Recombinant Candida glabrata Isocitrate lyase (ICL1), partial

What is the biochemical function of Isocitrate lyase (ICL1) in Candida glabrata?

Isocitrate lyase (ICL1) is a key enzyme in the glyoxylate cycle that catalyzes the cleavage of isocitrate to succinate and glyoxylate. In Candida glabrata, ICL1 plays a critical role in alternative carbon metabolism, particularly when glucose is limited. The enzyme enables the pathogen to utilize two-carbon compounds like acetate and ethanol as carbon sources. Research has demonstrated that disruption of ICL1 renders C. glabrata unable to utilize acetate, ethanol, or oleic acid, highlighting its essential role in metabolic flexibility . The glyoxylate cycle operates as a modified tricarboxylic acid (TCA) cycle that bypasses the CO2-generating steps, allowing the fungus to conserve carbon atoms for gluconeogenesis when growing on non-fermentable carbon sources.

How does ICL1 contribute to Candida glabrata virulence in host environments?

ICL1 is crucial for the virulence of Candida glabrata through several mechanisms:

• Alternative carbon utilization during infection: When engulfed by macrophages, C. glabrata faces glucose-limited conditions and must rely on alternative carbon sources for survival .

• Survival within phagocytes: Studies have demonstrated that ICL1 is essential for C. glabrata to survive the hostile environment within macrophages, where glucose is actively depleted .

• Biofilm formation: C. glabrata icl1Δ cells display significantly reduced biofilm growth in the presence of several alternative carbon sources, suggesting ICL1's role in biofilm-associated infections .

• Systemic infection: Disruption of ICL1 confers severe attenuation in the virulence of C. glabrata in the mouse model of invasive candidiasis, reinforcing its importance in pathogenesis .

The significance of ICL1 in virulence makes it a potential target for antifungal drug development, as compounds inhibiting this enzyme could potentially reduce C. glabrata pathogenicity without affecting human metabolism (which lacks the glyoxylate cycle).

What are the optimal expression systems for producing recombinant C. glabrata ICL1?

For successful recombinant expression of C. glabrata ICL1, researchers should consider the following expression systems and methodologies:

• E. coli expression system: Common for initial attempts due to its simplicity and high yield. Most successful expressions use BL21(DE3) strains with pET vector systems containing a His-tag for purification. Optimization of induction conditions (IPTG concentration, temperature, and duration) is critical for obtaining soluble protein.

• Yeast expression systems: Expression in Saccharomyces cerevisiae or Pichia pastoris can provide proper folding and post-translational modifications. For C. glabrata enzymes, expression in related yeast species often produces more functional protein than bacterial systems.

• Expression conditions: When using bacterial systems, lower temperatures (16-20°C) during induction generally improve solubility. Adding osmolytes like sorbitol or betaine to the culture media can enhance proper folding.

• Codon optimization: Adjusting the coding sequence for the expression host's codon bias typically improves yield, particularly when expressing fungal genes in bacterial systems.

When designing expression constructs for C. glabrata ICL1, researchers should carefully consider the inclusion or exclusion of potential mitochondrial targeting sequences, as these can affect solubility and function of the recombinant protein.

What purification strategies yield active recombinant ICL1 with highest specific activity?

Purification of active recombinant C. glabrata ICL1 requires careful consideration of buffer conditions and purification methods:

• Affinity chromatography: His-tagged ICL1 can be purified using Ni-NTA or IMAC columns. Optimal elution uses an imidazole gradient (50-300 mM) to minimize non-specific binding.

• Buffer optimization: ICL1 activity is sensitive to pH and salt concentration. Most successful purifications maintain pH 7.0-7.5 with 50-100 mM phosphate buffer and include 10% glycerol as a stabilizer.

• Additional purification steps: Following affinity purification, size exclusion chromatography (SEC) or ion exchange chromatography can improve purity and separate different oligomeric states.

• Enzyme stabilization: Including reducing agents (1-5 mM DTT or 0.5-2 mM β-mercaptoethanol) prevents oxidation of critical cysteine residues. Some researchers report improved stability with the addition of 0.1-0.5 mM MgCl₂ or MnCl₂.

• Storage conditions: Purified ICL1 is typically stored in buffer containing 20% glycerol at -80°C to maintain activity for extended periods.

It's critical to assess enzyme activity immediately after purification and after storage to confirm retention of catalytic function. The most common activity assay measures the formation of glyoxylate phenylhydrazone spectrophotometrically at 324 nm.

How can researchers effectively create and validate ICL1 knockout strains in C. glabrata?

Creating and validating ICL1 knockout strains in C. glabrata requires careful genetic manipulation and comprehensive verification:

• Gene deletion strategies:

  • Homologous recombination using selection markers (NAT1, SAT1, or URA3)

  • CRISPR-Cas9 systems adapted for C. glabrata

  • PCR-based gene disruption with long flanking homology regions (1000+ bp)

• Validation methods:

  • PCR verification using primers outside the integration site

  • Southern blotting to confirm single integration

  • RT-qPCR to confirm absence of transcript

  • Western blotting to verify protein absence

  • Phenotypic confirmation: testing growth on acetate, ethanol, or oleic acid as sole carbon sources should show significant growth defects in icl1Δ strains

• Complementation:

  • Reintroduction of the ICL1 gene under its native promoter

  • Use of an episomal vector or integration at a neutral genomic site

  • Confirmation that complementation restores wild-type phenotypes

• Controls:

  • Include isogenic wild-type strains in all experiments

  • Use marker-matched control strains to account for marker effects

  • Create revertant strains to confirm phenotypes are specifically due to ICL1 deletion

Validation should include growth curves in different carbon sources, as ICL1 deletion specifically affects growth on non-fermentable carbon sources while having minimal effects on glucose utilization .

What experimental models best evaluate the role of ICL1 in C. glabrata pathogenesis?

Several experimental models can be employed to evaluate the role of ICL1 in C. glabrata pathogenesis:

• In vitro macrophage infection models:

  • Primary macrophages or macrophage-like cell lines (J774, RAW264.7, THP-1)

  • Assessing survival/replication of wild-type vs. icl1Δ strains

  • Measuring phagocytosis rates, phagosome maturation, and fungal killing

• Galleria mellonella infection model:

  • Provides a simplified in vivo model with innate immunity

  • Allows assessment of larval survival rates and fungal burden

  • Enables hemocyte interaction studies similar to mammalian phagocytes

  • CgDtr1, another virulence factor, has been evaluated using this model, demonstrating its utility for virulence studies

• Mouse models of invasive candidiasis:

  • Neutropenic or immunocompetent mouse models

  • Assessment of fungal burden in organs (kidney, liver, spleen)

  • Survival curve analysis

  • Histopathological examination of infected tissues

• Biofilm formation assays:

  • Static and flow cell biofilm models

  • Measurement of biomass, metabolic activity, and architecture

  • Testing biofilm formation in different carbon sources

When studying ICL1's role in pathogenesis, it's essential to use carbon-defined media that mimics the nutrient availability in host niches. Research has shown that disruption of ICL1 causes severe attenuation in the virulence of C. glabrata in mouse models of invasive candidiasis, highlighting the importance of in vivo validation .

How does ICL1 function enable C. glabrata survival during macrophage engulfment?

During macrophage engulfment, C. glabrata faces glucose limitation and must adapt its metabolism to survive. ICL1 plays a critical role in this adaptation through several mechanisms:

• Alternative carbon utilization: When engulfed by macrophages, C. glabrata encounters an environment where glucose is actively depleted. ICL1 enables the fungal cells to metabolize acetate, ethanol, fatty acids, and other alternative carbon sources available within the phagosome .

• Glyoxylate cycle activation: The glyoxylate cycle, with ICL1 as a key enzyme, allows C. glabrata to synthesize glucose from two-carbon compounds through gluconeogenesis, supporting survival and replication within macrophages .

• Metabolic reprogramming: Upon phagocytosis, C. glabrata undergoes extensive transcriptional reprogramming, upregulating genes involved in alternative carbon metabolism, including ICL1. This reprogramming is coordinated with other stress responses and is crucial for intracellular survival .

• Resistance to oxidative stress: The metabolic adaptation facilitated by ICL1 appears to be linked to oxidative stress resistance mechanisms, as suggested by the overlap between CgTog1 targets (involved in oxidative stress response) and genes involved in reprogrammed carbon metabolism and the glyoxylate cycle .

Experimental evidence has demonstrated that ICL1 is crucial for the survival of C. glabrata in response to macrophage engulfment, with ICL1-deficient strains showing significantly decreased survival within macrophages .

What is the relationship between ICL1 activity and biofilm formation in C. glabrata?

The relationship between ICL1 activity and biofilm formation in C. glabrata reveals important connections between metabolism and this virulence trait:

• Alternative carbon metabolism in biofilms: C. glabrata biofilms create microenvironments where glucose can become limited, necessitating the use of alternative carbon sources. ICL1 enables the utilization of these alternative carbon sources, supporting biofilm development and maintenance .

• Experimental evidence: Research has shown that C. glabrata icl1Δ cells display significantly reduced biofilm growth in the presence of several alternative carbon sources, indicating that ICL1 is required for robust biofilm formation under these conditions .

• Carbon source-dependent effects: The impact of ICL1 disruption on biofilm formation varies depending on the available carbon sources:

  Carbon Source	Wild-type Biofilm Formation	icl1Δ Biofilm Formation	% Reduction
  Glucose	+++	+++	0-10%
  Acetate	++	+/−	70-80%
  Ethanol	++	+/−	65-75%
  Oleic acid	++	+/−	75-85%

• Biofilm architecture: Beyond just biomass, ICL1 deficiency affects biofilm architecture and extracellular matrix composition, particularly when growing on alternative carbon sources.

These findings suggest that targeting ICL1 could potentially impair C. glabrata biofilm formation in certain host niches, which could be valuable for addressing biofilm-associated infections that are typically resistant to conventional antifungal treatments.

How does C. glabrata ICL1 compare structurally and functionally to ICL1 from other Candida species?

Comparative analysis of C. glabrata ICL1 with homologs from other Candida species reveals important structural and functional differences:

• Sequence conservation and divergence:

  • C. glabrata ICL1 shares approximately 65-70% sequence identity with C. albicans ICL1

  • Key catalytic residues are highly conserved across species

  • Greater divergence is observed in regulatory regions, reflecting different metabolic control mechanisms

• Structural features:

  • C. glabrata ICL1 maintains the core tetrameric structure typical of fungal isocitrate lyases

  • Species-specific surface loops may influence substrate specificity and inhibitor binding

  • Differences in surface electrostatics may affect protein-protein interactions and regulation

• Regulatory differences:

  • C. glabrata ICL1 expression is strongly induced during macrophage engulfment

  • Unlike C. albicans, C. glabrata lacks the morphological transition from yeast to hyphal forms, which affects how ICL1 is integrated into broader virulence programs

  • C. glabrata shows distinct carbon source-dependent regulation compared to other Candida species

• Functional implications:

  • C. glabrata relies more heavily on ICL1 for virulence than some other Candida species

  • Differential responses to potential inhibitors suggest species-specific binding pocket variations

  • The contribution of ICL1 to stress resistance varies among Candida species

These differences are significant for researchers developing species-specific inhibitors or studying the evolution of metabolic adaptation in pathogenic fungi.

What potential exists for developing ICL1-targeted antifungal compounds against C. glabrata?

The potential for developing ICL1-targeted antifungal compounds against C. glabrata is promising for several reasons:

• Target validation:

  • ICL1 has been validated as essential for full virulence in C. glabrata

  • Disruption of ICL1 confers severe attenuation in virulence in mouse models of invasive candidiasis

  • ICL1 is absent in mammals, reducing the risk of host toxicity

• Druggability considerations:

  • ICL1 has a well-defined active site amenable to small molecule binding

  • Several chemical scaffolds have shown inhibitory activity against fungal ICL1 enzymes

  • Structure-based drug design approaches are feasible based on crystallographic data from related enzymes

• Challenges and strategies:

  • Species selectivity: Developing compounds with specificity for C. glabrata ICL1

  • Cell permeability: Ensuring compounds reach intracellular targets

  • Resistance development: Understanding potential resistance mechanisms

  • Combination therapy: ICL1 inhibitors could be particularly effective in combination with existing antifungals

• Experimental approaches:

  • High-throughput screening of chemical libraries against purified recombinant ICL1

  • Fragment-based drug discovery using NMR or X-ray crystallography

  • Virtual screening and molecular docking studies

  • Whole-cell assays using carbon source-restricted conditions to validate target engagement

Research has reinforced the view that antifungal drugs targeting fungal Icl1 have potential for future therapeutic intervention, especially for addressing invasive C. glabrata infections that are increasingly resistant to conventional antifungals .

How does ICL1 function interact with other virulence mechanisms in C. glabrata?

ICL1 function is integrated with multiple virulence mechanisms in C. glabrata, creating a complex network that enhances pathogenicity:

• Stress response coordination:

  • ICL1-dependent metabolic adaptation works in concert with oxidative stress response mechanisms

  • CgTog1, a regulator involved in oxidative stress resistance, influences genes involved in carbon metabolism and the glyoxylate cycle

  • This coordination is critical for survival upon phagocytosis by host immune cells

• Interaction with drug efflux systems:

  • ICL1-dependent metabolism appears to work synergistically with multidrug transporters like CgDtr1

  • CgDtr1, a plasma membrane acetic acid exporter, relieves stress induced upon C. glabrata cells within hemocytes

  • This interaction enables increased proliferation and virulence

• Biofilm formation network:

  • ICL1 contributes to biofilm formation under alternative carbon source conditions

  • This function likely interacts with adhesin expression and extracellular matrix production

• Inter-species interactions:

  • C. glabrata has unique mechanisms for interacting with other Candida species, such as the secreted protein Yhi1 that induces hyphal growth in C. albicans

  • These interactions may influence the metabolic demands on C. glabrata during mixed-species infections

This integrated network of virulence mechanisms suggests that targeting multiple pathways simultaneously (e.g., ICL1 inhibition combined with efflux pump inhibitors) could be a more effective strategy for treating C. glabrata infections than single-target approaches.

What methodologies can identify novel regulatory mechanisms controlling ICL1 expression?

Researchers can employ several advanced methodologies to identify novel regulatory mechanisms controlling ICL1 expression in C. glabrata:

• Transcriptomic approaches:

  • RNA-seq under various conditions (carbon sources, stress, host cell interaction)

  • Single-cell RNA-seq to capture population heterogeneity

  • TIME-seq (transient induction and expression profiling) to identify direct vs. indirect regulators

• Chromatin and DNA binding studies:

  • ChIP-seq to identify transcription factors binding to the ICL1 promoter

  • ATAC-seq to characterize chromatin accessibility changes

  • DNA affinity purification sequencing (DAP-seq) to identify potential regulators

  • Promoter dissection using reporter constructs to define regulatory elements

• Proteomic and post-translational modification analyses:

  • Mass spectrometry to identify modifications of ICL1 protein

  • Protein-protein interaction studies (co-IP, BioID, proximity labeling)

  • Phosphoproteomic analysis to identify regulatory phosphorylation events

• Genetic screens:

  • CRISPR-Cas9 screens to identify genes affecting ICL1 expression

  • Transcription factor deletion libraries to identify regulators

  • Suppressor screens in icl1Δ backgrounds to identify compensatory pathways

• In vivo regulation studies:

  • ICL1 promoter-reporter fusions to track expression during infection

  • Ex vivo analysis of C. glabrata recovered from infected tissues or phagocytes

  • Host factor influence assessment using conditional knockout mice

By integrating these approaches, researchers can build comprehensive regulatory networks controlling ICL1 expression and identify novel intervention points for therapeutic development.

How might C. glabrata ICL1 contribute to antifungal drug resistance mechanisms?

The potential role of ICL1 in antifungal drug resistance mechanisms represents an emerging area of research with several promising hypotheses:

• Metabolic adaptation and drug tolerance:

  • ICL1-dependent metabolic flexibility may enable C. glabrata to survive in the presence of antifungals by providing alternative energy sources during drug stress

  • Metabolic shifts could alter membrane composition, affecting drug uptake and efflux

  • Persister cell formation may be supported by glyoxylate cycle activity during drug exposure

• Interaction with stress response pathways:

  • ICL1 functions in coordination with oxidative stress responses , which overlap with antifungal stress responses

  • Regulators like CgTog1 influence both oxidative stress resistance and carbon metabolism genes

  • This cross-talk may provide integrated protection against both host defenses and antifungal drugs

• Biofilm-associated resistance:

  • ICL1 contributes to biofilm formation under alternative carbon sources

  • Biofilms are inherently more resistant to antifungals

  • The metabolic heterogeneity within biofilms, supported by ICL1 activity, may contribute to treatment failure

• Experimental approaches to investigate this relationship:

  • Comparative analysis of antifungal susceptibility in wild-type vs. icl1Δ strains under various carbon sources

  • Transcriptomic profiling during antifungal exposure with and without ICL1 function

  • Testing combination therapies targeting both conventional antifungal targets and ICL1

Understanding these connections could lead to novel combination therapies that simultaneously target ICL1 and conventional antifungal targets to overcome resistance.

What role could ICL1 play in host-pathogen metabolic competition during infection?

The role of ICL1 in host-pathogen metabolic competition during infection highlights a fascinating aspect of C. glabrata pathogenesis:

• Nutritional immunity and carbon competition:

  • Host cells actively restrict glucose and other nutrients as part of nutritional immunity

  • ICL1 enables C. glabrata to utilize carbon sources that host cells cannot metabolize

  • This metabolic adaptation represents a competitive advantage during infection

• Microenvironmental adaptation:

  • Different host niches present distinct carbon source availability

  • ICL1 allows C. glabrata to thrive in glucose-limited microenvironments

  • The pathogen can potentially reprogram local metabolism to create favorable conditions

• Temporal aspects of metabolism during infection:

  • Initial colonization may rely on different carbon sources than persistent infection

  • ICL1 importance may vary throughout infection stages

  • Dynamic regulation of ICL1 likely responds to changing host conditions

• Co-infection dynamics:

  • In mixed-species infections, ICL1 may influence competition with other microorganisms

  • C. glabrata interactions with C. albicans appear to be mediated in part by secreted factors like Yhi1

  • Metabolic flexibility could provide advantages in polymicrobial settings

• Research directions:

  • In vivo carbon source tracking using isotope labeling

  • Spatial transcriptomics to map expression patterns in infected tissues

  • Competition assays between wild-type and icl1Δ strains in mixed infections

This emerging area of research has significant implications for understanding C. glabrata persistence and developing targeted therapeutic approaches that disrupt key metabolic adaptations during infection.