Recombinant Candida glabrata Vacuolar membrane protein CAGL0J10076g (CAGL0J10076g)

Basic Research Questions

• What is CAGL0J10076g and what is its cellular localization?

  CAGL0J10076g is a vacuolar membrane protein found in Candida glabrata (strain ATCC 2001/CBS 138/JCM 3761/NBRC 0622/NRRL Y-65), an opportunistic fungal pathogen. As indicated by its annotation, this protein is localized to the vacuolar membrane, suggesting potential roles in vacuolar functions such as ion homeostasis, protein degradation, or metabolite storage. According to UniProt data (Q6FNP6), the full-length protein consists of 378 amino acids with characteristic membrane-spanning domains .

• How should recombinant CAGL0J10076g be stored to maintain optimal activity?

  For maximum stability and activity retention, recombinant CAGL0J10076g should be stored following this protocol:

  • Primary storage buffer: Tris-based buffer with 50% glycerol optimized for this specific protein

  • Storage temperature: -20°C for routine storage; -80°C recommended for extended preservation

  • Working aliquots: Can be maintained at 4°C for up to one week

  • Handling precautions: Repeated freeze-thaw cycles should be avoided as they compromise protein integrity

  This storage protocol helps maintain the native conformation and functional properties of the recombinant protein during experimental timeframes .

• What expression systems are suitable for producing recombinant CAGL0J10076g?

  Successful expression of functional CAGL0J10076g requires selecting appropriate systems based on experimental needs:

  Expression System	Advantages	Considerations	Yield Expectations
  E. coli	Rapid growth, easy manipulation	May lack appropriate post-translational modifications	Medium (requires optimization)
  Yeast (P. pastoris)	Eukaryotic processing, higher similarity to native folding	Longer cultivation time	High
  Insect cells	Advanced eukaryotic modifications	Complex media requirements	Medium-High

  For membrane proteins like CAGL0J10076g, expression tags are critical for purification. While the specific tag type may be determined during the production process, common options include His6, GST, or MBP tags positioned to minimize interference with protein function .

Advanced Research Questions

• How does CAGL0J10076g potentially contribute to Candida glabrata pathogenicity?

  While direct evidence for CAGL0J10076g's role in virulence is not fully characterized, its vacuolar membrane localization suggests several potential contributions to pathogenicity:

  1. Stress response regulation: Vacuolar proteins often mediate adaptation to host-induced stresses

  2. pH homeostasis: Maintaining appropriate internal pH despite environmental fluctuations

  3. Nutrient acquisition and storage: Supporting survival in nutrient-limited host environments

  Methodological approach for investigating these functions:

  • Create CAGL0J10076g knockout strains using CRISPR-Cas9 or traditional homologous recombination

  • Evaluate virulence in models such as Galleria mellonella following protocols similar to those used for CgDTR1

  • Assess phenotypes under specific stressors (oxidative, pH, nutritional)

  • Measure vacuolar function in wild-type versus mutant strains using pH-sensitive fluorescent probes

• What methodological approaches are most effective for studying CAGL0J10076g localization?

  Confirming and characterizing CAGL0J10076g localization requires multi-faceted approaches:

  1. Fluorescent protein tagging:

     • C-terminal or N-terminal GFP/mCherry fusion constructs

     • Validation by co-localization with established vacuolar markers (e.g., FM4-64)

     • Live-cell imaging under various environmental conditions

  2. Immunolocalization:

     • Generation of specific antibodies against CAGL0J10076g epitopes

     • Optimization of fixation methods for membrane protein preservation

     • Double-labeling with organelle-specific antibodies

  3. Subcellular fractionation:

     • Differential centrifugation to isolate vacuolar membrane fractions

     • Western blot analysis of fractions using protein-specific antibodies

     • Mass spectrometry verification of protein presence in isolated fractions

  These approaches should be performed under both standard laboratory conditions and infection-relevant stressors to determine if localization changes during pathogenesis .

• How can researchers effectively design knockout and complementation studies for CAGL0J10076g?

  Rigorous genetic manipulation studies require careful experimental design:

  1. Knockout strategy:

     • Design deletion constructs with 500-1000bp homology regions flanking CAGL0J10076g

     • Include selectable markers appropriate for C. glabrata (e.g., NAT1, SAT1)

     • Confirm deletion by PCR, Southern blotting, and RT-PCR/Western blotting

  2. Complementation approach:

     • Reintroduce CAGL0J10076g under native promoter control

     • Alternative: use controllable promoters (e.g., MET3, TET-OFF) for regulated expression

     • Include epitope tags if antibodies are unavailable

  3. Phenotypic analysis matrix:

     Phenotype	Wild-type	Δcagl0j10076g	Complemented	Methodology
     Growth rate	Baseline	Assess impact	Should restore	Growth curves in various media
     Stress tolerance	Baseline	Potential decrease	Should restore	Spot assays with stressors
     Vacuolar function	Normal	Potentially altered	Should restore	Vacuolar pH and morphology assays
     Virulence	Baseline	Hypothesized decrease	Should restore	G. mellonella infection model

  This systematic approach allows attribution of phenotypes specifically to CAGL0J10076g function while controlling for potential secondary effects of genetic manipulation .

• How might CAGL0J10076g interact with other virulence factors in Candida glabrata?

  Investigating potential functional relationships between CAGL0J10076g and other virulence determinants requires integrated approaches:

  1. Transcriptomic analysis:

     • Compare gene expression profiles of wild-type and Δcagl0j10076g strains under infection-relevant conditions

     • Identify co-regulated genes using RNA-seq

     • Validate key findings with qRT-PCR

  2. Epistasis studies:

     • Generate double mutants with known virulence genes (e.g., CgDTR1)

     • Assess phenotypes to determine additive, synergistic, or epistatic relationships

     • Analyze in both in vitro and infection models

  3. Protein-protein interaction studies:

     • Co-immunoprecipitation with membrane-compatible detergents

     • Proximity-dependent labeling (BioID or APEX)

     • Yeast two-hybrid adapted for membrane proteins

  These approaches could reveal functional connections between CAGL0J10076g and other virulence determinants such as CgDtr1, which has been established as an important factor in C. glabrata pathogenesis through similar methodological approaches .

• What experimental design is optimal for studying CAGL0J10076g role in host-pathogen interactions?

  A comprehensive investigation of CAGL0J10076g in host-pathogen interactions should include:

  1. Macrophage interaction model:

     • Infect human/murine macrophages with wild-type and Δcagl0j10076g strains

     • Assess phagocytosis rates, intracellular survival, and replication

     • Measure host cytokine responses and macrophage activation markers

     • Compare with known virulence mutants like Δcgdtr1, which shows impaired proliferation within hemocytes

  2. Invertebrate infection model:

     • G. mellonella larvae as cost-effective in vivo system

     • Inject standardized inoculum (5×10^7 CFU/larvae)

     • Monitor survival using Kaplan-Meier analysis

     • Quantify fungal burden in hemolymph at defined timepoints (1h, 24h, 48h)

     • Assess interaction with hemocytes in ex vivo co-culture systems

  3. Advanced analysis techniques:

     • Live-cell microscopy tracking labeled C. glabrata within phagocytes

     • Transcriptional profiling of both pathogen and host during interaction

     • Measurement of vacuolar pH and function during phagocytosis

  This multi-level approach parallels successful studies of other C. glabrata virulence factors like CgDtr1, which demonstrated increased virulence and proliferation in G. mellonella through similar methodological frameworks .

• How can researchers investigate the potential role of CAGL0J10076g in antifungal resistance?

  Given C. glabrata's known propensity for developing antifungal resistance, CAGL0J10076g's contribution should be investigated through:

  1. Susceptibility testing:

     • Determine minimum inhibitory concentrations (MICs) for multiple antifungal classes

     • Compare wild-type, knockout, and overexpression strains

     • Assess development of resistance under selective pressure

  2. Mechanistic investigations:

     • Measure intracellular drug accumulation using fluorescent antifungal analogs

     • Assess vacuolar sequestration of antifungals

     • Monitor membrane potential and drug efflux activity

  3. Resistance marker correlation study:

     • Analyze CAGL0J10076g expression in clinical isolates with defined resistance profiles

     • Sequence CAGL0J10076g from resistant isolates to identify potential mutations

     • Test contribution to resistance in heterologous expression systems

  4. Data integration:

     Strain	Azole MIC	Echinocandin MIC	Intracellular Drug Accumulation	CAGL0J10076g Expression
     Wild-type	Baseline	Baseline	Baseline	Normal
     Δcagl0j10076g	Hypothesized change	Hypothesized change	To be measured	Absent
     Overexpression	Hypothesized change	Hypothesized change	To be measured	Elevated
     Resistant isolates	Elevated	Variable	Often decreased	To be determined

  This approach provides a comprehensive assessment of CAGL0J10076g's potential role in antifungal resistance mechanisms, particularly as vacuolar proteins may contribute to drug sequestration or altered membrane properties .

• What comparative genomic approaches can reveal about CAGL0J10076g evolution and conservation?

  Understanding the evolutionary context of CAGL0J10076g requires systematic comparative analysis:

  1. Ortholog identification:

     • BLAST searches against other Candida species and fungi

     • Synteny analysis to confirm orthologous relationships

     • Phylogenetic reconstruction to trace evolutionary history

  2. Sequence conservation analysis:

     • Multiple sequence alignment of identified orthologs

     • Identification of conserved domains and critical residues

     • Selection pressure analysis (dN/dS ratios)

  3. Functional conservation testing:

     • Heterologous expression of orthologs in Δcagl0j10076g background

     • Assessment of phenotypic complementation

     • Domain swapping experiments to identify functionally critical regions

  4. Correlation with pathogenicity:

     • Compare conservation patterns between pathogenic and non-pathogenic species

     • Identify pathogen-specific features or adaptations

     • Correlate with known virulence characteristics across species

  These approaches place CAGL0J10076g in an evolutionary context that may reveal its functional significance and potential as a species-specific virulence factor or conserved fungal protein with broader biological importance .