Recombinant Candida glabrata Glutamine synthetase (GLN1)

What is the role of glutamine synthetase (GLN1) in Candida glabrata metabolism?

Glutamine synthetase (GLN1) in C. glabrata catalyzes the ATP-dependent conversion of glutamate and ammonium to glutamine, serving as a critical component of nitrogen assimilation pathways. This enzyme fulfills several essential metabolic functions:

• Primary nitrogen incorporation when ammonium is the sole nitrogen source

• Glutamine biosynthesis for protein synthesis and as a nitrogen donor for various biosynthetic pathways

• Maintenance of nitrogen homeostasis through integration with nitrogen catabolite repression (NCR) pathways

• Contribution to stress resistance by facilitating adaptation to nitrogen-limited environments

In C. glabrata, glutamine metabolism is particularly important for pathogenicity and survival within host tissues, where nitrogen sources can be limited or variable. The enzyme functions within a network of nitrogen-responsive elements, including transcription factors like Gln3 that regulate nitrogen assimilation pathways .

How does GLN1 relate to nitrogen catabolite repression in C. glabrata?

In C. glabrata, GLN1 is subject to nitrogen catabolite repression (NCR), a regulatory mechanism that prioritizes preferred nitrogen sources. This relationship is multifaceted:

• GLN1 expression is repressed when preferred nitrogen sources like glutamine are abundant

• Under nitrogen limitation, NCR is relieved and GLN1 expression increases

• Transcription factors including Gln3 directly regulate GLN1 expression

Research has demonstrated that Gln3 plays a significant role in nitrogen assimilation in C. glabrata, with its absence affecting cell growth when glutamine is involved . This regulatory network ensures that C. glabrata can efficiently adapt to changing nitrogen environments during colonization and infection.

The interplay between GLN1 and NCR contributes to metabolic flexibility, allowing C. glabrata to rapidly assimilate different nitrogen compounds from the host. This adaptability is considered an important factor in C. glabrata's success as a pathogen in various host niches.

How does GLN1 expression change under different environmental conditions?

GLN1 expression in C. glabrata responds dynamically to environmental conditions through a complex regulatory network:

Under preferred nitrogen source conditions (e.g., glutamine), GLN1 expression is repressed through the TOR signaling pathway and Ure2-mediated sequestration of transcription factors. When cells encounter nitrogen limitation, particularly when ammonium is the primary nitrogen source, Gln3 translocates to the nucleus and activates GLN1 transcription .

Additionally, during stress conditions such as oxidative stress or macrophage phagocytosis, GLN1 expression can be induced through stress-response pathways independent of nitrogen availability, highlighting the integration of nitrogen metabolism with stress response mechanisms in this pathogen.

What are the optimal conditions for expressing recombinant C. glabrata GLN1 in heterologous systems?

Successful expression of recombinant C. glabrata GLN1 requires optimization of several parameters depending on the expression system chosen:

For E. coli Expression:

• Recommended strain: BL21(DE3) or Rosetta(DE3) for improved codon usage

• Expression vector: pET28a(+) with N-terminal His-tag for purification

• Culture conditions: Growth at 37°C until OD600 reaches 0.6-0.8, followed by induction at 18-20°C for 16-20 hours

• Induction: 0.1-0.5 mM IPTG at reduced temperature (18-20°C)

• Buffer optimization: Include 5 mM MgCl2 and 1 mM DTT to maintain enzyme stability

For Expression in Yeast Systems:

• S. cerevisiae or C. glabrata: Use vectors with constitutive promoters (TEF1, PGK1) or inducible promoters (GAL1, CUP1)

• Selection: Complement auxotrophic markers (URA3, LEU2) for stable expression

• Copper-inducible systems: The MTI promoter provides tightly controlled expression in C. glabrata

• Secretion: Include a signal sequence if extracellular production is desired

Critical Parameters for GLN1 Expression:

• Lower temperatures during induction significantly improve soluble protein yield

• Addition of stabilizing agents (glycerol, MgCl2) enhances enzyme stability

• Codon optimization can improve yields by 3-5 fold in E. coli

• Monitor for potential toxicity if overexpressed in the native host

These approaches can be adapted from methods described for expressing other C. glabrata proteins, such as those used for CgDtr1 expression systems .

How can I create a C. glabrata GLN1 deletion mutant for functional studies?

Creating a C. glabrata GLN1 deletion mutant requires a targeted gene replacement strategy. The following methodology is recommended:

Materials Required:

• C. glabrata wild-type strain (KUE100, L5U1, or CBS138 are commonly used lab strains)

• Selection marker (typically URA3 for prototrophic selection)

• PCR primers for amplifying flanking regions of GLN1

• Transformation reagents

Step-by-Step Protocol:

• Create deletion cassette: Use fusion PCR or restriction enzyme-based cloning to join the 5' homology region, selection marker, and 3' homology region

• Transform C. glabrata: Use lithium acetate/polyethylene glycol method with modifications for C. glabrata

• Select transformants: Plate on selective medium lacking uracil (if using URA3 marker)

• Verify deletion: Confirm GLN1 deletion by PCR and sequencing

Special Considerations:

• Since GLN1 may be essential under certain conditions, provide glutamine supplementation (0.5-1 g/L) in the medium during transformation and initial selection

• For complementation studies, the reintroduction of GLN1 can be performed using plasmids like pGREG576 with the copper-inducible MTI promoter, similar to methods described for other C. glabrata genes

• Consider creating conditional mutants if complete deletion proves lethal

Similar gene deletion strategies have been successfully employed for studying other metabolic genes in C. glabrata, such as CgDTR1 .

What purification strategy works best for recombinant C. glabrata glutamine synthetase?

Purifying recombinant C. glabrata glutamine synthetase requires a multi-step approach to achieve high purity while maintaining enzymatic activity:

Recommended Purification Strategy:

• Cell Lysis:

  • Resuspend cells in lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM imidazole, 5 mM MgCl2, 1 mM DTT, 10% glycerol, protease inhibitor cocktail)

  • Lyse cells by sonication or using a French press

  • Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Immobilized Metal Affinity Chromatography (IMAC):

  • Load clarified lysate onto Ni-NTA column pre-equilibrated with lysis buffer

  • Wash with 10-20 column volumes of wash buffer (lysis buffer with 30 mM imidazole)

  • Elute protein with elution buffer (lysis buffer with 250 mM imidazole)

• Size Exclusion Chromatography:

  • Apply concentrated IMAC eluate to a Superdex 200 column

  • Elute with SEC buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol)

Purification Performance Metrics:

Critical Considerations:

• Maintain 5 mM MgCl2 throughout purification to stabilize the oligomeric structure

• Include 1 mM DTT to prevent oxidation of sensitive cysteine residues

• Avoid freeze-thaw cycles; store purified enzyme at 4°C for short-term or in 50% glycerol at -20°C for long-term storage

• Activity is typically highest at pH 7.0-7.5 and temperature of 30°C

These purification methods can be adapted from protocols used for other recombinant C. glabrata proteins, with modifications specific to maintain glutamine synthetase activity.

What assays can be used to measure glutamine synthetase activity in C. glabrata?

Several assays can be employed to measure glutamine synthetase activity in C. glabrata samples:

1. γ-Glutamyl Transferase Assay:

• Principle: Measures the transferase activity of glutamine synthetase by following the formation of γ-glutamylhydroxamate

• Reaction: Glutamine + hydroxylamine → γ-glutamylhydroxamate + NH3

• Detection: Colorimetric measurement at 540 nm after reaction with ferric chloride

• Sensitivity: 0.1-5 μmol/min/mg protein

2. Coupled Enzyme Assay:

• Principle: Couples glutamine synthesis to NADH oxidation through auxiliary enzymes

• Reaction: Glutamate + NH4+ + ATP → Glutamine + ADP + Pi; coupled to NADH → NAD+

• Detection: Spectrophotometric measurement of NADH oxidation at 340 nm

• Sensitivity: 0.05-1 μmol/min/mg protein

3. Radiometric Assay:

• Principle: Measures incorporation of labeled substrates into glutamine

• Detection: Scintillation counting or mass spectrometry

• Sensitivity: 0.01-0.1 μmol/min/mg protein

• Advantage: Highest sensitivity for measuring activity in crude extracts

Sample Preparation Protocol:

• Harvest C. glabrata cells in mid-log phase

• Wash cells with ice-cold buffer (50 mM HEPES pH 7.0)

• Resuspend in extraction buffer (50 mM HEPES pH 7.0, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, protease inhibitors)

• Disrupt cells using glass beads (8 cycles of 30 sec vortexing followed by 30 sec on ice)

• Centrifuge at 15,000 × g for 15 min at 4°C

• Use supernatant for activity assays

For gene expression analysis, real-time RT-PCR can be performed using methods similar to those described for CgDTR1, with appropriate primers specific for GLN1 and reference genes like CgACT1 .

How can I distinguish between glutamine synthetase activity and other ammonium-assimilating enzymes?

Distinguishing glutamine synthetase (GLN1) activity from other ammonium-assimilating enzymes in C. glabrata requires selective assay conditions and inhibitors:

Differential Inhibition Approach:

• Measure total ammonium-assimilating activity in cell extracts

• Add methionine sulfoximine (MSO, 1-5 mM), a specific inhibitor of glutamine synthetase

• The difference between total activity and activity with MSO represents glutamine synthetase contribution

Enzyme-Specific Conditions:

Data Interpretation Guidelines:

• In nitrogen-rich conditions, GDH activity typically dominates ammonium assimilation

• Under nitrogen limitation, GLN1 activity becomes predominant

• During specific stress conditions, all three pathways may be simultaneously active

• Consider using GLN1 deletion mutants (complemented with glutamine) as negative controls

When analyzing gene expression patterns, normalize to appropriate reference genes such as CgACT1, as demonstrated for the analysis of other C. glabrata genes .

What statistical approaches are recommended for analyzing GLN1 expression data in different conditions?

Recommended Statistical Approaches:

• For RT-PCR Data:

  • Normalize GLN1 expression to reference genes (ACT1 is commonly used in C. glabrata)

  • Apply the 2^-ΔΔCt method for relative quantification

  • Use ANOVA with post-hoc tests (Tukey's HSD) for comparing multiple conditions

  • For time-course experiments, consider repeated measures ANOVA

• For RNA-Seq Data:

  • Normalize using RPKM/FPKM or TPM methods

  • Apply DESeq2 or edgeR for differential expression analysis

  • Use false discovery rate (FDR) correction for multiple testing

  • Consider gene set enrichment analysis to identify coordinated changes in nitrogen metabolism pathways

Sample Size and Replication Recommendations:

• Minimum of 3 biological replicates per condition

• 2-3 technical replicates per biological sample

• Power analysis to determine adequate sample size (typically n=5-6 for detecting 1.5-fold changes with 80% power)

Visualization of GLN1 Expression Data:

These statistical approaches align with methods used in published C. glabrata gene expression analyses, such as those described for analyzing expression of other genes involved in stress responses and virulence .

How should I interpret changes in GLN1 activity during infection models?

Interpreting GLN1 activity changes during infection models requires consideration of multiple factors specific to host-pathogen interactions:

Interpretation Framework for Infection Models:

• Baseline Considerations:

  • Establish GLN1 activity baseline in pre-infection C. glabrata cells

  • Account for variability in host immune responses

  • Consider the influence of temperature and pH on enzyme activity

• Temporal Analysis:

  • Early infection phase (1-4h): Initial adaptation to host environment

  • Mid infection phase (24h): Established infection and immune response

  • Late infection phase (48-72h): Persistent infection or clearance

• Correlating GLN1 Activity with Infection Parameters:

Methodological Considerations:

• Extract C. glabrata cells from infection sites at defined timepoints

• Immediately stabilize samples to prevent ex vivo activity changes

• Use both enzymatic assays and transcript analysis to distinguish between expression and activity changes

• Include appropriate controls for each timepoint

In G. mellonella infection models, similar to those used for studying CgDtr1 function , increased GLN1 activity during later infection stages (48h) would suggest that glutamine synthesis becomes important for sustained proliferation within the host. This interpretation would be strengthened if GLN1 deletion mutants show reduced proliferation and virulence at comparable timepoints.

How does GLN1 contribute to C. glabrata virulence and survival within host macrophages?

GLN1 plays several critical roles in C. glabrata virulence and intramacrophage survival through both direct and indirect mechanisms:

Direct Contributions to Virulence:

• Nitrogen Source Adaptation: GLN1 enables utilization of limited nitrogen sources within macrophage phagosomes

• Glutamine Provision: Supplies glutamine for cell wall remodeling needed during stress adaptation

• pH Homeostasis: Contributes to cytoplasmic pH regulation during exposure to phagosomal acidification

Indirect Contributions via Metabolic Integration:

• Stress Response Coordination: GLN1 activity is linked to oxidative stress resistance pathways

• Energy Metabolism: Glutamine synthesis affects TCA cycle activity under glucose-limited conditions

• Cell Wall Integrity: GLN1-derived glutamine is required for chitin and mannan biosynthesis

These mechanisms parallel observations with other metabolic enzymes in C. glabrata, such as CgDtr1, which contributes to virulence by protecting cells from stress agents present in macrophages .

Mechanistic Model of GLN1 in Macrophage Survival:

• Following phagocytosis, phagosomal acidification and reactive oxygen species production create a hostile environment

• C. glabrata upregulates GLN1 to maintain glutamine pools despite limited nitrogen availability

• GLN1-derived glutamine feeds into glutathione synthesis, enhancing protection against oxidative damage

• Sustained GLN1 activity enables prolonged survival and eventual replication within macrophages

This model suggests that targeting GLN1 could potentially enhance macrophage clearance of C. glabrata, representing a novel approach to antifungal therapy development.

What structural differences exist between C. glabrata glutamine synthetase and human glutamine synthetase?

Structural differences between C. glabrata and human glutamine synthetases provide several opportunities for understanding species-specific functions and potential selective inhibitor development:

Key Structural Differences:

• Active Site Architecture:

  • C. glabrata GLN1 has a more constricted ATP-binding pocket

  • Different metal coordination geometry (Mn2+ vs. Mg2+ preference)

  • C. glabrata enzyme contains unique cysteine residues near the glutamate binding site

• Quaternary Structure:

  • C. glabrata GLN1: Octameric structure (8 identical subunits)

  • Human GS: Decameric structure (10 identical subunits)

  • Different subunit interface residues affect allosteric regulation

• Regulatory Regions:

  • C. glabrata GLN1 lacks the extended C-terminal regulatory domain found in human GS

  • Different patterns of post-translational modification sites (phosphorylation, acetylation)

  • Species-specific allosteric binding pockets

Comparative Structural Analysis:

These structural differences not only explain the biochemical properties of C. glabrata GLN1 but also provide insights into its evolutionary adaptation to the host environment and potential vulnerabilities that could be exploited for research and therapeutic purposes.

How does the interplay between GLN1 and other nitrogen metabolism genes affect adaptation to host environments?

The adaptive capacity of C. glabrata in diverse host niches depends on coordinated regulation of nitrogen metabolism genes, with GLN1 playing a central role:

Key Regulatory Interactions:

• GLN1-GDH1/3 Balance:

  • During nitrogen abundance: GDH1/3 (glutamate dehydrogenases) are preferentially expressed

  • During nitrogen limitation: GLN1 activity increases via Gln3-mediated transcriptional activation

  • This reciprocal regulation optimizes nitrogen incorporation efficiency

• GLN1-GLN3 Regulatory Circuit:

  • Gln3 activates GLN1 transcription under nitrogen limitation

  • GLN1-derived glutamine feeds back to regulate Gln3 nuclear localization

  • This feedback loop enables rapid adaptation to changing nitrogen availability

• Integration with Stress Response Pathways:

  • Oxidative stress triggers GLN1 upregulation independent of nitrogen status

  • pH stress modulates the balance between GLN1 and GDH activities

  • Carbon source availability affects GLN1 expression through retrograde signaling

Host Environment-Specific Adaptations:

This metabolic flexibility parallels adaptation mechanisms observed with other transporters and metabolic enzymes in C. glabrata, such as CgDtr1 which helps the organism adapt to acidic environments .

What are the challenges in developing selective inhibitors targeting C. glabrata GLN1?

Developing selective inhibitors against C. glabrata GLN1 presents several challenges that researchers must address:

Target Validation Challenges:

• Metabolic Redundancy: Alternative nitrogen assimilation pathways may compensate for GLN1 inhibition

• Essentiality Assessment: Determining conditions under which GLN1 is essential for survival

• Host Environment Effects: Glutamine availability in host tissues may bypass inhibitor effects

Biochemical and Structural Challenges:

• Enzyme Conservation: High degree of conservation in catalytic domains across species

• Specificity Design: Achieving sufficient selectivity over human glutamine synthetase

• Binding Site Access: Designing molecules that can access the active site within the octameric structure

Pharmacological Challenges:

• Penetration Barriers: Overcoming the cell wall and membrane barriers of C. glabrata

• Efflux Systems: Addressing potential efflux by multidrug transporters like CgDtr1

• Bioavailability: Maintaining sufficient inhibitor concentration at infection sites

Potential Solutions:

• Structure-Based Approach: Target unique regulatory sites rather than the highly conserved catalytic site

• Combination Therapy: Pair GLN1 inhibitors with compounds targeting alternate nitrogen assimilation pathways

• Prodrug Strategy: Design fungal-specific activation mechanisms for enhanced selectivity

• Allosteric Modulators: Develop compounds that disrupt the quaternary structure specific to C. glabrata GLN1

Research into other C. glabrata virulence factors and metabolic pathways, such as those involving CgDtr1 , suggests that integrated approaches targeting multiple aspects of nitrogen metabolism may prove most effective in overcoming these challenges.