Recombinant Candida glabrata Glucose-repressible alcohol dehydrogenase transcriptional effector (CCR4), partial

What is CCR4 and what are its primary functions in Candida glabrata?

CCR4 (Carbon Catabolite Repressor 4) in Candida glabrata functions as a glucose-repressible alcohol dehydrogenase transcriptional effector and as a component of the Ccr4-Not complex, which serves as the major cytoplasmic mRNA deadenylase. This complex plays critical roles in post-transcriptional gene regulation through control of mRNA poly(A) tail length . The protein contains a characteristic leucine-rich repeat (LRR) domain that is essential for its regulatory functions and protein-protein interactions, similar to its homolog in Saccharomyces cerevisiae . In C. glabrata, CCR4 contributes to the regulation of cell wall integrity (CWI) signaling pathway by modulating mRNA levels of specific Rho1 regulators .

What is the relationship between CCR4 and alcohol dehydrogenase regulation in C. glabrata?

Similar to its S. cerevisiae counterpart, C. glabrata CCR4 likely plays a role in regulating alcohol dehydrogenase genes, though the specific mechanisms may differ. In C. glabrata, alcohol dehydrogenase (ADH) activity influences biofilm formation capabilities, with ADH downregulation associated with enhanced biofilm formation . The transcriptional regulation of ADH genes by CCR4 represents a potential control point for this phenotype. The functional significance of this relationship is that modulation of alcohol dehydrogenase activity affects the production of ethanol, which inhibits biofilm formation at concentrations of ≥20% . Understanding CCR4's role in this regulatory network could provide insights into C. glabrata's pathogenicity mechanisms and potential therapeutic targets.

What are the recommended methods for generating recombinant CCR4 from C. glabrata for functional studies?

For generating functional recombinant CCR4 from C. glabrata, we recommend a multi-step approach:

• Gene cloning: Amplify the CCR4 gene using PCR with high-fidelity polymerase and primers designed with appropriate restriction sites for subsequent cloning.

• Expression system selection: For yeast proteins like CCR4, expression in Pichia pastoris or S. cerevisiae often provides proper post-translational modifications. For studies requiring higher protein yields, E. coli systems with optimized codons can be effective.

• Purification strategy: Include a His-tag or GST-tag for affinity purification, followed by size exclusion chromatography to ensure protein homogeneity.

• Functional validation: Test the deadenylase activity of purified CCR4 using synthetic RNA substrates with poly(A) tails and analyze the products by denaturing gel electrophoresis.

To ensure proper protein folding, expression conditions should be optimized for temperature, induction time, and media composition. For C. glabrata proteins, codon optimization may be necessary when using heterologous expression systems .

What genetic manipulation techniques are most effective for studying CCR4 function in C. glabrata?

For genetic manipulation of CCR4 in C. glabrata, consider these methodological approaches:

• Gene deletion strategy: The LIG4 deletion strain of C. glabrata significantly increases correct gene targeting efficiency, making it an excellent background for generating CCR4 knockout strains. The absence of Lig4 reduces non-homologous end joining (NHEJ) without the undesired side effects observed in ku80 mutants .

• Domain-specific mutations: Targeted mutagenesis of the leucine-rich repeat domain is particularly informative, as this region is crucial for CCR4's regulatory functions and protein-protein interactions .

• Complementation assays: After generating a ccr4Δ strain, complementation with wild-type and mutant versions can identify functional domains. This approach has successfully identified the roles of CCR4 in regulating LRG1 expression in the cell wall integrity pathway .

• Reporter systems: Developing fluorescent reporter constructs for downstream targets allows real-time monitoring of CCR4-dependent regulation.

These approaches can be combined with phenotypic assays to correlate molecular function with cellular physiology .

How can researchers effectively measure CCR4 deadenylase activity in vitro and in vivo?

To comprehensively assess CCR4 deadenylase activity, researchers should employ both in vitro and in vivo approaches:

In vitro measurement:

• Synthetic RNA substrate assay: Synthesize RNA oligonucleotides with defined poly(A) tails and fluorescent labels. Incubate with purified CCR4 protein and analyze deadenylation by denaturing PAGE.

• Kinetic analysis: Determine enzyme kinetics by varying substrate concentration and measuring initial reaction rates.

• Inhibitor studies: Test potential inhibitors to characterize catalytic mechanisms and regulatory features.

In vivo measurement:

• Reporter mRNA analysis: Express reporter genes with defined 3'UTRs and measure poly(A) tail length dynamics using the ePAT (extension Poly(A) Test) method.

• Transcriptome-wide analysis: Apply TAIL-seq or PAL-seq to quantify poly(A) tail lengths genome-wide in wild-type and ccr4Δ strains.

• Targeted mRNA analysis: For specific mRNAs like LRG1, use qRT-PCR with oligo(dT) fractionation to quantify poly(A) tail length changes .

For both approaches, controls with catalytically inactive CCR4 mutants should be included to confirm specificity .

How does CCR4 integrate into the cell wall integrity (CWI) signaling network in C. glabrata?

CCR4 plays a crucial role in the cell wall integrity (CWI) signaling pathway by regulating the expression of key modulators of Rho1 activity. Specifically, CCR4 affects mRNA levels of LRG1, which encodes a GTPase-activating protein (GAP) for Rho1 . This regulation involves:

• Post-transcriptional control: CCR4, as part of the Ccr4-Not complex, regulates LRG1 mRNA levels through its deadenylase activity. The ccr4Δ mutant shows increased LRG1 mRNA, suggesting CCR4 normally promotes LRG1 mRNA degradation .

• Functional consequences: Loss of CCR4 leads to increased Lrg1 levels, which consequently reduces Rho1 activity, resulting in cell wall defects and potential cell lysis .

• Regulatory partners: In this process, CCR4 functions together with Pop2 and Dhh1, as the pop2Δ and dhh1Δ mutants also show increased LRG1 mRNA levels .

• Specificity of regulation: Interestingly, while CCR4 regulates both ROM2 (encoding a Rho1 guanine nucleotide exchange factor) and LRG1 expression, Pop2 and Dhh1 specifically affect LRG1 but not ROM2 expression, highlighting the complexity and specificity of this regulatory network .

These findings establish CCR4 as a crucial post-transcriptional regulator in the CWI pathway, with implications for understanding cell wall homeostasis and potential antifungal targets .

What is the relationship between CCR4 and stress response mechanisms in C. glabrata?

CCR4 in C. glabrata likely contributes to stress response mechanisms through its role in post-transcriptional gene regulation, similar to its function in related yeast species. Several lines of evidence support this role:

• Transcriptional adaptation: In S. cerevisiae, Ccr4 contributes to replication stress tolerance through control of CRT1 mRNA poly(A) tail length, suggesting a similar function may exist in C. glabrata .

• Cell wall stress response: The ccr4Δ mutant in yeast shows cell lysis phenotypes, indicating compromised cell wall integrity under stress conditions. This phenotype becomes more severe in ccr4Δ khd1Δ double mutants, suggesting CCR4 works in parallel with other regulators to maintain cell wall integrity during stress .

• Potential role in azole resistance: While not directly shown for CCR4, the regulatory network involving transcription factors in C. glabrata affects azole resistance. Specifically, transcription factors like Zcf27 and Zcf4 regulate ERG genes involved in ergosterol biosynthesis, the target of azole antifungals .

• Metabolic adaptation: As a glucose-repressible regulator, CCR4 likely participates in metabolic adaptation to changing carbon sources, an important stress response mechanism for C. glabrata during host colonization .

Understanding these connections could provide insights into C. glabrata's remarkable ability to survive various host environments and develop drug resistance .

How does the Ccr4-Not complex composition in C. glabrata compare to other fungal species, and what are the functional implications?

The Ccr4-Not complex composition in C. glabrata shares core components with other fungi but may have unique functional adaptations reflective of its evolutionary history and pathogenic lifestyle:

• Core components: Like S. cerevisiae, the C. glabrata Ccr4-Not complex includes Ccr4, Pop2, and Not proteins, with associated factors like Dhh1 . These components work together in post-transcriptional regulation through mRNA deadenylation.

• Differential regulation: Analysis of C. glabrata mutants reveals that while ccr4Δ affects both ROM2 and LRG1 mRNA levels, pop2Δ and dhh1Δ specifically affect LRG1 but not ROM2 expression . This suggests C. glabrata may have evolved target-specific regulatory mechanisms that differ from other fungi.

• Pathogenesis implications: Given C. glabrata's clinical importance as an opportunistic pathogen, its Ccr4-Not complex may have evolved to regulate virulence-associated genes. This contrasts with S. cerevisiae, where the complex primarily regulates metabolic adaptation genes .

• Stress response specialization: C. glabrata inhabits diverse host niches and faces unique stresses including antifungal exposure. Its Ccr4-Not complex composition may reflect adaptations to these environmental challenges .

These differences likely contribute to C. glabrata's distinct biology, including its notable azole resistance and biofilm formation capabilities compared to other Candida species .

What are the current challenges in resolving the three-dimensional structure of C. glabrata CCR4, and how might structural insights inform function?

Determining the three-dimensional structure of C. glabrata CCR4 presents several technical challenges:

• Domain complexity: CCR4 contains multiple functional domains, including a leucine-rich repeat (LRR) region and a catalytic nuclease domain . Crystallizing the full-length protein while maintaining native conformation is challenging.

• Protein-protein interactions: As part of the larger Ccr4-Not complex, CCR4 engages in numerous protein-protein interactions that may stabilize its structure. Purifying CCR4 in isolation might not capture its physiologically relevant conformation.

• Post-translational modifications: Potential yeast-specific modifications may affect structure and function, necessitating expression in eukaryotic systems rather than bacterial systems.

• Conformational flexibility: The deadenylase activity likely requires conformational changes during catalysis, making it difficult to capture a single representative structure.

Structural insights would significantly advance our understanding by:

• Identifying catalytic residues for targeted mutagenesis studies

• Revealing substrate binding mechanisms and specificity determinants

• Providing a foundation for rational drug design targeting CCR4 function

• Explaining species-specific differences in CCR4 activity and regulation

Cryo-electron microscopy combined with computational approaches may overcome some of these challenges by capturing multiple conformational states .

How do transcriptional and post-transcriptional regulatory networks involving CCR4 contribute to azole resistance in C. glabrata?

The contribution of CCR4 to azole resistance in C. glabrata likely involves complex interplay between transcriptional and post-transcriptional regulatory mechanisms:

• ERG gene regulation: C. glabrata has evolved specific transcription factors like Zcf27 and Zcf4 that regulate ERG genes involved in ergosterol biosynthesis, the target of azole antifungals . CCR4, as a transcriptional effector and mRNA deadenylase, may influence the stability of transcripts encoding these transcription factors or their target genes.

• Post-transcriptional control of stress response: CCR4's deadenylase activity affects mRNA stability and translation efficiency. In S. cerevisiae, Ccr4 contributes to stress tolerance through control of mRNA poly(A) tail length . Similar mechanisms may help C. glabrata adapt to azole stress.

• Cell wall integrity modulation: CCR4 regulates the CWI pathway by controlling mRNA levels of Rho1 regulators . Since cell wall integrity and membrane composition are interconnected, this regulation may indirectly affect azole resistance, which targets membrane ergosterol.

• Regulatory network complexity: The finding that Zcf27 and Zcf4 have evolved distinct roles in C. glabrata's adaptation to host and environmental conditions suggests a complex regulatory network . CCR4 likely intersects with these pathways at multiple levels.

Understanding these regulatory networks could identify novel therapeutic targets or strategies to overcome azole resistance in this clinically significant pathogen .

What phenotypic changes are observed in CCR4 knockout strains of C. glabrata?

Based on research findings, CCR4 knockout strains of C. glabrata exhibit several significant phenotypic alterations:

These phenotypes can be partially explained by CCR4's role in regulating LRG1 mRNA levels, as evidenced by the observation that lrg1Δ mutation can suppress some growth defects caused by ccr4Δ mutation . This suggests that the increased LRG1 expression in ccr4Δ strains contributes significantly to the observed phenotypes through dysregulation of the Rho1 GTPase, a central regulator of cell wall integrity signaling .

How does recombinant CCR4 expression affect cellular metabolism and gene expression profiles?

Recombinant expression or overexpression of CCR4 in C. glabrata and related yeast species produces complex effects on cellular metabolism and gene expression:

• Altered carbon metabolism: As a glucose-repressible alcohol dehydrogenase transcriptional effector, modulating CCR4 levels affects the expression of genes involved in carbon utilization, particularly alcohol dehydrogenase genes . This influences the balance between fermentative and respiratory metabolism.

• Global transcriptome changes: Transcriptome analysis reveals that CCR4 affects numerous mRNAs beyond its direct targets, creating a cascade effect through:

  • Direct deadenylation of specific mRNAs

  • Altered expression of other transcriptional regulators

  • Feedback loops involving stress response pathways

• Cell wall-related genes: Expression profiling shows significant changes in genes encoding cell wall proteins and enzymes involved in cell wall biosynthesis and remodeling, consistent with CCR4's role in the cell wall integrity pathway .

• Stress response gene regulation: Genes involved in various stress responses show altered expression patterns, reflecting CCR4's contribution to stress adaptation mechanisms .

These findings highlight CCR4's role as a multifunctional regulator affecting diverse cellular processes through both its transcriptional effector activity and its function in the Ccr4-Not deadenylase complex .

What novel therapeutic approaches could target CCR4 function in pathogenic Candida species?

Several promising therapeutic strategies targeting CCR4 function could be developed:

• Small molecule inhibitors of deadenylase activity: Structure-based drug design targeting the catalytic domain of CCR4 could yield specific inhibitors that reduce pathogen viability without affecting host deadenylases. Such inhibitors would disrupt post-transcriptional regulation of numerous virulence-associated genes .

• Peptide-based disruption of protein-protein interactions: Peptides mimicking interaction surfaces between CCR4 and other components of the Ccr4-Not complex could selectively disrupt complex formation. This approach takes advantage of potential structural differences between fungal and human complexes .

• Combination therapy targeting CCR4 and cell wall integrity: Since CCR4 regulates the cell wall integrity pathway, combining CCR4 inhibitors with existing cell wall-targeting antifungals could produce synergistic effects, potentially overcoming resistance mechanisms .

• RNA-based therapeutics: Antisense oligonucleotides or RNA aptamers designed to bind CCR4 mRNA or protein could provide an alternative approach to inhibition with potentially higher specificity .

• Biofilm prevention: Given the relationship between alcohol dehydrogenase regulation and biofilm formation, targeting CCR4's regulatory function could impair biofilm development on medical devices, reducing a major source of persistent infections .

These approaches would require extensive validation in vitro and in vivo to ensure efficacy and specificity for fungal rather than human targets .

How might systems biology approaches advance our understanding of CCR4's role in fungal regulatory networks?

Systems biology approaches offer powerful frameworks for elucidating CCR4's complex roles in fungal regulatory networks:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data from wild-type, ccr4Δ, and CCR4-overexpressing strains would provide a comprehensive view of CCR4's impact across biological scales. This integration could reveal emergent properties not evident from single-omics approaches .

• Network inference and modeling: Computational inference of gene regulatory networks incorporating CCR4 would identify direct and indirect targets, feedback loops, and network motifs. Mathematical modeling of these networks could predict system responses to perturbations and identify critical nodes for therapeutic targeting .

• Comparative systems analysis: Applying systems approaches across multiple Candida species and S. cerevisiae would elucidate how CCR4 regulatory networks have evolved, potentially explaining species-specific pathogenicity traits and drug resistance mechanisms .

• Single-cell approaches: Single-cell transcriptomics could reveal population heterogeneity in CCR4-regulated processes, potentially explaining phenomena like heteroresistance to antifungals or variable biofilm formation capacity .

• Temporal dynamics analysis: Time-series experiments capturing the dynamics of CCR4-dependent regulation during stress responses or host infection would provide insights into the temporal organization of adaptive responses .

These approaches would advance our understanding beyond individual pathways to a systems-level comprehension of CCR4's role in fungal biology and pathogenesis .