Recombinant Candida glabrata Protein transport protein SEC13-2 (SEC132)

What is the function of protein transport protein SEC13-2 in Candida glabrata?

Protein transport proteins like SEC13-2 in C. glabrata are likely involved in intracellular trafficking, particularly in vesicle-mediated transport between cellular compartments. While specific research on SEC13-2 is limited, studies on other transport proteins in C. glabrata demonstrate their importance in cellular functions. For example, the CgDtr1 transporter has been identified as a plasma membrane exporter that contributes to resistance against stressors like acetic acid, which is relevant during phagocytosis by host immune cells . The methodological approach to characterizing SEC13-2 function would involve gene deletion studies similar to those conducted for CgDTR1, followed by phenotypic analysis under various stress conditions to determine functional consequences.

How does the genetic diversity of Candida glabrata affect protein expression studies?

C. glabrata exhibits remarkable genetic diversity with numerous sequence types (STs) identified globally. Recent genomic analysis of 68 clinical isolates from Scotland alone revealed 20 separate sequence types, including one previously unidentified ST . This genetic heterogeneity directly impacts protein expression studies, as different strains may exhibit variations in protein coding sequences, expression levels, and post-translational modifications. When planning recombinant protein studies, researchers should carefully document the specific strain used and consider how strain-specific genetic variations might influence experimental results. A comprehensive approach would involve comparing protein sequences across multiple reference strains and potentially conducting expression studies in different genetic backgrounds.

What expression systems are suitable for producing recombinant Candida glabrata proteins?

Based on methodologies employed in C. glabrata research, several expression systems have proven effective. For homologous expression, the copper-inducible C. glabrata MTI promoter has been successfully used, as demonstrated in studies of the CgDtr1 transporter . For heterologous expression, Saccharomyces cerevisiae often serves as an appropriate host due to its genetic similarity to C. glabrata. This approach was used for CgDtr1, where the CAGL0M06281g gene was expressed in S. cerevisiae using the pGREG576 vector system with subsequent promoter replacement . E. coli systems may also be suitable for expressing protein domains, though proper folding of eukaryotic proteins can be challenging in prokaryotic hosts. Selection of an appropriate expression system should be guided by the specific research questions and downstream applications.

How can protein-protein interactions of SEC13-2 be effectively studied in Candida glabrata?

Investigating protein-protein interactions of SEC13-2 requires specialized approaches adapted to C. glabrata's unique biology. A multi-faceted strategy would include:

• Affinity purification coupled with mass spectrometry (AP-MS): Express SEC13-2 with epitope tags (such as FLAG or HA) to enable purification of protein complexes.

• Yeast two-hybrid screening: Using SEC13-2 as bait against a C. glabrata cDNA library.

• Co-immunoprecipitation with suspected interaction partners: Following up on candidates identified through genomic approaches.

• Bimolecular fluorescence complementation (BiFC): For visualizing interactions in living cells.

The choice of vector systems is critical. Studies on C. glabrata proteins have successfully employed the pGREG576 vector system with promoter replacement strategies to enable gene expression in both S. cerevisiae and C. glabrata . When designing fusion proteins, careful attention must be paid to the potential impact of tags on protein folding and function. Control experiments should include testing the function of tagged versions of SEC13-2 to ensure they behave similarly to the native protein.

What approaches can resolve the oligomerization state of recombinant SEC13-2?

Determining the oligomerization state of SEC13-2 would follow methodologies similar to those used for other C. glabrata proteins. For example, research on Cdc13 has revealed important insights into how domain interactions affect function through oligomerization . A comprehensive approach would include:

• Size exclusion chromatography (SEC) of purified recombinant protein.

• Analytical ultracentrifugation to determine native molecular weight.

• Chemical crosslinking followed by SDS-PAGE analysis.

• Native PAGE analysis under various buffer conditions.

• Multi-angle light scattering (MALS) for absolute molecular weight determination.

Additionally, structural biology approaches such as X-ray crystallography or cryo-electron microscopy could provide direct visualization of oligomeric assemblies. Functional studies correlating oligomerization state with activity would be essential to understand the biological relevance of any observed oligomeric states. Researchers should consider how environmental factors such as pH, salt concentration, and the presence of binding partners might influence oligomerization, as these factors have been shown to affect the behavior of other C. glabrata proteins .

How do post-translational modifications affect SEC13-2 function in Candida glabrata?

Analysis of post-translational modifications (PTMs) on SEC13-2 requires sophisticated proteomics approaches. Research methodology should include:

• Expression and purification of recombinant SEC13-2 from C. glabrata.

• Mass spectrometry analysis to identify PTMs (phosphorylation, glycosylation, ubiquitination, etc.).

• Site-directed mutagenesis of modified residues to determine functional significance.

• Comparison of PTM patterns under different growth conditions and stressors.

The importance of these modifications may be context-dependent. For example, stress response in C. glabrata often involves altered protein modifications as part of adaptation mechanisms. Studies of C. glabrata response to oxidative stress, which is relevant during host-pathogen interactions, have demonstrated how protein modifications contribute to resistance mechanisms . The presence of PTMs on SEC13-2 might similarly regulate its function during specific cellular responses or environmental conditions. Researchers should design experiments to compare PTM patterns in different growth phases and stress conditions to determine their regulatory significance.

How does SEC13-2 potentially contribute to Candida glabrata virulence?

While specific information about SEC13-2's role in virulence is not available, research on other C. glabrata transport proteins provides a framework for investigation. The multidrug transporter CgDtr1 has been identified as a determinant of C. glabrata virulence in the Galleria mellonella infection model, affecting the organism's ability to proliferate in host hemolymph and tolerate hemocyte attack . To investigate SEC13-2's potential role in virulence, researchers should:

• Create SEC13-2 deletion mutants in C. glabrata.

• Assess growth and survival in infection models such as G. mellonella larvae.

• Measure proliferation in the presence of host immune cells.

• Test resistance to stressors encountered during infection (oxidative stress, acidic environment, etc.).

• Evaluate SEC13-2 expression levels during different stages of infection.

Gene expression analysis using quantitative RT-PCR, similar to methods used for CgDTR1 , would help determine if SEC13-2 is upregulated during stress conditions or infection, providing evidence for its potential role in pathogenicity.

How do genomic variations in SEC13-2 correlate with antifungal resistance patterns?

Genomic diversity in C. glabrata has been linked to variations in drug resistance. A methodological approach to investigating SEC13-2's potential role would include:

• Sequence analysis of SEC13-2 across multiple clinical isolates with varying drug susceptibility profiles.

• Correlation analysis between specific genetic variants and minimum inhibitory concentrations (MICs).

• Functional validation through heterologous expression of variant SEC13-2 alleles.

• Transcriptional analysis of SEC13-2 in response to antifungal exposure.

Research on C. glabrata has identified microevolution within patients that affects cell surface proteins and genes involved in drug resistance, including the ergosterol synthesis gene ERG4 and the echinocandin target FKS1/2 . This suggests that transport proteins may undergo similar selective pressure during infection and treatment. A comprehensive study would examine SEC13-2 sequence in paired isolates from patients before and after antifungal therapy to identify treatment-associated mutations.

What in vivo models are most appropriate for studying SEC13-2 function in host-pathogen interactions?

Based on existing research on C. glabrata virulence factors, several model systems could be appropriate:

• Galleria mellonella larvae model: G. mellonella has been effectively used to study virulence determinants in C. glabrata, including the role of the CgDtr1 transporter . This model allows for assessment of fungal killing capacity, proliferation in hemolymph, and interaction with hemocytes.

• Murine models: For more complex host-pathogen interactions, murine models of disseminated candidiasis provide a mammalian system that better recapitulates human infection.

• Ex vivo human cell models: Interactions with human macrophages or epithelial cells can be studied using tissue culture systems.

The G. mellonella model offers several methodological advantages, including ease of use, low cost, and ethical considerations. The protocol involves injecting yeast cell suspensions (~5 × 10^7 cells) into the last left proleg of larvae, followed by monitoring survival over 72 hours . Proliferation can be assessed by collecting hemolymph at different time points post-infection. For more detailed analysis of SEC13-2's role in immune evasion, in vitro assays with isolated hemocytes can be performed as described in the literature .

How can CRISPR-Cas9 technology be optimized for studying SEC13-2 in Candida glabrata?

CRISPR-Cas9 editing in C. glabrata requires specific considerations due to its haploid nature and distinct genetic properties. A methodological approach would include:

• Design of guide RNAs specific to SEC13-2 with minimal off-target effects.

• Selection of appropriate promoters for Cas9 and gRNA expression in C. glabrata.

• Development of efficient transformation protocols optimized for CRISPR components.

• Creation of repair templates for gene deletion, tagging, or point mutations.

• Screening strategies to identify successful editing events.

Traditional gene deletion approaches in C. glabrata have used URA3 and LEU2 markers , but CRISPR-Cas9 offers advantages for marker-free editing. Researchers should consider using episomal expression of Cas9 followed by plasmid curing to minimize off-target effects. When designing experiments, it's important to include appropriate controls and validation of edited strains through PCR, sequencing, and phenotypic characterization to ensure the specificity of observed effects.

What strategies can overcome challenges in crystallizing recombinant Candida glabrata membrane proteins?

Crystallization of membrane proteins like SEC13-2 presents significant challenges that require specialized approaches:

• Construct optimization: Design multiple constructs with varying N- and C-terminal boundaries to identify stable, crystallizable fragments.

• Expression system selection: While E. coli is commonly used, eukaryotic expression systems like Pichia pastoris or insect cells may provide better folding for C. glabrata proteins.

• Detergent screening: Systematic testing of different detergents for protein extraction and stability during purification.

• Lipidic cubic phase (LCP) crystallization: This method has proven successful for many recalcitrant membrane proteins.

• Co-crystallization with antibody fragments or binding partners to stabilize the protein.

Studies of C. glabrata protein structure, such as those examining Cdc13 molecular architecture , provide useful precedents for structural biology approaches. Researchers should monitor protein stability through techniques like size-exclusion chromatography and thermal shift assays during optimization. Alternative approaches such as cryo-electron microscopy should be considered if crystallization proves challenging, as this technique has revolutionized structural studies of membrane proteins in recent years.

How can high-throughput screening be designed to identify small molecule modulators of SEC13-2 function?

Developing a high-throughput screening (HTS) assay for SEC13-2 modulators would require:

• Functional assay development: Create a robust, reproducible assay that measures SEC13-2 activity. This could be based on:

  • Transport assays if SEC13-2 is involved in direct transport

  • Growth phenotypes in SEC13-2 mutants under specific conditions

  • Protein-protein interaction disruption assays

• Assay optimization for HTS format:

  • Miniaturization to 384- or 1536-well format

  • Automation compatibility

  • Robust statistical parameters (Z'-factor > 0.5)

  • Low variability between replicates

• Compound library selection:

  • Focused libraries based on known modulators of similar proteins

  • Diversity-oriented collections for novel scaffold discovery

  • Natural product libraries that might contain fungal-specific inhibitors

• Secondary assay development for hit validation:

  • Orthogonal assays to confirm mechanism of action

  • Cytotoxicity assessment in human cells

  • Specificity testing against related proteins

Studies on C. glabrata drug susceptibility have employed methods like spot assays and liquid growth medium cultivation to evaluate responses to toxic compounds . These approaches could be adapted and optimized for higher throughput to screen for SEC13-2 modulators. Phenotypic screens based on growth under specific stress conditions relevant to SEC13-2 function could provide a functionally relevant readout without requiring purified protein.

How does the study of SEC13-2 contribute to our understanding of Candida glabrata evolution?

Research on C. glabrata proteins in an evolutionary context reveals important insights into adaptation mechanisms. Analysis of SEC13-2 could contribute to understanding:

• Evolutionary conservation and divergence: Comparative analysis of SEC13-2 across Candida species and other fungi can reveal evolutionary pressures on protein transport systems.

• Adaptation to host environments: SEC13-2 variants might reflect adaptations to specific host niches or immune pressures.

• Genetic exchange and recombination: Evidence of recombination affecting SEC13-2 could provide insights into C. glabrata's cryptic sexual cycle.

Population genomics studies have revealed that C. glabrata isolates demonstrate evidence of ancestral recombination and genetic exchange between geographically distinct populations . This suggests that transport proteins may have evolved through similar mechanisms. A methodological approach would involve phylogenetic analysis of SEC13-2 sequences across clinical isolates from different geographical regions and correlation with phenotypic characteristics relevant to virulence and stress resistance.

What computational approaches can best predict SEC13-2 structure-function relationships?

Modern computational methods offer powerful tools for predicting protein structure and function:

• Homology modeling: Using structures of related proteins as templates for SEC13-2 structure prediction.

• Molecular dynamics simulations: To explore conformational dynamics and potential transport mechanisms.

• Protein-protein interaction prediction: Using algorithms that consider sequence conservation, co-evolution patterns, and structural compatibility.

• Machine learning approaches: Training on known transport protein structures to identify functional motifs in SEC13-2.

• Molecular docking: To predict potential binding sites for small molecules or interaction partners.

The effectiveness of these approaches would depend on the degree of conservation between SEC13-2 and proteins of known structure. Research on C. glabrata proteins has demonstrated how structural analysis can reveal functional mechanisms, as illustrated by studies of Cdc13 domain organization and its impact on telomeric DNA binding . Researchers should validate computational predictions through experimental approaches such as site-directed mutagenesis of predicted functional residues.

How might single-cell technologies advance our understanding of SEC13-2 function during infection?

Single-cell technologies offer unprecedented insights into cellular heterogeneity and could reveal important aspects of SEC13-2 function:

• Single-cell RNA-sequencing (scRNA-seq): To identify subpopulations of C. glabrata with differential SEC13-2 expression during infection.

• CyTOF or spectral flow cytometry: For multiparameter analysis of SEC13-2 protein levels and localization across populations.

• Microfluidic devices: To study SEC13-2 dynamics in individual cells under controlled conditions.

• Single-cell proteomics: To measure changes in the broader proteome associated with SEC13-2 function.