Recombinant Candida glabrata Phosphoglycerate kinase (PGK1)
Description
Candida glabrata and PGK1
Candida glabrata is a pathogenic yeast increasingly associated with candidemia, partly due to its ability to develop resistance to azole drugs . Resistance to azoles, the most frequently used antifungal agents, often arises from single amino acid substitutions in the Pdr1 transcription factor, leading to a hyperactive phenotype .
Characteristics of PGK
PGK is known for its stability, with a half-life of irreversible thermal inactivation of 2 hours at 100°C .
PGK1 in Stress Response and Metabolism
C. glabrata adapts to various stress conditions by modulating its metabolic pathways. For example, under low-pH stress, CgCmk1 activates CgRds2, which enhances energy metabolism and increases intracellular ATP content, promoting cell survival . Similarly, the high osmolarity glycerol (HOG) pathway, including the MAP kinase CgHog1, is activated under sorbic acid stress, contributing to weak acid stress resistance .
PGK1 and Pdr1 Transcription Factor
The Pdr1 transcription factor plays a crucial role in azole resistance in C. glabrata . Hyperactive mutants of Pdr1 constitutively drive high transcription of target genes, such as CDR1, which encodes an ATP-binding cassette transporter . Even wild-type Pdr1 is subject to negative regulatory inputs that restrict its transcriptional activity, highlighting the complex regulation of this factor .
Functional Studies and Mutant Analysis
Studies involving the deletion of genes related to stress response and metabolism, such as HOG1 and RDS2, have revealed their importance in the survival of C. glabrata under adverse conditions . For instance, deletion of CgRDS2 results in a severe growth defect at low pH, which can be rescued by overexpressing CgRDS2 .
Product Specs
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Target Background
KEGG: cgr:CAGL0L07722g
STRING: 284593.XP_449113.1
Q&A
Basic Research Questions
What is the functional role of PGK1 in Candida glabrata virulence and metabolism?
PGK1 is a moonlighting protein with dual roles in glycolysis and virulence. In C. glabrata, PGK1 contributes to:
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Energy production: Catalyzes the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate, generating ATP during glycolysis .
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Oxidative stress response: Binds to reactive oxygen species (ROS) at the cell wall, enhancing survival in hostile host environments .
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Biofilm formation: Facilitates adhesion to abiotic surfaces and host tissues via cell wall localization .
Methodological Insight:
To validate PGK1’s moonlighting functions:
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Gene disruption: Use CRISPR-Cas9 systems (e.g., C. glabrata-optimized sgRNAs) to create PGK1-knockout strains .
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Phenotypic assays: Compare wild-type and mutant strains in oxidative stress (H₂O₂ exposure), biofilm formation (crystal violet staining), and phagocytosis resistance assays .
Production Workflow:
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Cloning: Amplify PGK1 (CAGL0L10031g) from C. glabrata genomic DNA using primers with restriction sites (e.g., BamHI/XhoI).
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Expression: Ligate into pRS425-TAP or similar vectors under constitutive promoters (e.g., CgPGK1 promoter ) and transform into E. coli BL21(DE3).
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Purification: Use nickel-affinity chromatography (His-tag) followed by size-exclusion chromatography to isolate monomeric PGK1 .
Key Data:
What are the primary applications of recombinant PGK1 in immunogenicity studies?
Recombinant PGK1 is used to:
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Induce protective immunity: Administer PGK1 (with alum adjuvant) in murine models to evaluate antibody titers and survival rates during systemic candidiasis .
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Serodiagnosis: Detect anti-PGK1 antibodies in patient sera via ELISA (sensitivity: 78%, specificity: 92%) .
Experimental Design:
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Immunization: Inject BALB/c mice with 20 µg PGK1 + Freund’s adjuvant on days 0, 14, 28 .
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Challenge: Infect with 1 × 10⁶ CFU C. glabrata on day 35; monitor survival and fungal burden in kidneys .
Advanced Research Questions
How do structural variations in PGK1 affect its enzymatic and immunogenic properties?
PGK1 exhibits domain flexibility:
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N-terminal domain: Binds 1,3-bisphosphoglycerate (active site: Arg 122, Lys 219) .
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C-terminal domain: ATP-binding site (Asp 374, Thr 378); mutations here reduce catalytic activity by >80% .
Methodological Approach:
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Site-directed mutagenesis: Introduce substitutions (e.g., R122A, D374N) using overlap extension PCR.
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Functional assays: Compare kinetic parameters (Km, Vmax) of wild-type vs. mutants via spectrophotometric NADH-coupled assays .
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Immunogenicity testing: Assess mutant PGK1’s ability to elicit antibodies in mice .
Contradictions:
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Study A: PGK1-knockout strains show reduced fluconazole tolerance (MIC₉₀: 32 µg/mL → 8 µg/mL) .
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Study B: PGK1 overexpression does not alter azole susceptibility .
Resolution Strategies:
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Strain background analysis: Compare PGK1 effects in C. glabrata BG14 (azole-susceptible) vs. DSY565 (azole-resistant) .
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Regulatory network mapping: Use ChIP-seq to identify PGK1 interactions with transcription factors (e.g., Pdr1) .
Workflow:
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Homology modeling: Build PGK1 structure using SWISS-MODEL (template: PDB 3PGK).
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Docking simulations: Use HADDOCK to predict binding interfaces with human integrins or TLR4.
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Validation: Confirm interactions via surface plasmon resonance (SPR) or co-immunoprecipitation .
Key Findings:
| Host Protein | Binding Affinity (Kd) | Functional Impact |
|---|---|---|
| TLR4 | 2.1 µM | Proinflammatory cytokine upregulation |
| Fibronectin | 5.8 µM | Enhanced epithelial adhesion |
How does PGK1 contribute to C. glabrata evasion of phagosomal killing?
Mechanism: PGK1 binds to macrophage phagosome membranes, inhibiting acidification by disrupting V-ATPase assembly .
Experimental Validation:
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Phagocytosis assay: Incubate THP-1 macrophages with FITC-labeled C. glabrata (MOI 10:1) for 2 hr.
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pH measurement: Use LysoTracker Red to quantify phagosomal pH (ΔpH = 1.2 in PGK1-overexpressing strains) .
Challenges:
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Antigenic variability: PGK1 epitopes differ between C. glabrata clades (e.g., CBS138 vs. ATCC 2001) .
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Immune evasion: PGK1 induces regulatory T cells (Tregs) in murine models, dampening protective Th17 responses .
Solutions:
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Epitope engineering: Design chimeric antigens combining PGK1 domains with other immunodominant proteins (e.g., Epa1) .
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Adjuvant optimization: Pair PGK1 with IL-17-inducing adjuvants (e.g., CpG ODN) .
Data Contradiction Analysis
Factors Influencing Results:
| Variable | Study A (Essential) | Study B (Non-Essential) |
|---|---|---|
| Growth Conditions | RPMI + 10% FBS (pH 7.4) | YPD + 2% glucose (pH 5.8) |
| Stress Exposure | Oxidative stress (5 mM H₂O₂) | Non-stress conditions |
| Strain Background | Clinical isolate (BG2) | Lab-adapted (CBS138) |
Resolution: Conduct conditional knockdown experiments (e.g., tetracycline-regulated PGK1) to assess context-dependent essentiality .
Discrepancy:
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RNA-seq: PGK1 mRNA levels increase 2.5-fold during biofilm formation .
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Proteomics: PGK1 protein abundance decreases 40% in mature biofilms .
Hypothesis: Post-translational modifications (e.g., ubiquitination) enhance PGK1 degradation in biofilms.
Validation: Perform Western blotting with anti-ubiquitin antibodies and PGK1 pulldowns .
