Recombinant Candida glabrata Acetyl-CoA hydrolase (ACH1), partial

What is the role of Acetyl-CoA hydrolase (ACH1) in Candida glabrata metabolism?

Acetyl-CoA hydrolase (ACH1) in C. glabrata catalyzes the hydrolysis of acetyl-CoA to acetate and coenzyme A, playing a crucial role in alternative carbon metabolism. This enzyme is particularly important when C. glabrata must utilize non-glucose carbon sources such as acetate, ethanol, or fatty acids. In such conditions, the glyoxylate cycle becomes essential, and ACH1 helps regulate acetyl-CoA pools that connect this cycle with other metabolic pathways . The enzyme facilitates carbon flux between different cellular compartments, which is vital for metabolic adaptation during growth in glucose-limited environments such as those encountered during host infection.

How does ACH1 contribute to alternative carbon source utilization in C. glabrata?

ACH1 plays a significant role in alternative carbon metabolism by regulating intracellular acetyl-CoA levels. When C. glabrata utilizes carbon sources like ethanol or acetate, these are ultimately converted to acetyl-CoA via acetyl-CoA synthetase (Acs1) in the cytosol . ACH1 helps balance acetyl-CoA concentrations between different metabolic pathways, including:

• Directing acetyl-CoA toward the glyoxylate cycle for gluconeogenesis

• Regulating acetyl-CoA flux between cytosol and mitochondria

• Facilitating acetyl-CoA utilization when fatty acids are metabolized

The enzyme becomes particularly important during carbon source shifts, allowing C. glabrata to thrive in microenvironments with poor glucose availability, such as within macrophages or neutrophils where acetate may be more abundant .

How is ACH1 expression regulated in response to environmental conditions?

ACH1 expression in C. glabrata is regulated through complex transcriptional networks that respond to carbon source availability. While not directly mentioned in the search results, we can infer from related systems that ACH1 likely shows similar regulation patterns to other genes involved in alternative carbon metabolism. In environments where glucose is limited, ACH1 expression is upregulated alongside other enzymes of the glyoxylate cycle. This regulation may involve transcription factors similar to Pdr1, which has been shown to control numerous genes in C. glabrata in response to environmental stressors . The enzyme's expression is particularly important during host-pathogen interactions, where C. glabrata must adapt to changing carbon landscapes within different host niches.

What expression systems are most effective for producing recombinant C. glabrata ACH1?

For optimal expression of recombinant C. glabrata ACH1, several expression systems can be employed, each with specific advantages:

The methodological approach should include:

• Gene optimization based on codon usage of the expression host

• Addition of affinity tags (His6 or GST) to facilitate purification

• Temperature optimization (typically 18-25°C for E. coli) to enhance soluble protein yield

• Induction protocol optimization (IPTG concentration for E. coli or methanol for P. pastoris)

For a valid experimental design, include appropriate controls and systematically test expression variables as outlined in standard experimental design principles .

What purification strategies yield the highest purity and activity for recombinant ACH1?

A multi-step purification strategy is recommended for obtaining high-purity, active recombinant ACH1:

• Initial Capture: Affinity chromatography using Ni-NTA for His-tagged protein or glutathione resin for GST-tagged constructs

• Intermediate Purification: Ion exchange chromatography (typically Q-Sepharose at pH 8.0)

• Polishing: Size exclusion chromatography to remove aggregates and obtain homogeneous protein

Throughout purification, maintain these critical parameters:

• Buffer composition: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT

• Temperature: Maintain samples at 4°C throughout purification

• Protease inhibitors: Add complete protease inhibitor cocktail to prevent degradation

• Activity assays: Monitor enzyme activity after each purification step using a spectrophotometric assay measuring CoA release

This methodological approach ensures both high purity and preserved enzymatic activity, which is essential for subsequent functional studies.

How can I verify the structural integrity and functional activity of purified recombinant ACH1?

Multiple complementary techniques should be employed to verify both structural integrity and functional activity:

Structural Integrity Assessment:

• SDS-PAGE: Confirms molecular weight and purity

• Circular Dichroism (CD): Analyzes secondary structure elements

• Thermal Shift Assay: Determines protein stability and proper folding

• Dynamic Light Scattering (DLS): Assesses homogeneity and aggregation state

Functional Activity Verification:

• Spectrophotometric Assay: Measure CoA release using DTNB (Ellman's reagent)

• HPLC Analysis: Quantify acetate production

• Isothermal Titration Calorimetry (ITC): Determine binding parameters to substrates and inhibitors

The combination of these techniques provides comprehensive validation of your recombinant protein preparation before proceeding to more complex experiments.

How does ACH1 contribute to C. glabrata virulence and pathogenicity?

ACH1 contributes to C. glabrata virulence through its role in alternative carbon metabolism, which is essential for survival in diverse host microenvironments. While not directly mentioned in the search results, we can infer from the information about carbon metabolism that ACH1 likely contributes to pathogenicity through:

• Metabolic Flexibility: By participating in acetyl-CoA metabolism, ACH1 enables C. glabrata to utilize alternative carbon sources like acetate when glucose is limited, particularly within phagocytic cells .

• Survival in Immune Cells: The upregulation of acetate permease gene ADY2 in C. glabrata during engulfment by macrophages and neutrophils suggests that acetate metabolism, which involves ACH1, is crucial for survival within these immune cells .

• Adaptation to Host Niches: Similar to observations in C. albicans, the glyoxylate cycle and related enzymes like ACH1 likely enable C. glabrata to colonize different anatomical sites with varying nutrient availability.

This metabolic adaptation strategy is a significant virulence factor that allows C. glabrata to persist during infection despite nutrient limitations imposed by the host.

Is there evidence linking ACH1 function to antifungal drug resistance mechanisms?

While direct evidence linking ACH1 to antifungal resistance is not provided in the search results, there are several plausible connections:

• Metabolic Adaptation: ACH1's role in alternative carbon metabolism may indirectly contribute to stress resistance, including response to antifungal drugs. Metabolic flexibility often correlates with enhanced stress tolerance.

• Transcriptional Regulation: In C. glabrata, antifungal resistance is often mediated through transcription factors like Pdr1, which controls numerous genes . If ACH1 falls under similar regulatory networks, its expression might be altered in resistant strains.

• Energy Homeostasis: By regulating acetyl-CoA pools, ACH1 influences energy metabolism, which may affect the cell's ability to activate efflux pumps like Cdr1 that require ATP and are primary mediators of azole resistance .

To investigate these potential connections, experiments should compare ACH1 expression and activity between drug-susceptible and resistant isolates, and assess whether ACH1 deletion affects minimum inhibitory concentrations (MICs) of various antifungals.

How does ACH1 activity differ between commensal and invasive C. glabrata states?

The activity and expression of ACH1 likely differ between commensal and invasive states of C. glabrata, reflecting adaptation to different host environments:

In the commensal state, C. glabrata encounters varying levels of glucose and must maintain metabolic flexibility. During invasion, the pathogen faces increased nutrient competition and host-imposed stresses, requiring upregulation of alternative metabolic pathways involving ACH1. When phagocytosed, C. glabrata must rapidly adapt to the glucose-poor environment inside immune cells, where acetate metabolism becomes crucial for survival .

To experimentally verify these differences, researchers should compare ACH1 expression and activity in C. glabrata isolated from different host niches or growth conditions mimicking these environments.

How can site-directed mutagenesis be used to elucidate ACH1 catalytic mechanism?

Site-directed mutagenesis represents a powerful approach to investigate the catalytic mechanism of C. glabrata ACH1. The methodology involves:

• Identification of Key Residues: Based on sequence alignment with homologous enzymes and structural predictions, identify conserved residues likely involved in substrate binding and catalysis.

• Mutation Design Strategy:

  • Conservative mutations: Replace residues with similarly charged/sized amino acids to assess specific chemical properties

  • Non-conservative mutations: Replace with functionally distinct residues to confirm essentiality

  • Alanine scanning: Systematically replace residues with alanine to map the active site

• Experimental Protocol:

  • Use PCR-based mutagenesis with complementary primers containing the desired mutation

  • Verify mutations by DNA sequencing

  • Express and purify mutant proteins following the same protocol as wild-type

• Functional Characterization:

  • Determine kinetic parameters (kcat, Km) for each mutant

  • Assess pH-dependence profiles to identify changes in ionizable groups

  • Perform substrate specificity assays to detect altered recognition patterns

The experimental design should follow established principles for controlling variables, with wild-type enzyme serving as the control and multiple independent protein preparations to ensure reproducibility .

What structural and functional differences exist between C. glabrata ACH1 and homologs in related species?

Comparative analysis of ACH1 across Candida species reveals significant structural and functional adaptations:

C. glabrata ACH1 likely shares core catalytic mechanisms with its homologs, but with specific adaptations reflecting its unique ecological niche and metabolic requirements. While C. albicans relies exclusively on the carnitine shuttle for acetyl-CoA transportation due to the absence of peroxisomal citrate synthase (Cit2) , C. glabrata appears to possess both transportation systems similar to S. cerevisiae. This metabolic difference may influence the regulation and function of ACH1 in C. glabrata compared to other Candida species.

To experimentally characterize these differences, researchers should express and purify homologs from different species under identical conditions and compare their biochemical properties, substrate specificities, and responses to inhibitors.

How can computational approaches enhance our understanding of ACH1 substrate interactions?

Computational approaches offer valuable insights into ACH1 substrate interactions through multiple methodologies:

• Homology Modeling and Molecular Docking:

  • Generate a 3D structural model of C. glabrata ACH1 using closely related crystal structures as templates

  • Perform molecular docking simulations with acetyl-CoA and potential inhibitors

  • Calculate binding energies and identify key interaction residues

• Molecular Dynamics Simulations:

  • Simulate enzyme-substrate complex in explicit solvent over nanosecond timescales

  • Analyze conformational changes upon substrate binding

  • Identify water-mediated interactions in the active site

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Model the reaction mechanism at the electronic level

  • Calculate activation energies for different mechanistic hypotheses

  • Predict the effects of mutations on catalysis

• Machine Learning Applications:

  • Develop models to predict substrate specificity based on sequence features

  • Identify potential inhibitors through virtual screening of compound libraries

  • Classify ACH1 variants based on predicted functional properties

These computational approaches should be validated with experimental data, creating an iterative process where computational predictions guide experimental design, and experimental results refine computational models.

What are common challenges in obtaining active recombinant C. glabrata ACH1 and their solutions?

Researchers frequently encounter several challenges when working with recombinant C. glabrata ACH1:

When designing experiments to address these challenges, apply systematic troubleshooting by changing one variable at a time and including appropriate controls . Document all optimization steps meticulously to ensure reproducibility.

How can isotope labeling be used to track ACH1 activity in metabolic studies?

Isotope labeling represents a powerful approach for tracking ACH1 activity in metabolic studies:

• Experimental Design Methodology:

  • Label acetate or acetyl-CoA with 13C or 14C at specific carbon positions

  • Introduce labeled substrates to cell cultures or enzyme reactions

  • Track the fate of labeled carbon atoms through various metabolic pathways

• Analytical Techniques:

  • 13C-NMR Spectroscopy: Detects position-specific incorporation of 13C labels

  • Mass Spectrometry: Measures mass shifts in metabolites containing incorporated labels

  • Scintillation Counting: Quantifies 14C incorporation into various metabolic fractions

• Experimental Applications:

  • Determine the contribution of ACH1 to acetate metabolism using knockout strains

  • Track carbon flux through glyoxylate cycle versus TCA cycle

  • Measure compartment-specific acetyl-CoA pools affected by ACH1 activity

This methodology allows researchers to quantitatively assess ACH1's contribution to C. glabrata metabolism under different growth conditions or during interaction with host cells, providing insights not obtainable through static measurements.

What considerations are important when designing inhibitor studies for C. glabrata ACH1?

When designing inhibitor studies for C. glabrata ACH1, several critical methodological considerations must be addressed:

• Inhibitor Selection Strategy:

  • Structure-based: Design compounds based on substrate analogs or transition state mimics

  • Screening-based: Test libraries of compounds with established antifungal activity

  • Natural product approach: Evaluate plant extracts or microbial metabolites

• In Vitro Inhibition Characterization:

  • Determine inhibition mechanism (competitive, non-competitive, uncompetitive)

  • Calculate Ki values under standardized conditions

  • Assess time-dependence and reversibility of inhibition

• Selectivity Assessment:

  • Test against human homologs to evaluate potential toxicity

  • Compare inhibition against ACH1 from non-pathogenic yeast species

  • Evaluate effects on other acetyl-CoA utilizing enzymes

• Cellular Studies Design:

  • Measure growth inhibition in media with different carbon sources

  • Assess effects on acetate utilization in wild-type versus ACH1-overexpressing strains

  • Evaluate synergy with established antifungal drugs

• Controls and Validation:

  • Include enzyme-free and substrate-free controls

  • Verify inhibitor purity and stability under assay conditions

  • Use multiple, complementary assay methods to confirm results

These methodological considerations ensure that inhibitor studies provide reliable, reproducible data that can guide the development of potential therapeutic strategies targeting C. glabrata metabolism.

What are the most promising research directions for understanding C. glabrata ACH1 in pathogenesis?

Future research on C. glabrata ACH1 should focus on several promising directions:

• Host-Pathogen Interaction Studies: Investigate ACH1 expression and activity during interaction with different host cell types, particularly during phagocytosis by macrophages and neutrophils where acetate metabolism appears important .

• Genetic Regulation Networks: Determine whether ACH1 is regulated by transcription factors like Pdr1 that control virulence and drug resistance genes in C. glabrata .

• In Vivo Significance: Develop animal models to assess the contribution of ACH1 to colonization, persistence, and virulence in different anatomical niches.

• Metabolic Adaptation: Explore how ACH1 contributes to metabolic flexibility during shifts between commensal and pathogenic lifestyles, particularly in relation to carbon source utilization.

• Inhibitor Development: Design and test specific inhibitors of ACH1 as potential antifungal leads, especially compounds that might sensitize C. glabrata to existing antifungals.

These research directions have significant potential to advance our understanding of C. glabrata pathogenesis and identify novel therapeutic targets for this emerging fungal pathogen.

How might ACH1 research contribute to novel antifungal development strategies?

Research on C. glabrata ACH1 could contribute to novel antifungal strategies through several mechanisms:

• Metabolic Vulnerability Targeting: By understanding ACH1's role in alternative carbon metabolism, researchers could develop compounds that inhibit growth under the nutrient-limited conditions found during infection.

• Biofilm Disruption: If ACH1 contributes to biofilm formation through its metabolic functions, inhibitors could potentially disrupt this virulence trait.

• Combination Therapy Approaches: ACH1 inhibitors might sensitize C. glabrata to existing antifungals, particularly in strains with activated Pdr1 that show increased virulence and drug resistance .

• Host-Directed Therapy: Understanding how host conditions affect ACH1 expression could lead to strategies that modify the host environment to disadvantage the pathogen.

• Broad-Spectrum Potential: Due to the conservation of acetyl-CoA metabolism across fungal pathogens, ACH1-targeted strategies might be effective against multiple Candida species.