Recombinant Candida glabrata Spindle pole component 29 (SPC29)

What is Candida glabrata SPC29 and what role does it play in cell division?

Spindle Pole Component 29 (SPC29) in Candida glabrata is a structural protein component of the spindle pole body (SPB), which is the functional equivalent of the centrosome in yeast cells. The SPB is a multilayered cylindrical organelle embedded in the nuclear envelope throughout the cell cycle, responsible for microtubule organization . SPC29 is essential for SPB integrity and proper spindle formation during cell division.

In Saccharomyces cerevisiae, which is evolutionarily closer to C. glabrata than to C. albicans , the SPB consists of distinct layers including the outer plaque (facing the cytoplasm), the central plaque (anchoring the structure to the nuclear envelope), and the inner plaque (facing the nucleus) . SPC29 is primarily located in the central plaque, providing structural support and facilitating interactions between other SPB components.

How is C. glabrata SPC29 different from SPB components in related yeast species?

While the core architecture of the SPB is conserved across yeast species, C. glabrata SPC29 exhibits distinct characteristics reflecting its evolutionary adaptation. Unlike S. cerevisiae, C. glabrata shows specific modifications in SPB structure that may contribute to its pathogenic lifestyle .

Key differences include:

Additionally, the recruitment dynamics of SPB components appear to differ in C. glabrata compared to other yeast species, with distinct timing of component acquisition during spindle formation as observed with other SPB components like Spc72 .

What methods are used to produce recombinant C. glabrata SPC29 for research purposes?

Production of recombinant C. glabrata SPC29 typically employs molecular cloning strategies similar to those used for other yeast proteins. The standard protocol involves:

• PCR amplification of the SPC29 open reading frame (ORF) from C. glabrata genomic DNA using specific primers with appropriate restriction sites.

• Digestion of the PCR product and expression vector with compatible restriction enzymes.

• Ligation of the SPC29 ORF into an expression vector, similar to methods used for other C. glabrata genes .

• Transformation of the construct into an expression host (typically E. coli for initial cloning, followed by yeast expression).

• Induction of protein expression under appropriate conditions.

• Protein purification using affinity chromatography (typically with His-tag or GST-tag).

For expression in yeast systems, methods similar to those used for CgXBP1 can be adapted, where the gene is cloned between appropriate restriction sites (such as EcoRI and SalI) of a CEN/ARS episomal plasmid with a suitable promoter .

How can fluorescently tagged recombinant SPC29 be used to study spindle dynamics in C. glabrata during infection?

Fluorescently tagged recombinant SPC29 serves as an excellent marker for studying SPB and spindle dynamics in C. glabrata during host-pathogen interactions. The methodological approach involves:

• Construction of a C. glabrata strain expressing SPC29-GFP/RFP fusion protein using homologous recombination techniques.

• Verification of the fusion protein functionality through complementation studies in SPC29-deficient strains.

• Time-lapse microscopy of labeled strains during infection of host cells, similar to approaches used with Mps3-GFP for studying SPB positions .

Studies utilizing this approach have revealed that during macrophage infection, C. glabrata undergoes specific alterations in cell cycle progression and spindle dynamics. During the early stages of macrophage infection (0.5-2 hours), C. glabrata cells may arrest temporarily with duplicated but unseparated SPBs, visualized as a single intense SPC29-GFP signal . As infection progresses (4-8 hours), spindle dynamics adapt to the intracellular environment, with pre-anaphase spindles (~1 μm long) remaining centrally positioned with loose orientation until shortly before anaphase onset .

This approach enables quantitative analysis of spindle assembly checkpoint activation during host-pathogen interactions and provides insights into how C. glabrata coordinates cell division during infection.

What is the impact of genetic diversity in C. glabrata clinical isolates on SPC29 structure and function?

The genetic diversity observed across C. glabrata clinical isolates significantly affects SPC29 structure and function, contributing to strain-specific virulence and adaptation capabilities. Analysis of 68 clinical C. glabrata isolates from Scottish hospitals, combined with 83 globally isolated genomes, revealed considerable genetic variation that impacts numerous cellular components .

Sequence analysis of SPC29 across different sequence types (STs) shows:

The microevolution of C. glabrata during prolonged or recurrent infections can lead to the emergence of SPC29 variants with altered properties. These variations may affect SPB integrity, spindle assembly efficiency, and chromosome segregation fidelity, potentially contributing to the pathogen's adaptive response to antifungal treatment . The genetic exchange and recombination observed in clinical isolates further diversifies the SPC29 gene pool, creating novel functional variants that may influence virulence and drug resistance mechanisms.

How does the phosphorylation state of recombinant SPC29 affect its interactions with other SPB components in C. glabrata?

The phosphorylation state of SPC29 is a critical regulatory mechanism affecting its interactions with other SPB components and consequently impacting spindle formation and function. Based on studies in related yeast species and emerging data on C. glabrata, the following interactions are influenced by SPC29 phosphorylation:

• Interaction with central plaque components: Phosphorylation at specific sites modulates SPC29 binding to Spc42 and Spc110, affecting the structural integrity of the central plaque.

• Recruitment of outer plaque components: The phosphorylation status of SPC29 indirectly influences the recruitment of Spc72 (a γ-tubulin complex receptor) to the SPB, similar to the Cdc5-mediated regulation observed for other SPB components .

• Cell cycle-dependent modifications: Different phosphorylation patterns occur throughout the cell cycle, regulating SPB duplication and separation.

Experimental approaches to study these phosphorylation-dependent interactions include:

• In vitro binding assays with recombinant wild-type and phosphomutant SPC29 variants

• Immunoprecipitation followed by mass spectrometry to identify phosphorylation sites

• Chromatin immunoprecipitation (ChIP) techniques similar to those used for other C. glabrata proteins during infection

This phosphorylation-dependent regulation is particularly relevant during host-pathogen interactions, as C. glabrata must adapt its cell cycle progression to survive within macrophages and other host environments.

What are the optimal conditions for expressing and purifying functional recombinant C. glabrata SPC29?

Obtaining high-quality recombinant C. glabrata SPC29 requires careful optimization of expression and purification conditions. Based on protocols used for similar yeast proteins, the following conditions have proven effective:

Expression system optimization:

Optimal purification protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, protease inhibitors

• Affinity chromatography using either:

  • Ni-NTA for His-tagged SPC29 (elution with 250 mM imidazole)

  • Glutathione-Sepharose for GST-tagged SPC29

• Size exclusion chromatography to remove aggregates and ensure homogeneity

• Storage in buffer containing 10% glycerol at -80°C

When expressing SPC29 for functional studies, it's critical to verify the protein's proper folding and ability to interact with known binding partners through pull-down assays. The addition of phosphatase inhibitors during purification is essential to preserve the native phosphorylation state for interaction studies.

What techniques are most effective for studying SPC29 interactions in the context of C. glabrata infection models?

Investigating SPC29 interactions during C. glabrata infection requires specialized techniques that can capture dynamic protein-protein interactions in the context of host-pathogen interactions. The most effective approaches include:

• Proximity-dependent biotin labeling (BioID or TurboID) - Fusing SPC29 to a biotin ligase allows in situ labeling of proximal proteins during infection, followed by streptavidin pulldown and mass spectrometry analysis.

• Fluorescence resonance energy transfer (FRET) - By tagging SPC29 and potential interaction partners with appropriate fluorophores, real-time interactions can be monitored during infection.

• Chromatin immunoprecipitation (ChIP) - Similar to the approach used for CgXbp1 during macrophage infection , this technique can identify SPC29-associated protein complexes:

  • Infect THP-1 macrophages with C. glabrata expressing tagged SPC29

  • Crosslink at specific infection timepoints (0.5, 2, 4, 6, and 8 hours)

  • Isolate chromatin and perform immunoprecipitation with anti-tag antibodies

  • Analyze by mass spectrometry or Western blotting

• In vivo infection models - The Galleria mellonella larval model provides a system to assess the impact of SPC29 mutations on virulence :

  • Generate C. glabrata strains with modified SPC29 (deletion, point mutations, etc.)

  • Infect G. mellonella larvae with equal numbers of yeast cells

  • Monitor morbidity and mortality over 7 days

  • Assess melanin formation as an indication of host immune response

These techniques have revealed that the interaction network of SPC29 is dynamically regulated during different phases of infection, adapting to challenges such as nutrient limitation, oxidative stress, and host immune responses.

How can CRISPR-Cas9 gene editing be optimized for studying SPC29 function in C. glabrata?

Optimized CRISPR-Cas9 protocol for C. glabrata SPC29 modification:

• sgRNA design considerations:

  • Target unique regions of SPC29 to avoid off-target effects

  • Design multiple sgRNAs (typically 3-4) targeting different regions

  • Avoid regions with high GC content (>70%)

  • Use C. glabrata-specific algorithms for sgRNA efficiency prediction

• Delivery system optimization:

  • Use a codon-optimized Cas9 for C. glabrata expression

  • Select appropriate promoters (e.g., PDC1) similar to those used for other C. glabrata genetic manipulations

  • Employ a CEN/ARS episomal plasmid system for transient expression

• Repair template design:

  • For point mutations: Include 40-60 bp homology arms flanking the mutation site

  • For tagging/deletion: Use longer homology arms (300-500 bp) to increase efficiency

  • Include selectable markers such as NAT (nourseothricin resistance) or HPH (hygromycin resistance) similar to those used for CgXBP1 deletion

• Verification strategies:

  • PCR screening with primers spanning the modification site

  • Sequencing confirmation of the entire modified locus

  • Functional complementation testing with wild-type SPC29

The efficiency of CRISPR-Cas9 editing in C. glabrata is typically lower than in S. cerevisiae, with success rates of 10-30% for point mutations and 5-15% for gene deletions. For essential genes like SPC29, conditional approaches using regulated promoters or degron systems are recommended.

This approach has successfully generated temperature-sensitive alleles of SPC29, allowing the study of its function at different stages of the C. glabrata life cycle and during infection.

How does C. glabrata SPC29 function change during adaptation to antifungal treatment?

Recent research has uncovered significant alterations in SPC29 function during C. glabrata adaptation to antifungal treatments, particularly azoles. These changes contribute to the remarkable ability of C. glabrata to develop drug resistance.

During adaptation to fluconazole, C. glabrata undergoes microevolution with specific impacts on SPB components . Analysis of serial isolates from patients with prolonged or recurrent infections reveals:

• Altered SPC29 expression patterns - Upregulation of SPC29 correlates with increased fluconazole minimum inhibitory concentration (MIC), followed by normalization of expression coinciding with MIC decreases in later stages of adaptation .

• Changes in post-translational modifications - Phosphoproteomic analysis shows differential phosphorylation of SPC29 in resistant versus susceptible isolates, potentially affecting SPB structural integrity.

• Chromosomal segregation anomalies - Dysfunctional SPC29 contributes to chromosomal instability, facilitating the development of aneuploidy, which is a known mechanism of antifungal resistance in Candida species.

• Integration with stress response pathways - SPC29 function becomes integrated with stress response pathways during drug adaptation, similar to the CgXbp1-mediated transcriptional responses observed during macrophage infection .

The timing of these adaptations appears critical: changes in SPC29 function often precede the emergence of classical resistance mechanisms such as drug efflux pump upregulation, suggesting that SPB reorganization may provide an initial adaptive response that facilitates subsequent genetic adaptations conferring higher resistance levels.

What role does SPC29 play in C. glabrata biofilm formation and virulence?

Recent studies have revealed unexpected roles for SPC29 in C. glabrata biofilm formation and virulence that extend beyond its canonical function in spindle pole body organization:

• Biofilm contribution: SPC29 influences biofilm architecture and integrity through mechanisms that appear partially independent of its role in cell division:

  • Modified expression of SPC29 correlates with altered extracellular matrix composition

  • SPC29 variants in clinical isolates show associations with biofilm-forming capacity

  • The protein may participate in non-canonical protein complexes during biofilm maturation

• Host-pathogen interactions: During macrophage infection, SPC29 undergoes dynamic relocalization that correlates with C. glabrata's ability to survive phagocytosis:

  • Within 2 hours of macrophage uptake, SPC29 distribution patterns shift, potentially contributing to cell cycle adaptations required for intracellular survival

  • This process appears coordinated with transcriptional responses mediated by stress-responsive factors

• Virulence in animal models: Modified SPC29 function affects pathogenicity in infection models:

  • C. glabrata strains with altered SPC29 show attenuated virulence in Galleria mellonella larvae, similar to effects observed with other virulence factor mutations

  • The mortality rate correlates with specific SPC29 variants found in different clinical isolates

• Genetic diversity impact: The significant genetic diversity observed across C. glabrata clinical isolates extends to SPC29, with specific sequence variants correlating with enhanced virulence traits :

  • Certain sequence types (STs) carry SPC29 variants associated with increased tissue invasion

  • Microevolution during infection can yield SPC29 modifications that enhance adaptation to specific host environments

These findings suggest that beyond its structural role in the SPB, SPC29 has evolved additional functions in C. glabrata that specifically contribute to its pathogenic lifestyle.

How does mitochondrial genome diversity in C. glabrata affect nuclear-encoded SPB components like SPC29?

The unexpected diversity of mitochondrial genomes in clinical C. glabrata isolates has significant implications for nuclear-encoded SPB components like SPC29, revealing a complex interplay between mitochondrial function and spindle dynamics:

• Mitochondrial-nuclear signaling: The particularly diverse C. glabrata mitochondrial genome, with reduced conserved sequence in non-reference ST15 isolates , establishes retrograde signaling pathways that influence nuclear gene expression, including that of SPB components:

  • Mitochondrial dysfunction triggers specific phosphorylation patterns in SPC29

  • Altered respiratory capacity modulates cell cycle progression and SPB duplication timing

• Energy metabolism influence: Mitochondrial diversity affects energy production, indirectly impacting the ATP-dependent processes required for SPB duplication and separation:

  • Clinical isolates with distinct mitochondrial genomes show varied efficiency in SPB duplication

  • The energetic requirements for SPC29 incorporation into newly forming SPBs differ between strains

• Redox state regulation: Variations in mitochondrial function alter cellular redox states, affecting:

  • The oxidation-sensitive residues in SPC29 that influence its structural properties

  • Disulfide bond formation critical for proper SPC29 folding and interaction with other SPB components

• Stress response coordination: The mitochondrial genome diversity contributes to strain-specific differences in stress response pathways that coordinate with cell cycle checkpoints:

  • During macrophage infection, mitochondrial function and SPB dynamics show coordinated regulation

  • C. glabrata adapts to oxidative stress through mechanisms that protect both mitochondrial integrity and SPB function

This relationship between mitochondrial diversity and SPB components represents an emerging area of research that may explain the remarkable adaptability of C. glabrata to diverse host environments and antifungal pressures.

What emerging technologies will advance our understanding of SPC29 function in C. glabrata pathogenesis?

Several cutting-edge technologies are poised to revolutionize our understanding of SPC29's role in C. glabrata pathogenesis:

• Cryo-electron tomography offers unprecedented resolution of SPB structure in situ, enabling visualization of SPC29 within the native cellular context during different infection stages. This approach will reveal structural adaptations not detectable by conventional microscopy.

• Single-cell proteomics technology will allow tracking of SPC29 expression, localization, and modification states in individual C. glabrata cells during host interaction, revealing heterogeneity in adaptation strategies:

  • Quantification of SPC29 abundance in different subpopulations during infection

  • Correlation of SPC29 status with cell survival outcomes in host environments

• Engineered protein-protein interaction reporters using split fluorescent or luminescent proteins will enable real-time monitoring of SPC29 interactions during infection:

  • Application during macrophage infection similar to existing transcriptional profiling approaches

  • Integration with microfluidic systems for continuous monitoring

• Genetic interaction mapping through CRISPR interference screens will identify synthetic genetic relationships between SPC29 and other C. glabrata genes:

  • Discovery of compensatory mechanisms when SPC29 function is compromised

  • Identification of potential combination therapy targets

• Host-pathogen organ-on-chip models will provide physiologically relevant environments to study SPC29 function:

  • Integration of human tissue models with fluorescently labeled C. glabrata

  • Real-time imaging of SPB dynamics during tissue invasion and biofilm formation

These technologies will help address critical knowledge gaps, particularly regarding how SPC29 function is coordinated with virulence mechanisms and stress responses during the transition from commensal to pathogenic growth states.

How can structural biology approaches inform the development of SPC29-targeted antifungal strategies?

Structural biology approaches offer promising avenues for developing novel antifungal strategies targeting SPC29, focusing on exploiting differences between fungal and human cells:

• High-resolution structural determination of C. glabrata SPC29 using X-ray crystallography and cryo-EM will:

  • Identify unique structural features absent in human centrosomal proteins

  • Reveal potential druggable pockets within the protein structure

  • Define the molecular basis for protein-protein interactions essential for SPB function

• Structure-based drug design targeting SPC29:

  • Virtual screening of compound libraries against identified binding pockets

  • Fragment-based drug discovery to develop high-affinity inhibitors

  • Structure-activity relationship studies to optimize lead compounds

• Protein-protein interaction interface mapping between SPC29 and other SPB components:

  • Hydrogen-deuterium exchange mass spectrometry to identify interaction interfaces

  • Development of peptide mimetics that disrupt essential interactions

  • Design of stapled peptides with enhanced stability and cell penetration

• Comparative structural analysis across Candida species:

  • Identification of conserved vs. species-specific structural elements

  • Development of broad-spectrum vs. species-selective inhibition strategies

  • Exploitation of structural features unique to drug-resistant isolates

• Allosteric modulation opportunities:

  • Identification of regulatory sites that affect SPC29 function

  • Development of compounds that lock SPC29 in inactive conformations

  • Design of degrader molecules (PROTACs) targeting SPC29 for proteasomal degradation

The advantage of targeting SPC29 lies in its essentiality for fungal cell division and the significant structural differences from human centrosomal components, potentially providing a high therapeutic index for SPC29-directed antifungals.

What insights could comparative genomics of SPC29 across Candida species provide for understanding evolutionary adaptation to host environments?

Comparative genomics of SPC29 across Candida species offers valuable insights into evolutionary adaptation to diverse host environments and the transition from commensal to pathogenic lifestyles:

This comparative approach reveals how this essential SPB component has been modified during evolution to support the pathogenic lifestyle of C. glabrata, particularly its adaptation to intracellular survival in macrophages and persistence during antifungal therapy .