Recombinant Candida glabrata Polyadenylation factor subunit 2 (PFS2)

What is Polyadenylation Factor Subunit 2 (PFS2) in Candida glabrata and what is its primary function?

Polyadenylation Factor Subunit 2 (PFS2) in Candida glabrata is an essential component of the Cleavage and Polyadenylation Factor (CPF) complex that executes the cotranscriptional 3' processing of RNA polymerase II transcripts. Based on orthologous proteins in related yeasts, PFS2 is crucial for pre-mRNA cleavage and polyadenylation, a process that precedes transcription termination . The PFS2 protein likely functions as part of a multiprotein complex that recognizes specific signal sequences in the 3' untranslated region of pre-mRNAs and coordinates the cleavage and subsequent addition of the poly(A) tail. In fission yeast, Pfs2 has been shown to be fundamentally important for chromosome segregation and cell cycle progression, suggesting that the C. glabrata ortholog may have similar critical roles in cellular processes beyond RNA processing .

How does PFS2 contribute to C. glabrata pathogenicity and stress responses?

PFS2 likely contributes to C. glabrata pathogenicity through its essential role in RNA processing, which affects the expression of numerous genes involved in virulence and stress responses. Given that C. glabrata is known for its ability to withstand various environmental stresses (including low pH, oxidative stress, and antifungal exposure), proper mRNA processing is crucial for mounting appropriate stress responses .

By analogy to other systems, disruption of PFS2 function would be expected to impair the cell's ability to respond to stressful conditions encountered during host invasion. For example, in C. glabrata, the Med2 subunit of the Mediator complex has been shown to be essential for low-pH stress tolerance , and similar regulatory mechanisms might involve PFS2-dependent RNA processing. The SWI/SNF chromatin remodeling complex in C. glabrata is crucial for intracellular survival in macrophages , suggesting that proper gene expression regulation, including RNA processing, is critical during infection.

How conserved is PFS2 across Candida species and other yeasts?

PFS2 is highly conserved across fungal species, reflecting its essential function in RNA processing. In Schizosaccharomyces pombe, Pfs2 has been characterized as an essential gene required for pre-mRNA 3' end processing . The orthologous gene in Saccharomyces cerevisiae (also called PFS2) encodes a component of the CPF complex with similar functions.

What are the optimal expression systems for producing recombinant C. glabrata PFS2 protein?

For recombinant expression of C. glabrata PFS2, several systems can be employed depending on the research objectives:

E. coli expression system:

• Advantages: Rapid growth, high protein yields, and cost-effectiveness

• Considerations: May lack post-translational modifications; codon optimization might be necessary due to GC content differences between C. glabrata and E. coli

• Recommended vectors: pET series for IPTG-inducible expression with His-tag or GST-tag for purification

Yeast expression systems:

• S. cerevisiae: Provides a eukaryotic environment closer to the native context

• Vectors: pY26 with GPD promoter has been successfully used for overexpression of C. glabrata genes

• Expression verification: qRT-PCR to confirm expression levels (typically aiming for 4-5 fold overexpression compared to wild-type)

Table 1: Comparison of Expression Systems for C. glabrata PFS2

How can I generate and characterize C. glabrata PFS2 mutants?

To generate and characterize C. glabrata PFS2 mutants, follow these methodological approaches:

Generation of deletion mutants:

• Construct deletion cassettes containing selectable markers (e.g., NAT1 for nourseothricin resistance) flanked by homologous regions of the PFS2 gene

• Transform C. glabrata cells using electroporation or lithium acetate methods

• Select transformants on appropriate selective media

• Confirm gene deletion by PCR and/or Southern blotting

Generation of conditional mutants (essential for studying essential genes like PFS2):

• Replace the native promoter with a regulatable promoter (e.g., MET3 or tetO promoters)

• For temperature-sensitive mutants, use error-prone PCR to generate a library of mutated PFS2 sequences, as demonstrated for S. pombe Pfs2

• Transform into a strain with wild-type PFS2 under a repressible promoter

• Screen for conditional phenotypes (e.g., temperature sensitivity)

Characterization approaches:

• Growth assays under various conditions (different temperatures, pH levels, stressors)

• Transcriptome analysis (RNA-seq) to assess global effects on gene expression

• Protein-protein interaction studies (co-immunoprecipitation, yeast two-hybrid)

• Subcellular localization using fluorescent protein fusions

• Phenotypic profiling under stress conditions relevant to pathogenicity

For point mutations, low-fidelity PCR with primers flanking the PFS2 locus can be used, followed by transformation to replace the native gene through homologous recombination, as demonstrated for S. pombe Pfs2 .

What purification methods are most effective for recombinant PFS2 protein?

For efficient purification of recombinant C. glabrata PFS2 protein, a multi-step approach is recommended:

Affinity chromatography (first step):

• Hexahistidine (His6) tag purification:

  • Use Ni-NTA or TALON resin

  • Employ imidazole gradient elution (20-250 mM)

  • Include 1-2 mM DTT in buffers if the protein contains cysteines

  • pH 7.5-8.0 is optimal for His-tagged protein binding

• GST-tag purification (alternative):

  • Use glutathione-sepharose resin

  • Elute with reduced glutathione (10-20 mM)

  • Consider on-column cleavage with PreScission protease

Secondary purification steps:

• Ion exchange chromatography:

  • Analyze protein pI to determine appropriate resin (cation or anion exchange)

  • Use a salt gradient (typically 50-500 mM NaCl) for elution

• Size exclusion chromatography (final polishing):

  • Select column based on expected molecular weight

  • Use buffer conditions that maintain protein stability

  • Analyze fractions by SDS-PAGE for purity

Buffer optimization considerations:

• Include glycerol (10-15%) to improve protein stability

• Add reducing agents (DTT or TCEP) to prevent oxidation

• Consider the addition of specific metal ions if PFS2 requires them for stability

• Perform thermal shift assays to identify optimal buffer conditions

For structural studies requiring high-purity protein, additional steps such as hydrophobic interaction chromatography may be necessary to achieve >95% purity.

How does PFS2 interact with other components of the CPF complex in C. glabrata?

The interaction of PFS2 with other components of the Cleavage and Polyadenylation Factor (CPF) complex in C. glabrata likely follows patterns similar to those observed in related yeasts, though with species-specific variations. Based on studies in fission yeast, PFS2 likely interacts with multiple CPF subunits, forming a bridge between different functional modules of the complex.

Predicted key interactions:

• Core CPF components: Direct interactions with the catalytic subunits responsible for the cleavage and polyadenylation activities

• RNA recognition components: Coordination with subunits that recognize polyadenylation signal sequences

• Connection to transcription machinery: Interactions that couple 3' end processing with transcription termination

Experimental approaches to study these interactions:

• Co-immunoprecipitation (Co-IP): Using tagged versions of PFS2 to pull down interacting partners, followed by mass spectrometry analysis

• Yeast two-hybrid (Y2H) assays: Similar to approaches used for Ppn1 in fission yeast , where specific domains of PFS2 can be tested against other CPF components

• Proximity-dependent biotin labeling: Using BioID or TurboID fused to PFS2 to identify proteins in close proximity in vivo

• Crosslinking mass spectrometry: To map specific interaction surfaces between PFS2 and other CPF components

Drawing from the study of the fission yeast CPF subunit Ppn1, which mediates association of a subcomplex with the CPF core, specific domains of PFS2 likely mediate distinct protein-protein interactions . Yeast two-hybrid assays can be particularly effective for mapping these interaction domains, as demonstrated for the Ppn1 interactions with Dis2 and Swd22 .

What phenotypes result from PFS2 disruption or mutation in C. glabrata?

Disruption or mutation of PFS2 in C. glabrata is expected to result in diverse phenotypes affecting multiple cellular processes. Based on data from orthologous proteins in related yeasts and the essential nature of RNA processing:

Expected phenotypes of PFS2 disruption:

• Growth defects: Complete deletion likely results in inviability due to the essential nature of proper mRNA processing

• Cell cycle abnormalities: Based on the S. pombe ortholog, defects in chromosome segregation and cell division

• Stress response deficiencies: Impaired ability to respond to environmental stresses (pH, oxidative, nutrient limitation)

• Reduced virulence: Decreased ability to survive in host environments and overcome host defenses

Conditional mutant phenotypes:

• Temperature sensitivity: Growth defects at elevated temperatures (37°C)

• pH sensitivity: Impaired growth under acidic conditions (pH 2.0-4.0), similar to observations with Med2 mutants

• Transcriptome alterations: Global changes in gene expression patterns, particularly affecting genes with complex processing requirements

Experimental approaches to characterize phenotypes:

• Growth assays on various media and conditions

• Microscopy to examine cell morphology and division patterns

• Stress survival assays (H₂O₂, acids, antifungals)

• Virulence assays using infection models such as Galleria mellonella larvae

• RNA-seq to assess global transcriptome changes

These phenotypic assays should be performed with conditional mutants (temperature-sensitive alleles or promoter shut-off strains) since complete deletion is likely lethal, as observed with the orthologous gene in S. pombe .

How does PFS2 function impact C. glabrata virulence in infection models?

The impact of PFS2 function on C. glabrata virulence would need to be assessed using conditional mutants, as complete deletion is likely lethal. Based on the role of RNA processing in stress responses and adaptation to host environments, several aspects of virulence are likely affected:

Expected virulence phenotypes:

• Survival in macrophages: Impaired ability to persist within phagocytic cells, similar to the importance of the SWI/SNF chromatin remodeling complex for C. glabrata survival in macrophages

• Adaptation to host pH: Reduced capacity to adapt to acidic environments in the host, as seen with Med2 mutants that show pH sensitivity

• Stress tolerance: Decreased resistance to oxidative stress encountered during phagocytosis

• Biofilm formation: Potential defects in biofilm development on medical devices

Methodological approaches to assess virulence:

• Galleria mellonella infection model:

  • Inject larvae with ~5 × 10⁷ C. glabrata cells of wild-type vs. conditional PFS2 mutant strains

  • Monitor larval survival over 72 hours

  • Assess fungal proliferation by collecting hemolymph at defined time points

  • Examine interaction with hemocytes (insect immune cells)

• Macrophage survival assays:

  • Infect THP-1 macrophages with C. glabrata strains at MOI 1:5

  • Lyse macrophages at various time points (1, 4, 24, 48h)

  • Quantify viable fungal cells by colony counting

  • Assess cytokine production by macrophages

• Murine infection models:

  • Systemic infection via tail vein injection

  • Organ fungal burden determination at various time points

  • Histopathological analysis of infected tissues

  • Immune response characterization

• Transcriptional profiling during infection:

  • RNA-seq analysis of C. glabrata recovered from infection models

  • Identification of PFS2-dependent gene expression patterns during host interaction

When designing these experiments, it's essential to use appropriate controls, including complemented strains where the wild-type PFS2 gene is reintroduced to verify that observed phenotypes are specifically due to PFS2 disruption.

How does PFS2 contribute to antifungal resistance mechanisms in C. glabrata?

PFS2's contribution to antifungal resistance in C. glabrata is likely multifaceted, given the integral role of RNA processing in gene expression regulation. While there's no direct evidence in the search results specifically linking PFS2 to antifungal resistance, we can make informed predictions based on known resistance mechanisms in C. glabrata:

Potential mechanisms of PFS2-mediated resistance contributions:

• Regulation of drug efflux transporters:

  • PFS2 may influence the expression of ABC transporters like CDR1, PDH1, and SNQ2, which are known to confer azole resistance

  • Proper 3' end processing of these transporter mRNAs is crucial for their expression and function

• Interaction with stress response pathways:

  • PFS2 could affect the expression of transcription factors like Pdr1 and Upc2A, which regulate genes involved in azole resistance

  • Alterations in PFS2 function might disrupt the expression of stress response genes required for antifungal tolerance

• Cell wall remodeling gene expression:

  • Echinocandin resistance involves cell wall modifications, and genes encoding cell wall components require proper mRNA processing

  • Mutations affecting PFS2 function could alter the expression of genes involved in β-glucan synthesis, impacting echinocandin susceptibility

Experimental approaches to investigate this relationship:

• Transcriptome analysis:

  • Compare RNA-seq profiles of wild-type and PFS2 conditional mutants with and without antifungal exposure

  • Focus on differential expression of known resistance genes

  • Analyze alterations in 3' UTR usage and polyadenylation site selection

• Antifungal susceptibility testing:

  • Determine minimum inhibitory concentrations (MICs) for various antifungal drugs

  • Perform time-kill assays to assess the rate of fungicidal activity

  • Test for synergistic effects between PFS2 inhibition and antifungal treatment

• Selection of resistant mutants:

  • Subject PFS2 conditional mutants to increasing antifungal concentrations

  • Sequence evolved strains to identify compensatory mutations

  • Characterize the mechanism of acquired resistance

Given C. glabrata's intrinsic resistance to azoles and increasing resistance to echinocandins , understanding how fundamental cellular processes like RNA processing contribute to resistance mechanisms could reveal new therapeutic targets or strategies to overcome resistance.

What is the structure-function relationship of PFS2 domains in C. glabrata?

Understanding the structure-function relationship of PFS2 domains in C. glabrata requires a combination of computational, biochemical, and genetic approaches. While specific structural information for C. glabrata PFS2 is not available in the search results, we can predict domain functions based on orthologous proteins and design experiments to test these predictions:

Predicted domain organization of C. glabrata PFS2:

• N-terminal domain: Likely involved in protein-protein interactions with other CPF components

• WD40 repeats: Predicted based on orthologous proteins; typically forms a β-propeller structure that serves as a platform for multiple protein interactions

• C-terminal region: Potentially contains regulatory elements or interaction surfaces specific to C. glabrata

Experimental approaches to elucidate domain functions:

• Domain deletion and mutation analysis:

  • Generate a series of truncation mutants removing specific domains

  • Create point mutations in conserved residues within each domain

  • Test functionality through complementation of conditional PFS2 mutants

  • This approach parallels the successful strategy used for Ppn1 in fission yeast

• Yeast two-hybrid domain mapping:

  • Test individual domains for interactions with other CPF components

  • Identify critical residues through alanine scanning mutagenesis

  • For example, in the analysis of Ppn1, alanine substitutions identified critical residues (V508A, W510A) that abolished protein-protein interactions

• Structural biology approaches:

  • X-ray crystallography or cryo-EM of recombinant domains

  • Small-angle X-ray scattering (SAXS) for solution structure

  • Cross-linking mass spectrometry to identify interaction surfaces

Table 2: Predicted Functional Domains of C. glabrata PFS2 and Experimental Approaches

By systematically characterizing these domains, researchers can develop a comprehensive understanding of how PFS2 functions within the CPF complex and potentially identify regions that could be targeted for antifungal development.

How can PFS2 be targeted for potential antifungal drug development?

Targeting PFS2 for antifungal drug development presents both opportunities and challenges, given its essential role in RNA processing. A strategic approach would involve:

Rationale for targeting PFS2:

• Essential function makes it a potential high-value target

• RNA processing machinery may have sufficient fungal-specific features to allow selective targeting

• Novel mechanism of action would address resistance to current antifungals

Drug development strategies:

• Structure-based drug design:

  • Obtain high-resolution structures of C. glabrata PFS2 alone and in complex with interacting partners

  • Identify potential binding pockets unique to fungal PFS2 proteins

  • Use in silico screening to identify small molecules that could disrupt critical interactions

  • Validate hits through binding assays and functional studies

• Protein-protein interaction inhibitors:

  • Target specific interactions between PFS2 and other CPF components

  • Develop peptide mimetics based on interaction motifs

  • Screen for small molecules that disrupt these interactions

  • This approach is supported by successful identification of specific binding interfaces in related proteins, such as the Ppn1-Dis2 interaction in fission yeast

• RNA-competitive inhibitors:

  • Design molecules that mimic RNA substrates but cannot be processed

  • Screen for compounds that bind to the RNA-binding pocket of PFS2

  • Test specificity against human RNA processing machinery

Validation approaches:

• In vitro assays:

  • Biochemical assays measuring RNA processing activity

  • Thermal shift assays to confirm compound binding

  • Surface plasmon resonance for binding kinetics

• Cellular assays:

  • Growth inhibition assays with C. glabrata

  • Specificity testing against human cell lines

  • Combination testing with existing antifungals

• Resistance development assessment:

  • Serial passage experiments to evaluate resistance emergence

  • Whole genome sequencing of resistant isolates

  • Cross-resistance testing with other antifungals

• In vivo efficacy studies:

  • Initial testing in G. mellonella infection model

  • Progression to murine models of candidiasis

  • Pharmacokinetic and toxicology studies

The significant challenge will be achieving selectivity for fungal over human RNA processing machinery. Success would depend on identifying structural or functional differences that can be exploited for selective inhibition of the fungal protein.

How does environmental stress affect PFS2 function and localization in C. glabrata?

Environmental stresses likely influence PFS2 function and localization in C. glabrata, given the importance of proper gene expression regulation during stress responses. While specific data on PFS2 is not provided in the search results, we can propose mechanisms and experimental approaches based on known stress responses in C. glabrata:

Potential stress-induced changes in PFS2:

• Relocalization within the nucleus:

  • Stress may trigger redistribution of PFS2 to specific nuclear domains

  • Association with stress-responsive genes might increase

  • Formation of stress-specific processing complexes

• Post-translational modifications:

  • Phosphorylation status might change in response to stress

  • Similar to the regulation of CgYap6 by CgYak1 phosphorylation under low-pH stress

  • These modifications could alter interaction partners or activity

• Alternative complex formation:

  • Stress might induce association with different subsets of CPF components

  • Formation of specialized RNA processing complexes for stress-responsive genes

Methodological approaches to study stress effects:

• Fluorescence microscopy:

  • Generate PFS2-GFP fusion protein expressed from the native locus

  • Expose cells to various stresses (pH, oxidative, antifungal)

  • Monitor changes in localization using live-cell imaging

  • Quantify nuclear distribution patterns

• Biochemical analysis:

  • Perform immunoprecipitation of PFS2 under various stress conditions

  • Identify differential interactors by mass spectrometry

  • Analyze post-translational modifications using phospho-specific antibodies or mass spectrometry

  • Compare complex composition under normal and stress conditions

• Transcriptome analysis:

  • Perform RNA-seq on wild-type and PFS2 conditional mutants under stress

  • Analyze alternative polyadenylation patterns using 3'-seq

  • Identify stress-responsive genes dependent on PFS2 function

• ChIP-seq analysis:

  • Map PFS2 association with chromatin under normal and stress conditions

  • Identify stress-specific binding patterns

  • Compare with the distribution of RNA polymerase II

Given that C. glabrata must adapt to various hostile environments during infection (low pH in the vaginal tract, oxidative stress in phagocytes, nutrient limitation in the bloodstream), understanding how PFS2 function is modulated during these stresses could provide insights into virulence mechanisms and potential therapeutic interventions.

What are the common challenges in expressing and purifying recombinant C. glabrata PFS2 and how can they be addressed?

Expressing and purifying recombinant C. glabrata PFS2 presents several challenges that researchers should anticipate and address:

Common challenges and solutions:

• Low expression levels:

  • Optimize codon usage for the expression host

  • Test multiple promoters (T7, tac, AOX1 depending on the system)

  • Try different fusion tags (His, GST, MBP, SUMO)

  • Optimize induction conditions (temperature, inducer concentration, duration)

  • For bacterial expression, co-express with chaperones like GroEL/GroES

• Protein insolubility:

  • Express at lower temperatures (16-20°C)

  • Use solubility-enhancing tags like MBP or SUMO

  • Add solubility enhancers to lysis buffer (10% glycerol, 0.1% Triton X-100)

  • Consider on-column refolding for proteins recovered from inclusion bodies

  • Try expression in insect or mammalian cells for improved folding

• Protein instability:

  • Include protease inhibitors during purification

  • Add stabilizing agents (glycerol, arginine, trehalose)

  • Determine optimal buffer conditions using thermal shift assays

  • Test different pH conditions (typically 7.0-8.0)

  • Consider co-expression with binding partners

• Low purity:

  • Implement multi-step purification strategy

  • Use stringent washing conditions during affinity chromatography

  • Apply size exclusion chromatography as a final polishing step

  • Consider ion exchange chromatography to separate closely related contaminants

Troubleshooting approach:

• Systematically test expression in different compartments (cytoplasmic, periplasmic, secreted)

• Express individual domains if full-length protein proves difficult

• Use Western blotting to track protein through purification process

• Assess protein quality using dynamic light scattering to check for aggregation

• Verify protein identity and integrity by mass spectrometry

Table 3: Optimization Parameters for Recombinant PFS2 Expression

When designing purification strategies, it's advisable to learn from successful approaches used for related proteins in the CPF complex, adapting them to the specific properties of PFS2.

How can I validate the functionality of recombinant C. glabrata PFS2 protein?

Validating the functionality of recombinant C. glabrata PFS2 protein is essential to ensure that the purified protein retains its native biological activities. Multiple complementary approaches should be employed:

Biochemical activity assays:

• RNA binding assays:

  • Electrophoretic mobility shift assays (EMSA) with polyadenylation signal-containing RNA

  • Fluorescence anisotropy with labeled RNA substrates

  • Surface plasmon resonance to determine binding kinetics

• In vitro processing assays:

  • Reconstitution of the CPF complex with recombinant components

  • 3' end cleavage assays using labeled pre-mRNA substrates

  • Polyadenylation assays measuring poly(A) tail addition

• Protein-protein interaction validation:

  • Pull-down assays with known CPF components

  • Size exclusion chromatography to detect complex formation

  • Isothermal titration calorimetry to measure binding thermodynamics

Genetic complementation:

• Heterologous complementation:

  • Express C. glabrata PFS2 in S. cerevisiae pfs2 mutants

  • Test for rescue of growth defects or RNA processing phenotypes

  • This approach has been successfully used for other C. glabrata genes

• Homologous complementation:

  • Introduce recombinant PFS2 into C. glabrata conditional mutants

  • Assess restoration of normal growth and stress responses

  • Measure correction of RNA processing defects

Structural validation:

• Circular dichroism (CD) spectroscopy:

  • Confirm proper secondary structure composition

  • Compare with predicted structural elements

• Thermal stability assays:

  • Differential scanning fluorimetry to assess protein folding

  • Compare stability of wild-type and mutant variants

• Limited proteolysis:

  • Analyze domain organization and folding

  • Compare digestion patterns with predicted domain boundaries

Activity in cellular extracts:

• In vitro complementation:

  • Add recombinant PFS2 to extracts from PFS2-depleted cells

  • Measure restoration of RNA processing activities

  • Compare with wild-type extracts

When validating recombinant PFS2 functionality, it's important to include appropriate controls, such as known inactive mutants (e.g., point mutations in critical residues) and proteins with similar biochemical properties but different functions.

How does PFS2 interact with stress response pathways specific to C. glabrata?

C. glabrata exhibits remarkable stress tolerance compared to other Candida species, and understanding how PFS2 interfaces with stress response pathways could reveal important insights into this pathogen's resilience. While direct evidence for PFS2's role is not provided in the search results, we can propose mechanisms and research directions:

Potential interactions with stress response pathways:

• Low pH stress response:

  • Similar to CgMed2's role in acid pH tolerance , PFS2 might regulate the processing of mRNAs encoding proteins involved in glycerophospholipid metabolism

  • PFS2 could affect the expression of transcription factors like CgYap6, which is involved in low pH adaptation

• Oxidative stress response:

  • Processing of mRNAs encoding antioxidant enzymes and repair proteins

  • Coordination with the SWI/SNF complex, which is critical for C. glabrata survival in the oxidative environment of macrophages

• Drug stress response:

  • Potential role in regulating expression of drug resistance genes like CDR1, similar to the role of transcription factors Pdr1 and Upc2A

  • Processing of mRNAs encoding membrane proteins and lipid biosynthesis enzymes that affect drug permeability

Experimental approaches:

• Genetic interaction mapping:

  • Generate double mutants combining PFS2 conditional alleles with mutations in stress response genes

  • Screen for synthetic phenotypes that indicate functional relationships

  • Use quantitative fitness analysis to measure genetic interactions

• Stress-specific transcriptome analysis:

  • RNA-seq comparing wild-type and PFS2 mutant responses to various stresses

  • Analysis of 3' UTR usage and polyadenylation site selection under stress

  • Integration with data on transcription factor binding sites

• Protein interactome in stress conditions:

  • Immunoprecipitation of PFS2 under different stress conditions

  • Identification of stress-specific interaction partners

  • Validation of key interactions through targeted approaches

• Chromatin association studies:

  • ChIP-seq to map PFS2 association with chromatin under stress

  • Correlation with binding sites of stress-responsive transcription factors

  • Analysis of co-occupancy patterns

Understanding these interactions could reveal how C. glabrata coordinates rapid and appropriate gene expression responses to stress, which is critical for its survival during infection and its notorious ability to develop drug resistance.

Can PFS2 function be modulated to enhance antifungal susceptibility in resistant C. glabrata strains?

Modulating PFS2 function represents a potential strategy to enhance antifungal susceptibility in resistant C. glabrata strains by targeting the fundamental process of RNA processing. While this approach is theoretical based on available information, several strategies warrant investigation:

Potential approaches to modulate PFS2 for enhancing antifungal efficacy:

• Partial inhibition strategy:

  • Develop small molecule inhibitors that partially impair PFS2 function

  • Target specific PFS2 interactions rather than core catalytic activity

  • This approach might sensitize cells to existing antifungals without being directly lethal

• Combination therapy rationale:

  • PFS2 inhibition could disrupt proper expression of resistance genes

  • For example, proper processing of CDR1 mRNA (encoding an efflux pump) is likely PFS2-dependent

  • Simultaneous targeting of PFS2 and treatment with azoles might overcome efflux-mediated resistance

• Stress response disruption:

  • PFS2 inhibition could prevent proper adaptation to drug-induced stress

  • This would be analogous to the increased azole susceptibility observed in upc2aΔ mutants

  • The goal would be to prevent compensatory gene expression responses

Experimental validation approaches:

• Genetic validation:

  • Generate conditional PFS2 mutants in drug-resistant backgrounds

  • Test antifungal susceptibility under partial PFS2 repression

  • Measure changes in expression of resistance genes

• Chemical biology approach:

  • Screen for compounds that bind to PFS2 or disrupt its interactions

  • Test these compounds for synergy with existing antifungals

  • Perform mechanism of action studies to confirm target engagement

• RNA processing analysis:

  • Compare 3' end processing of resistance genes in sensitive vs. resistant strains

  • Identify processing differences that could be targeted

  • Develop antisense oligonucleotides to interfere with specific mRNA processing events

• Proof of concept in animal models:

  • Test combination approaches in G. mellonella and murine infection models

  • Assess both efficacy and potential toxicity

  • Evaluate resistance development

How does the function of PFS2 differ between pathogenic C. glabrata and non-pathogenic yeasts?

Understanding the functional differences of PFS2 between pathogenic C. glabrata and non-pathogenic yeasts could reveal adaptations relevant to virulence and identify potential targets for species-specific interventions:

Comparative genomics and evolutionary analysis:

• Sequence and structural comparison:

  • Align PFS2 sequences from C. glabrata, S. cerevisiae, S. pombe, and other yeasts

  • Identify C. glabrata-specific insertions, deletions, or substitutions

  • Perform phylogenetic analysis to trace evolutionary relationships

  • Map variations onto predicted structural models

• Domain architecture analysis:

  • Compare domain organization across species

  • Identify pathogen-specific domains or motifs

  • Analyze conservation of interaction surfaces

Functional comparative studies:

• Cross-species complementation:

  • Express C. glabrata PFS2 in S. cerevisiae pfs2 mutants

  • Express S. cerevisiae PFS2 in C. glabrata conditional mutants

  • Test domain swaps to identify regions responsible for functional differences

  • This approach parallels successful cross-species complementation studies with other genes

• Comparative interactome analysis:

  • Compare protein interaction networks in different species

  • Identify C. glabrata-specific interaction partners

  • Map these differences to specific domains or motifs

• Stress response comparison:

  • Compare the role of PFS2 in stress responses across species

  • Test whether C. glabrata PFS2 confers enhanced stress tolerance when expressed in non-pathogenic yeasts

  • This approach is supported by observations that C. glabrata-specific adaptations can differ from those in C. albicans

Relevance to pathogenesis:

• Host adaptation signatures:

  • Analyze whether PFS2 sequence variations correlate with host range

  • Test whether these variations affect processing of virulence-associated genes

  • Examine whether C. glabrata PFS2 shows signs of positive selection

• Virulence gene regulation:

  • Compare processing of orthologous mRNAs between species

  • Focus on genes involved in stress response and host interaction

  • Identify C. glabrata-specific processing events

An important consideration is that C. glabrata is phylogenetically closer to S. cerevisiae than to C. albicans, despite both being classified as Candida species . Therefore, functional comparisons should include both S. cerevisiae (close relative) and C. albicans (distant relative but similar niche) to distinguish general yeast traits from pathogen-specific adaptations.