Recombinant Candida glabrata Restriction of telomere capping protein 4 (RTC4)

What is RTC4 and what is its significance in Candida glabrata biology?

RTC4 (Restriction of telomere capping protein 4) is a 339-amino acid protein expressed in Candida glabrata. The protein's sequence includes multiple serine-rich regions and potential phosphorylation sites that suggest regulatory functions . While specific RTC4 functions are still being elucidated, telomere-associated proteins in pathogenic fungi often play crucial roles in genomic stability, stress responses, and virulence mechanisms. RTC4 likely contributes to C. glabrata's adaptive capabilities, which is particularly significant given that C. glabrata ranks as the second most commonly isolated Candida species in the United States and northern Europe, and third most common in southern Europe and Asia .

How do you properly store and handle recombinant RTC4 protein for experimental use?

Recombinant RTC4 protein requires careful handling to maintain stability and functionality. The recommended storage conditions depend on the formulation:

• Liquid formulations: Store at -20°C/-80°C with a typical shelf life of 6 months

• Lyophilized formulations: Store at -20°C/-80°C with an extended shelf life of 12 months

For reconstitution:

• Briefly centrifuge the vial before opening

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

Repeated freezing and thawing should be avoided as it may compromise protein integrity and activity.

What expression systems are most effective for producing recombinant RTC4?

E. coli expression systems are demonstrably effective for RTC4 production, as evidenced by commercially available recombinant preparations . When establishing an expression protocol:

• Vector selection: Consider vectors with strong inducible promoters (T7, tac) for controlled expression

• E. coli strain optimization: BL21(DE3) derivatives often provide good yields for fungal proteins

• Expression conditions: Optimize temperature (often lowered to 16-25°C), induction timing, and IPTG concentration

• Purification strategy: Implement a tag system (His-tag, GST) compatible with downstream applications

• Quality control: Verify expression using SDS-PAGE (target >85% purity) and western blotting

For structural or functional studies requiring proper folding, consider eukaryotic expression systems like P. pastoris, though these typically yield lower protein quantities but potentially higher biological activity.

How might RTC4 contribute to antifungal resistance mechanisms in C. glabrata?

Understanding RTC4's potential role in antifungal resistance requires examining it within the context of C. glabrata's established resistance mechanisms:

• Ergosterol pathway regulation: While CgRpn4 has been identified as a transcription factor mediating fluconazole tolerance through regulation of ergosterol biosynthesis genes (ERG1, ERG2, ERG3, and ERG11) , RTC4 may function in parallel or complementary pathways affecting membrane composition.

• Telomere-associated stress responses: As a telomere capping protein, RTC4 may contribute to genomic stability under antifungal stress conditions, potentially enabling adaptation through mutation accumulation or chromosomal rearrangements.

• Experimental approach: To investigate RTC4's role in resistance:

  • Generate RTC4 deletion mutants and assess minimum inhibitory concentration (MIC) changes for various antifungals

  • Perform RNA-seq comparing wild-type and ∆rtc4 strains under antifungal stress to identify differentially regulated pathways

  • Examine localization changes of RTC4 during antifungal exposure using fluorescent tagging

  • Assess changes in ergosterol content and membrane permeability in RTC4 mutants, particularly during fluconazole stress

C. glabrata's ability to rapidly acquire azole resistance has contributed to its increased prevalence relative to C. albicans in clinical settings , making proteins potentially involved in resistance mechanisms high-priority research targets.

What approaches should be used to study RTC4's potential interactions with the proteasome system?

Given that the S. cerevisiae homolog Rpn4 regulates the ubiquitin-proteasome system and its deletion results in increased sensitivity to azoles , investigating RTC4's relationship with proteasome function requires:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with proteasome components

  • Yeast two-hybrid screening

  • Proximity-dependent biotin labeling (BioID)

  • FRET/BRET assays for in vivo interaction detection

• Functional assays:

  • Measure proteasome activity using fluorogenic substrates in wild-type vs. ∆rtc4 strains

  • Assess ubiquitinated protein accumulation during stress responses

  • Monitor protein degradation kinetics of known proteasome substrates

• Transcriptional regulation analysis:

  • ChIP-seq to identify binding sites in proteasome subunit promoters

  • Reporter assays using proteasome gene promoters

  • qRT-PCR to measure proteasome gene expression in RTC4 mutants

• Stress response investigation:

  • Compare growth under various stressors (oxidative, thermal, ER stress) between wild-type and ∆rtc4 strains

  • Assess genetic interactions through synthetic genetic array with proteasome components

How can researchers effectively study RTC4's role in C. glabrata virulence using in vivo models?

Investigating RTC4's contribution to virulence requires carefully designed in vivo experiments:

• Mouse model of disseminated candidiasis:

  • Compare wild-type, ∆rtc4, and complemented strains for:

    • Fungal burden in organs (kidney, liver, spleen)

    • Survival rates

    • Inflammatory marker profiles

  • Use immunocompromised models to mirror clinical scenarios

• Biofilm formation assessment:

  • C. glabrata forms biofilms on medical devices that can harbor fungal infections

  • Compare biofilm formation capacity of wild-type vs. ∆rtc4 strains

  • Evaluate biofilm structure using confocal microscopy

  • Assess biofilm drug resistance profiles

• Host cell interaction studies:

  • Macrophage engulfment and survival assays

  • Epithelial cell adhesion and invasion assays

  • Neutrophil killing resistance

• Transcriptional profiling during infection:

  • RNA-seq of fungal cells recovered from infected tissues

  • Host-pathogen interaction transcriptomics

  • Identification of RTC4-dependent virulence factors

C. glabrata typically grows in yeast form and has evolved an infection strategy based on stealth and evasion without causing severe damage in murine models , making subtle virulence phenotypes important to detect through multiple complementary approaches.

What techniques should be employed to study the relationship between RTC4 and mitochondrial genome variation in C. glabrata?

C. glabrata exhibits hypervariable mitochondrial genomes with reduced conserved sequence in non-reference sequence types . To investigate RTC4's potential relationship with mitochondrial variation:

• Comparative genomics approach:

  • Sequence mitochondrial genomes from wild-type and ∆rtc4 strains

  • Track mitochondrial genome evolution during passage with and without RTC4

  • Analyze mitochondrial DNA copy number and integrity

• Functional mitochondrial assessments:

  • Measure respiration rates and mitochondrial membrane potential

  • Evaluate reactive oxygen species production

  • Assess mitochondrial morphology and distribution

• Stress response profiling:

  • Test sensitivity to oxidative stressors and mitochondrial inhibitors

  • Evaluate growth on non-fermentable carbon sources

  • Monitor adaptation to mitochondrial stress conditions

• Localization studies:

  • Investigate potential mitochondrial localization of RTC4 under various conditions

  • Track mitochondrial protein import efficiency

This approach can help determine whether RTC4 contributes to the observed hypervariability in C. glabrata mitochondrial genomes, which may impact virulence and drug resistance phenotypes .

What are the critical quality control parameters for recombinant RTC4 preparations?

Ensuring consistent quality of recombinant RTC4 preparations requires rigorous quality control:

When preparing RTC4 for experimental use, aliquot the protein and avoid repeated freeze-thaw cycles to maintain stability . For long-term storage, adding glycerol to a final concentration of 50% is recommended, though researchers should optimize based on downstream applications .

How can researchers effectively design gene deletion and complementation systems for RTC4 in C. glabrata?

Creating precise genetic tools for RTC4 manipulation requires careful consideration of C. glabrata's genetic properties:

• Deletion strategy:

  • Use homologous recombination with long flanking regions (>500 bp) due to C. glabrata's lower recombination efficiency compared to S. cerevisiae

  • Selection markers: NAT1, HygB, or URA3 for primary transformations

  • Verify deletions by:

    • PCR across deletion junctions

    • Southern blotting

    • RT-PCR/qPCR to confirm transcript absence

• Complementation approaches:

  • Reintegration at native locus

  • Integration at neutral locus (e.g., HIS3)

  • Episomal expression with ARS/CEN plasmids

  • Use native promoter and terminator for physiological expression levels

• Expression validation:

  • Western blotting with specific antibodies

  • qRT-PCR to quantify transcript levels

  • GFP/epitope tagging for localization studies

• Conditional expression systems:

  • Tetracycline-regulatable promoters

  • Methionine-repressible MET3 promoter

  • Estradiol-inducible system for tight control

When planning these genetic manipulations, researchers should consider that C. glabrata has a haploid genome, making it easier to generate complete gene knockouts compared to diploid Candida species like C. albicans.

What approaches should be used to identify RTC4 binding partners and genomic targets?

Comprehensive identification of RTC4 interactions requires multi-faceted strategies:

• Protein-protein interactions:

  • Affinity purification-mass spectrometry (AP-MS)

  • Yeast two-hybrid screening

  • Proximity-dependent biotin identification (BioID)

  • Co-immunoprecipitation followed by western blotting for candidate partners

  • Protein microarrays

• DNA-protein interactions:

  • Chromatin immunoprecipitation sequencing (ChIP-seq)

  • DNA pull-down assays with recombinant RTC4

  • Electrophoretic mobility shift assays (EMSA)

  • DNase I footprinting

  • Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

• RNA-protein interactions:

  • RNA immunoprecipitation (RIP)

  • Crosslinking and immunoprecipitation (CLIP)

  • RNA Electrophoretic Mobility Shift Assay (REMSA)

• Functional validation:

  • Mutagenesis of binding domains

  • Competition assays

  • Reporter assays for transcriptional targets

  • Co-localization studies

If RTC4 functions similar to transcription factors like CgRpn4, which directly regulates ERG11 expression through the TTGCAAA binding motif , ChIP-seq would be particularly valuable for identifying genome-wide binding sites and regulatory networks.

How does RTC4 function relate to C. glabrata's unique infection strategy?

C. glabrata has evolved an infection strategy based on stealth and evasion without causing severe damage in murine models . Understanding RTC4's role within this context requires:

• Comparative virulence assessment:

  • Compare wild-type and ∆rtc4 strains in various infection models

  • Analyze host immune response patterns (cytokine profiles, neutrophil recruitment)

  • Measure tissue damage markers and fungal burden

• Evolutionary context analysis:

  • Compare RTC4 sequences across the Nakaseomyces clade

  • Identify selective pressure signatures (dN/dS ratios)

  • Analyze RTC4 in environmental vs. clinical isolates

• Host interaction profiling:

  • Assess phagocytosis rates by macrophages

  • Measure survival within phagocytes

  • Evaluate adhesion to host tissues

  • Test immune recognition using reporter cell lines

• Stress adaptation role:

  • Examine RTC4 expression during host-relevant stresses

  • Assess ∆rtc4 strain fitness in host-mimicking conditions

  • Analyze metabolic adaptation in the absence of RTC4

Since C. glabrata's ability to infect humans is thought to have evolved relatively recently , analyzing RTC4's evolutionary history might provide insights into the emergence of pathogenicity in this species.

How can researchers integrate RTC4 findings with population genetics data in C. glabrata?

C. glabrata exhibits notable genetic diversity, including recombinant sequence types and variation in virulence genes and drug targets . To place RTC4 research within this population genetics framework:

• Sequence diversity analysis:

  • Sequence RTC4 across clinical isolates representing different sequence types

  • Identify polymorphisms and their potential functional impacts

  • Perform population genetic analyses (Tajima's D, FST) to detect selection signatures

• Phenotypic correlation studies:

  • Associate RTC4 variants with virulence phenotypes

  • Correlate RTC4 sequence/expression with antifungal susceptibility

  • Link genetic variation to clinical outcomes

• Microevolution tracking:

  • Monitor RTC4 changes in serial clinical isolates from prolonged infections

  • Assess RTC4 expression changes during host adaptation

• Recombination analysis:

  • Investigate evidence of recombination in the RTC4 locus

  • Examine linkage disequilibrium patterns with nearby genes

  • Compare RTC4 phylogeny with whole-genome phylogenetic trees

This integrated approach can help determine whether RTC4 is involved in the observed population admixture suggesting a yet undiscovered sexual cycle in C. glabrata .

What emerging technologies could advance our understanding of RTC4 function?

Several cutting-edge technologies show promise for elucidating RTC4's role:

• CRISPR-Cas9 precise genome editing:

  • Create point mutations in functional domains

  • Introduce fluorescent tags at endogenous loci

  • Generate conditional alleles

  • Perform genome-wide screens for genetic interactions

• Single-cell technologies:

  • Single-cell RNA-seq to capture heterogeneity in RTC4 expression

  • Single-cell proteomics to measure protein abundance variations

  • Microfluidics for tracking individual cell responses

• Structural biology approaches:

  • Cryo-EM for complex structural determination

  • AlphaFold2/RoseTTAFold for structure prediction

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Host-pathogen interaction technologies:

  • Intravital microscopy for in vivo tracking

  • Organ-on-chip models for tissue-specific interactions

  • Dual RNA-seq for simultaneous host-pathogen transcriptomics

• Long-read sequencing:

  • Full-length transcript analysis

  • Structural variant detection

  • Epigenetic modification mapping

These technologies can help resolve the complex relationship between RTC4, telomere function, and C. glabrata pathobiology.

How might understanding RTC4 contribute to novel antifungal development strategies?

RTC4's potential as an antifungal target or resistance mechanism can be explored through:

• Target validation studies:

  • Essentiality assessment under various conditions

  • Chemical genetic profiling

  • In vivo requirement during infection

  • Specificity comparison with human proteins

• Small molecule screening:

  • High-throughput assays targeting RTC4 function

  • Fragment-based drug discovery

  • Virtual screening using structural models

  • Phenotypic screens with RTC4-dependent readouts

• Combination therapy approaches:

  • Synergy testing between RTC4 inhibitors and existing antifungals

  • Sensitization of resistant strains

  • Prevention of resistance development

• Therapeutic potential assessment:

  • Efficacy in animal models

  • Resistance development frequency

  • Toxicity and pharmacokinetic profiling

  • Spectrum of activity across Candida species