental design for stress conditions:

  • Compare nutrient limitation, oxidative stress, and antifungal exposure

  • Monitor acute versus chronic stress responses

  • Assess CDC123 post-translational modifications under different stress conditions

• Integration with other pathways:

  • Examine interactions between CDC123 and other stress response pathways

  • Investigate potential feedback loops between translation regulation and cell cycle control

  • Compare responses in C. glabrata to related species like S. cerevisiae

The conserved interaction between CDC123 and domain III of Gcd11 (eIF2γ) provides a specific molecular event to monitor . When Gcd11 variants unable to complete assembly permanently bind CDC123, they limit the available CDC123 pool and inhibit translation. This mechanism could be exploited to develop molecular probes of CDC123 activity.

What are the implications of post-translational modifications on CDC123 function in fungal pathogens?

CDC123 function is regulated through post-translational modifications that control its stability and activity:

• Ubiquitination/deubiquitination:
  Evidence from mammalian systems indicates CDC123 is regulated through ubiquitination pathways. USP9X has been identified as a deubiquitinase that stabilizes CDC123 by removing ubiquitin markers . Similar regulation likely exists in fungal systems, though specific deubiquitinases remain to be identified.

• Phosphorylation:
  Phosphorylation state likely influences CDC123 activity in response to nutrient availability and stress conditions. Researchers should investigate kinases and phosphatases that interact with CDC123 under different growth conditions.

• Research strategies:

  • Apply mass spectrometry to map modification sites

  • Use phospho-mimetic and phospho-deficient mutations to assess functional impact

  • Develop antibodies specific to modified forms of CDC123

  • Apply selective inhibitors of modification enzymes to assess dynamics

Understanding these modifications could provide insights into how C. glabrata adapts to changing host environments during infection. The integration of post-translational modifications with transcriptional control likely allows fine-tuning of CDC123 activity in response to environmental signals.

How might CDC123 interact with drug resistance mechanisms in C. glabrata?

CDC123 may intersect with drug resistance mechanisms through several potential pathways:

• Metabolic adaptation:
  CDC123's role in nutrient sensing may contribute to the metabolic flexibility that allows C. glabrata to survive in the presence of antifungals. The glyoxylate cycle gene ICL1, necessary for alternative carbon source utilization, promotes growth and survival during macrophage engulfment . If CDC123 regulates metabolic pathways, it could influence susceptibility to antifungals indirectly.

• Cell cycle checkpoint control:
  Antifungal drugs may trigger cell cycle checkpoints, where CDC123 could play a regulatory role. Understanding how CDC123 influences cell cycle progression during drug exposure could reveal mechanisms of tolerance or persistence.

• Translation regulation:
  CDC123's function in eIF2 assembly links it to translational control. Azole resistance in C. glabrata often involves upregulation of drug efflux pumps or ergosterol biosynthesis genes , processes requiring coordinated protein synthesis where CDC123 may be involved.

Experimental approaches should include:

• Comparing CDC123 expression in azole-resistant versus susceptible strains

• Assessing whether CDC123 deletion affects minimum inhibitory concentrations

• Investigating potential interactions between CDC123 and known resistance factors like Pdr1

Future research may determine whether CDC123 could serve as a target for combination therapies to overcome antifungal resistance in C. glabrata infections .

What research tools and strain collections are available for studying CDC123 in C. glabrata?

Several specialized tools and resources have been developed that facilitate CDC123 research:

Strain collections:

• A comprehensive C. glabrata deletion collection containing 416 deletion strains has been created and used to examine virulence factors

• Clinical isolate collections with genome sequence data provide resources for comparative studies

Genetic manipulation tools:

• Optimized gene disruption protocols for C. glabrata

• CRISPR-Cas9 systems adapted for C. glabrata genome editing

• Expression vectors with varying promoter strengths for controlled gene expression

Antibodies and detection reagents:

• Validated anti-CDC123 antibodies are commercially available for multiple applications including Western blot, ELISA, immunofluorescence, and immunohistochemistry

In vivo models:

• Immunodeficient Drosophila melanogaster model for virulence assessment

• Mouse models of systemic and vaginal C. glabrata infection

Researchers should also consider establishing collaborations with clinical microbiology laboratories to access diverse clinical isolates with varying drug resistance profiles and virulence characteristics for comparative studies of CDC123 expression and function.

What are the key challenges and future directions in C. glabrata CDC123 research?

Key challenges and future research directions include:

• Functional characterization:

  • Determining the precise molecular mechanisms by which CDC123 regulates cell cycle in C. glabrata

  • Mapping the CDC123 interactome under different physiological conditions

  • Understanding CDC123's role in stress response pathways specific to host environments

• Clinical relevance:

  • Establishing whether CDC123 sequence variations correlate with clinical outcomes

  • Determining if CDC123 expression levels predict antifungal drug responses

  • Assessing CDC123's contribution to persistence during chronic infections

• Therapeutic potential:

  • Evaluating CDC123 as a potential antifungal drug target

  • Developing small molecule inhibitors of CDC123-protein interactions

  • Investigating combination approaches targeting CDC123-dependent pathways

• Methodological challenges:

  • Creating conditional CDC123 mutants if complete deletion is lethal

  • Developing improved in vivo models that recapitulate host niches

  • Standardizing approaches for comparing results across different C. glabrata strains