Recombinant Candida glabrata ATP-dependent RNA helicase eIF4A (TIF1)

How does C. glabrata eIF4A (TIF1) differ structurally from its homologs in other Candida species?

C. glabrata eIF4A (TIF1) shares significant sequence homology with eIF4A proteins in other Candida species, but contains critical amino acid variations that affect its function and drug susceptibility. A notable difference exists at a key residue position corresponding to position 152 in C. auris and 153 in C. albicans. C. glabrata possesses a phenylalanine at this position (similar to C. auris), whereas C. albicans contains a leucine. This single amino acid difference significantly impacts binding pocket size and interaction with compounds like rocaglates. These structural differences may reflect evolutionary adaptations to different host environments or stress conditions encountered by various Candida species. Interestingly, despite the similarity in amino acid sequence at position 152 between C. glabrata and C. auris, C. glabrata displays intrinsic resistance to rocaglates, suggesting that additional factors such as cellular permeability or efflux mechanisms may contribute to this resistance .

What experimental systems are most suitable for expressing recombinant C. glabrata eIF4A (TIF1)?

For recombinant expression of C. glabrata eIF4A (TIF1), several experimental systems can be employed with varying advantages:

What are the most effective techniques for assessing eIF4A (TIF1) helicase activity in C. glabrata?

Several methodologies can be employed to assess the helicase activity of C. glabrata eIF4A (TIF1), each with specific advantages:

• Fluorescence-based translation assay: This technique incorporates an alkynylated methionine analog into newly translated proteins, which can then be detected by Click reaction with a green fluorescent azide. This approach allows visualization of active translation and can directly measure translation inhibition when eIF4A activity is compromised. This method has successfully demonstrated translation inhibition in C. auris by rocaglates and could be adapted for C. glabrata .

• RNA unwinding assays: These assays utilize dual-labeled RNA substrates with a fluorophore on one end and a quencher on the other. When the RNA is double-stranded, fluorescence is quenched, but becomes detectable when unwound by active helicase. This real-time measurement provides quantitative data on unwinding kinetics.

• ATP hydrolysis assays: Since eIF4A is ATP-dependent, measuring ATP hydrolysis using colorimetric assays (e.g., malachite green) can serve as an indirect measure of helicase activity. The rate of ATP hydrolysis correlates with helicase function when RNA substrates are present.

• RNA-binding assays: Techniques such as electrophoretic mobility shift assays (EMSA) or fluorescence anisotropy can assess RNA-binding capability, which is prerequisite for helicase function.

For comprehensive analysis, combining multiple techniques provides the most robust assessment of eIF4A activity in different experimental conditions .

How can researchers generate targeted mutations in C. glabrata eIF4A (TIF1) for structure-function analysis?

For structure-function analysis of C. glabrata eIF4A (TIF1), researchers can employ several gene editing approaches:

• CRISPR-Cas9 system: This has become the preferred method for introducing precise mutations into the C. glabrata genome. The technique requires:

  • Design of a guide RNA targeting the specific region of TIF1

  • Creation of a repair template containing the desired mutation

  • Transformation of cells with both components using electroporation

  • Selection of transformants followed by sequencing verification

• Traditional homologous recombination: For C. glabrata, this approach uses:

  • PCR-amplified cassettes containing selectable markers

  • 50-100 bp homology arms flanking the target site

  • Transformation via lithium acetate method or electroporation

• Plasmid-based expression: For testing multiple variants:

  • Creating expression vectors with copper-inducible MTI promoters

  • Replacing the native TIF1 with mutated versions

When targeting critical residues in eIF4A, researchers should focus on:

• The F152 position (equivalent to L153 in C. albicans), which affects rocaglate sensitivity

• ATP-binding motifs that are critical for helicase function

• RNA-binding domains that determine substrate specificity

These mutations can be used to investigate how specific residues contribute to helicase activity, stress response, drug resistance, and virulence .

What purification strategies yield the highest activity of recombinant C. glabrata eIF4A (TIF1)?

Optimizing purification protocols for recombinant C. glabrata eIF4A (TIF1) is crucial for obtaining functionally active protein. The recommended multi-step purification strategy includes:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is highly effective for His-tagged eIF4A. Critical buffer components include:

  • 20 mM HEPES (pH 7.5)

  • 150-300 mM NaCl (higher concentrations reduce non-specific binding)

  • 5% glycerol (maintains protein stability)

  • 1 mM DTT (prevents oxidation of cysteine residues)

  • 0.1 mM ATP (stabilizes the active conformation)

• Intermediate purification: Ion exchange chromatography using a Q Sepharose column effectively separates eIF4A from contaminants with different charge properties.

• Polishing step: Size exclusion chromatography (Superdex 200) resolves monomeric eIF4A from aggregates and remaining impurities.

Key considerations for maintaining activity:

• Keep temperature at 4°C throughout purification

• Include an ATP regeneration system (phosphoenolpyruvate and pyruvate kinase) for activity assays

• Avoid multiple freeze-thaw cycles by flash-freezing aliquots in liquid nitrogen

• Monitor activity using RNA-dependent ATPase assays after each purification step

This approach typically yields >95% pure protein with specific activity of approximately 120-150 pmol ATP hydrolyzed/min/pmol enzyme when tested with poly(U) RNA .

How does C. glabrata eIF4A (TIF1) activity change under different stress conditions?

C. glabrata eIF4A (TIF1) activity undergoes significant regulation under various stress conditions, representing a critical response mechanism:

Under oxidative stress, which C. glabrata encounters within phagocytes, eIF4A activity is typically downregulated to conserve energy while the cell mounts stress responses. This regulation may involve both post-translational modifications and changes in protein-protein interactions within the translation initiation complex. The stress-induced modulation of eIF4A activity represents a key adaptation mechanism that allows C. glabrata to survive hostile host environments and contributes to its pathogenicity .

What role does eIF4A (TIF1) play in stress granule formation and RNA condensation in C. glabrata?

eIF4A (TIF1) plays a critical regulatory role in stress granule (SG) dynamics in C. glabrata, functioning as an "RNA decondensase" to modulate RNA-RNA interactions. During cellular stress, mRNAs and proteins can condense through liquid-liquid phase separation to form membrane-less compartments called stress granules. eIF4A actively regulates this process through several mechanisms:

• Unwinding of structured RNA: eIF4A's helicase activity disrupts RNA secondary structures that promote intermolecular RNA-RNA interactions, thus limiting spontaneous condensation.

• Competition with RNA-binding proteins: By binding to mRNAs, eIF4A prevents interaction with RNA-binding proteins that promote SG assembly.

• ATP-dependent regulation: The cycle of ATP binding, hydrolysis, and release by eIF4A creates a dynamic interaction with RNA that prevents stable condensate formation.

This regulatory role is particularly important because excessive or persistent stress granule formation can disrupt normal cellular functions and translation regulation. In C. glabrata, the balance between stress granule formation and dissolution is crucial for adaptation to changing environmental conditions and host defense mechanisms. Interestingly, when translation is inhibited (such as by targeting eIF4A), the resulting stress response and potential stress granule formation can vary significantly between related Candida species, suggesting evolutionary divergence in stress response pathways .

How does C. glabrata eIF4A (TIF1) contribute to virulence and pathogenicity?

C. glabrata eIF4A (TIF1) contributes to virulence and pathogenicity through several mechanisms that enhance survival in host environments:

• Translational regulation of virulence factors: As a core component of the translation machinery, eIF4A regulates the synthesis of proteins involved in adhesion, biofilm formation, and stress resistance. The selective translation of structured mRNAs encoding virulence factors is particularly dependent on eIF4A helicase activity.

• Stress adaptation within host: eIF4A helps C. glabrata adapt to various stresses encountered within the host, including oxidative stress within phagocytes. This adaptation is critical for fungal survival and proliferation in hostile host environments.

• Metabolic flexibility: Through its role in translation regulation, eIF4A facilitates metabolic adaptations necessary for growth in diverse host niches with different nutrient availability.

• Immune evasion: Proper translation of factors involved in cell wall remodeling and immune evasion requires functional eIF4A activity.

The importance of eIF4A in pathogenicity is highlighted by the finding that translation inhibitors targeting this factor can significantly reduce virulence in infection models. Furthermore, the single amino acid difference at the critical position (phenylalanine in C. glabrata, similar to C. auris) may contribute to its characteristic drug resistance profile and virulence patterns, although additional factors like permeability and efflux mechanisms may also play important roles in determining drug susceptibility .

How do eIF4A (TIF1) homologs differ across pathogenic Candida species, and what are the functional implications?

eIF4A (TIF1) homologs across pathogenic Candida species show critical structural and functional variations that impact their roles in translation and drug susceptibility:

These differences have significant implications for:

• Drug development: Species-specific targeting is possible based on these structural differences, potentially allowing selective inhibition of particular pathogens.

• Evolution: The conservation pattern suggests divergent evolutionary pressures across Candida species, possibly related to their different ecological niches and host interactions.

• Translation regulation: These differences may reflect adaptations in translation machinery that contribute to species-specific stress responses and virulence mechanisms.

• Cell death pathways: Interestingly, even when translation is inhibited in different species (e.g., by engineering the sensitizing mutation), the downstream cellular responses differ significantly, suggesting divergence in programmed cell death pathways between related pathogenic fungi .

What methodologies are most effective for comparative analysis of eIF4A (TIF1) activity across Candida species?

For effective comparative analysis of eIF4A (TIF1) activity across different Candida species, researchers should implement a multi-faceted approach:

• Fluorescence-based translation assays: The incorporation of alkynylated methionine followed by Click chemistry with fluorescent azides provides a direct visualization of translation activity across species. This technique has successfully demonstrated the differential effects of translation inhibitors between C. auris and C. albicans .

• Sequence alignment and structural modeling:

  • Clustal Omega alignment of eIF4A sequences from multiple species

  • Homology modeling based on resolved eIF4A structures

  • Analysis of key conserved domains and species-specific variations

• Heterologous expression systems:

  • Creating chimeric proteins with domains swapped between species

  • Expression of one species' eIF4A in another species background

  • Site-directed mutagenesis to introduce specific residue changes (e.g., F152L or L153F)

• Biochemical characterization:

  • Parallel purification using identical protocols

  • Side-by-side comparison of ATP hydrolysis rates

  • RNA unwinding assays with standardized substrates

  • Thermal stability assays to assess structural robustness

• Drug susceptibility profiling:

  • Dose-response matrices (checkerboards) with translation inhibitors

  • Cross-resistance analysis

  • Selection and characterization of resistant mutants

The most valuable insights come from combining these approaches with in vivo virulence models to connect biochemical differences with pathogenic potential .

How can researchers engineer C. glabrata eIF4A (TIF1) to study species-specific differences in drug susceptibility?

To engineer C. glabrata eIF4A (TIF1) for studying species-specific differences in drug susceptibility, researchers can employ several targeted approaches:

• Site-directed mutagenesis: Based on sequence alignments, researchers can modify critical residues in C. glabrata eIF4A that differ across Candida species. The key targets include:

  • The phenylalanine residue (equivalent to F152 in C. auris) that affects rocaglate sensitivity

  • ATP-binding pocket residues that influence interaction with other translation inhibitors

  • RNA-binding interface residues that affect substrate recognition

• Domain swapping: Creating chimeric proteins by exchanging entire domains between C. glabrata and other Candida species:

  • N-terminal domain (typically involved in protein-protein interactions)

  • C-terminal domain (often crucial for RNA binding)

  • Linker regions that affect interdomain communication

• Plasmid-based expression systems:

  • Using copper-inducible MTI promoters for controlled expression

  • Complementing TIF1 deletion strains with variant constructs

  • Co-expressing with fluorescent tags for localization studies

• CRISPR-Cas9 genome editing:

  • Creating precise genomic modifications at the endogenous locus

  • Introducing mutations while maintaining native regulation

For experimental validation, researchers should employ:

• Translation assays to assess functional impacts

• Drug susceptibility testing using checkerboard assays

• Protein-protein interaction studies to identify altered binding partners

• Stress response assays to evaluate phenotypic consequences

This engineering approach has proven successful in studies of C. albicans, where introduction of the L153F mutation sensitized cells to rocaglates, demonstrating that this single amino acid substitution is sufficient to alter drug susceptibility .

How can researchers exploit C. glabrata eIF4A (TIF1) as a target for developing species-specific antifungal agents?

C. glabrata eIF4A (TIF1) presents a promising target for species-specific antifungal development through several strategic approaches:

• Structure-based drug design: Using crystal structures or homology models of C. glabrata eIF4A to identify unique binding pockets not present in human eIF4A:

  • Focus on regions surrounding the phenylalanine residue (equivalent to F152 in C. auris)

  • Target the interface between eIF4A and other translation initiation factors

  • Design compounds that exploit differences in ATP-binding pocket architecture

• Modification of existing inhibitors: Rocaglates provide an excellent starting point for developing derivatives with enhanced specificity:

  • Synthesize analogs that maximize interaction with the phenylalanine residue

  • Incorporate moieties that circumvent efflux mechanisms in C. glabrata

  • Develop prodrug approaches to improve cellular penetration

• Combination therapy approaches:

  • Pair eIF4A inhibitors with efflux pump inhibitors to overcome intrinsic resistance

  • Develop dual-targeting compounds that simultaneously inhibit eIF4A and stress response pathways

  • Test synergistic combinations with existing antifungals

• Screening and validation pipeline:

  • High-throughput screening against recombinant C. glabrata eIF4A

  • Secondary screening in cellular translation assays

  • Tertiary screening for species selectivity against human eIF4A

  • In vivo validation in infection models

The ideal compound would exploit the unique structural features of C. glabrata eIF4A while overcoming the permeability or efflux mechanisms that confer resistance to natural products like rocaglates .

What are the most effective approaches for studying the interaction between eIF4A (TIF1) and other translation initiation factors in C. glabrata?

Investigating interactions between C. glabrata eIF4A (TIF1) and other translation initiation factors requires sophisticated methodological approaches:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Tagged eIF4A expression followed by pulldown experiments

  • Quantitative proteomics to identify interaction partners

  • SILAC or TMT labeling to compare interactomes under different conditions

  • Crosslinking approaches to capture transient interactions

• Proximity-based labeling techniques:

  • BioID or TurboID fusion with eIF4A to identify proximal proteins

  • APEX2 tagging for rapid biotin labeling of neighbors

  • These methods capture weak or transient interactions missed by traditional co-IP

• Fluorescence-based interaction assays:

  • Förster Resonance Energy Transfer (FRET) between tagged translation factors

  • Fluorescence correlation spectroscopy (FCS) to measure binding kinetics

  • Split fluorescent protein complementation to visualize interactions in vivo

• Structural biology approaches:

  • Cryo-EM of translation initiation complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

  • Integrative modeling combining low and high-resolution structural data

• Functional validation techniques:

  • Mutational analysis of predicted interaction interfaces

  • Translation assays with reconstituted systems

  • Checkerboard inhibitor assays targeting different complex components

These approaches should be applied under various stress conditions to understand how the translation initiation complex remodels during adaptation to host environments, which may reveal potential vulnerabilities for therapeutic targeting .

How does post-translational modification of eIF4A (TIF1) regulate its function in C. glabrata under different physiological conditions?

Post-translational modifications (PTMs) of C. glabrata eIF4A (TIF1) constitute a complex regulatory network that modulates its function in response to changing physiological conditions:

• Phosphorylation sites and their effects:

  • Serine/threonine phosphorylation often occurs in response to stress conditions

  • Likely kinases include PKA, CK2, and stress-activated protein kinases

  • Phosphorylation can alter RNA binding affinity, ATP hydrolysis rates, and protein-protein interactions

  • Key regulatory sites are often found in the linker region between domains

• Methodology for studying PTMs:

  • Phosphoproteomic analysis using TiO₂ enrichment and mass spectrometry

  • Site-specific phospho-antibodies for Western blotting

  • Phosphomimetic and phospho-dead mutants (S→E/D or S→A) for functional studies

  • In vitro kinase assays to identify responsible enzymes

• Other potential modifications:

  • Acetylation may regulate nuclear-cytoplasmic shuttling

  • Ubiquitination likely controls protein stability and turnover

  • SUMOylation could affect stress granule association

  • Redox-sensitive cysteines may act as oxidative stress sensors

• Condition-specific regulation patterns:

Understanding these regulatory mechanisms could reveal potential points for therapeutic intervention and explain species-specific differences in stress responses and drug susceptibility .

What are the most promising future research directions for understanding C. glabrata eIF4A (TIF1) biology?

The field of C. glabrata eIF4A (TIF1) research offers several promising directions for future investigation:

• Structural biology:

  • High-resolution cryo-EM structures of the complete C. glabrata translation initiation complex

  • Comparative structural analysis with human eIF4A to identify targetable differences

  • Dynamic structural studies to understand conformational changes during the translation cycle

• Systems biology approaches:

  • Global translatomics to identify mRNAs most dependent on eIF4A activity

  • Integration of transcriptomics, proteomics, and metabolomics data to build comprehensive models

  • Network analysis to position eIF4A within the broader stress response pathways

• Host-pathogen interaction:

  • Investigation of eIF4A's role in adaptation to specific host microenvironments

  • Understanding how translation regulation contributes to immune evasion

  • Characterization of host factors that interact with or regulate fungal translation

• Drug discovery:

  • Development of C. glabrata-specific eIF4A inhibitors that overcome efflux mechanisms

  • Investigation of combination therapies targeting translation and stress response

  • Exploration of RNA-binding small molecules that specifically disrupt eIF4A-mRNA interactions

• Evolutionary biology:

  • Comprehensive analysis of eIF4A sequence and functional divergence across pathogenic fungi

  • Investigation of how translation regulation contributes to speciation and niche adaptation

  • Understanding the selective pressures that drive amino acid substitutions at key positions

These research directions will not only advance our understanding of C. glabrata biology but also potentially lead to novel therapeutic strategies against this increasingly important pathogen .

How can researchers effectively combine biochemical and genomic approaches to study C. glabrata eIF4A (TIF1)?

Effective integration of biochemical and genomic approaches provides a powerful framework for comprehensive understanding of C. glabrata eIF4A (TIF1):

• Parallel biochemical and genomic characterization:

  • Purify recombinant wild-type and variant eIF4A proteins for in vitro analysis

  • Generate corresponding genomic modifications in C. glabrata using CRISPR-Cas9

  • Compare biochemical properties with phenotypic outcomes

• Functional genomics strategies:

  • RNA-seq analysis of eIF4A mutants to identify differentially translated mRNAs

  • Ribosome profiling to map translation efficiency changes genome-wide

  • CRAC (crosslinking and analysis of cDNAs) to identify direct RNA targets

  • ChIP-seq of translation factors to map their genomic associations

• Integrative methodological workflow:

• Advanced technologies:

  • Single-molecule techniques to observe eIF4A dynamics on individual mRNAs

  • Cryo-electron tomography of translation complexes in situ

  • Synthetic genetic array analysis to map genetic interactions

  • Proteome-wide thermal stability assays to monitor drug engagement

This integrated approach allows researchers to connect molecular mechanisms to cellular phenotypes and organismal fitness, providing a more complete understanding of eIF4A biology in C. glabrata .

What are the challenges and potential solutions in developing selective inhibitors targeting C. glabrata eIF4A (TIF1)?

Developing selective inhibitors for C. glabrata eIF4A (TIF1) presents several challenges that require innovative solutions:

• Selectivity challenges:

  • High conservation between fungal and human eIF4A proteins

  • Potential off-target effects on other helicases

  • Need to distinguish between closely related Candida species

  Solutions:

  • Focus on unique pockets created by single amino acid differences

  • Design allosteric inhibitors targeting less conserved regulatory interfaces

  • Develop compounds that require bioactivation by fungal-specific enzymes

• Delivery and resistance challenges:

  • C. glabrata's robust drug efflux mechanisms

  • Limited penetration through the fungal cell wall

  • Potential for rapid resistance development

  Solutions:

  • Co-administration with efflux pump inhibitors

  • Development of pro-drug approaches with enhanced cell penetration

  • Combination therapies targeting multiple pathways to prevent resistance

• Validation challenges:

  • Limited translation from in vitro to in vivo efficacy

  • Difficulties in establishing relevant infection models

  • Complex pharmacokinetics in host environments

  Solutions:

  • Development of ex vivo human tissue models

  • Implementation of PKPD modeling early in development

  • Utilization of fluorescent-tagged compounds to track tissue distribution

• Technical challenges in drug discovery:

  • Limited availability of crystal structures for C. glabrata eIF4A

  • Difficulties in high-throughput screening for translation inhibitors

  • Complex assay development for species-specific activity

  Solutions:

  • Homology modeling based on related structures

  • Development of yeast-based screening systems with fluorescent reporters

  • Machine learning approaches to predict species-specific binding