Recombinant Candida glabrata 40S ribosomal protein S0 (RPS0)

What is Recombinant Candida glabrata 40S Ribosomal Protein S0 (RPS0)?

Recombinant Candida glabrata 40S ribosomal protein S0 (RPS0) is a purified protein component of the small ribosomal subunit from the pathogenic yeast Candida glabrata. It is produced through recombinant expression systems, typically in mammalian cells, to yield a protein with a purity of >85% as verified by SDS-PAGE. The protein consists of 250 amino acids (expression region 2-251) with a well-defined sequence and is associated with the UniProt number Q6FJX4 . RPS0 plays an essential role in ribosome assembly and protein synthesis in this opportunistic fungal pathogen.

How does RPS0 contribute to the biology of Candida glabrata?

RPS0 is an integral component of the ribosomal machinery in Candida glabrata, contributing to protein synthesis and cellular adaptation to various environmental conditions. Research suggests that ribosomal proteins in Candida species, including RPS0, may contribute to pathogenicity and stress responses. Gene duplication events have constrained and driven regulatory evolution of ribosomal proteins in Candida glabrata, indicating their importance in adaptation . Additionally, the RPS0 gene has been utilized for molecular identification of Candida species, suggesting the presence of species-specific sequences within this gene that have evolutionary significance .

How is RPS0 used for molecular identification of Candida species?

The RPS0 gene contains intron sequences that have proven valuable for species-specific identification of Candida glabrata and related species. Researchers have designed PCR primers targeting the RPS0 gene intron fragment that produce amplicons of distinct sizes: 406 bp for C. glabrata and 150 bp for C. parapsilosis . This molecular identification method is more reliable than traditional phenotypic identification, particularly for closely related Candida species like C. glabrata and C. parapsilosis. The technique has even demonstrated the ability to differentiate between C. parapsilosis and C. orthopsilosis, providing a valuable tool for early and accurate identification of Candida species responsible for candidiasis .

What role does RPS0 play in biofilm formation and antifungal resistance?

While the direct role of RPS0 in biofilm formation has not been explicitly described in the search results, transcriptome analyses of C. glabrata biofilm cells have revealed that ribosomal proteins, potentially including RPS0, undergo expression changes during biofilm formation and in response to antifungal treatments . C. glabrata biofilms exhibit high tolerance to antifungal treatments, and gene expression reprogramming occurs in response to fluconazole treatment in a carbon source and pH-dependent manner. This reprogramming affects genes involved in DNA replication, ergosterol biosynthesis, and ubiquinone biosynthesis, potentially including pathways that involve ribosomal proteins . Understanding how RPS0 expression changes during biofilm formation and antifungal treatment could provide insights into resistance mechanisms.

How does RPS0 expression change under different environmental conditions?

Transcriptomic analyses have shown that C. glabrata undergoes extensive gene expression changes in response to environmental conditions such as pH variations, carbon source availability, and exposure to antifungal agents . While specific data on RPS0 expression changes are not explicitly provided in the search results, studies have shown that ribosomal proteins in Candida species respond to heat shock. Specifically, research with C. glabrata grown at control (22°C) versus heat-shock temperatures (37°C or 42°C) has demonstrated alterations in gene expression patterns . Similarly, during macrophage infection, C. glabrata undergoes temporal transcriptional responses that likely involve ribosomal components . These studies suggest that RPS0 expression may be modulated during host-pathogen interactions and stress responses.

What are the optimal storage and handling conditions for recombinant RPS0?

Recombinant RPS0 protein has specific storage and handling requirements to maintain its stability and functionality. According to product information, the shelf life of the liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form can be stored for up to 12 months at -20°C/-80°C . To maintain protein integrity, repeated freezing and thawing should be avoided. Working aliquots can be stored at 4°C for up to one week .

For reconstitution, the protein vial should be briefly centrifuged prior to opening to bring contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol to a final concentration of 5-50% (with 50% being the recommended default) is advised for long-term storage at -20°C/-80°C .

What methods are used to express and purify recombinant RPS0?

The recombinant Candida glabrata 40S ribosomal protein S0 is typically expressed in mammalian cell systems . While detailed expression and purification protocols are not explicitly described in the search results, standard recombinant protein production procedures would involve:

• Cloning the RPS0 gene into an appropriate expression vector

• Transfection or transduction of mammalian host cells

• Cultivation of the cells under optimized conditions

• Cell lysis and initial clarification of the lysate

• Affinity chromatography based on the tag used (tag type is determined during the manufacturing process )

• Additional purification steps as needed

• Quality control, including purity assessment by SDS-PAGE (>85% purity standard )

How can researchers verify the authenticity and functionality of RPS0?

Verification of recombinant RPS0 authenticity and functionality can be approached through several methods:

• Sequence Verification: Comparing the amino acid sequence with the reference sequence (UniProt Q6FJX4) .

• Purity Assessment: SDS-PAGE analysis should confirm >85% purity .

• Molecular Weight Confirmation: Mass spectrometry to verify the expected molecular weight.

• Functional Assays: While not explicitly described in the search results, potential functional assays could include:

  • RNA binding assays

  • Ribosome assembly in vitro

  • Translation efficiency assays in reconstituted systems

• Immunological Detection: Using specific antibodies against RPS0 or against the tag present in the recombinant protein.

How can RPS0 be used to study host-pathogen interactions?

RPS0 can serve as a valuable tool for studying host-pathogen interactions in several ways:

• Infection Models: Studies have utilized THP-1 macrophage infection models with C. glabrata to analyze temporal transcriptional responses . RPS0 expression patterns or the protein itself could be monitored during such infections to understand ribosomal adaptation during phagocytosis.

• Biofilm Studies: C. glabrata biofilm formation is an important virulence factor, and transcriptional responses during biofilm formation under different environmental conditions have been analyzed . RPS0-specific antibodies or tagged versions of the protein could be used to track its localization and expression during biofilm development.

• Antifungal Response: The role of RPS0 in antifungal resistance mechanisms can be studied by analyzing its expression and potential modifications in response to drugs like fluconazole, particularly in the context of different carbon sources and pH environments .

• Species Identification during Infection: The species-specific nature of RPS0 gene sequences makes it useful for identifying C. glabrata in clinical samples or during mixed-species infections .

What is the relationship between RPS0 and mitochondrial function in antifungal resistance?

Research has shown that loss of mitochondrial function is associated with fluconazole resistance in C. glabrata, independent of growth conditions . While direct evidence linking RPS0 to mitochondrial function is not explicitly provided in the search results, there are potential connections worth investigating:

• The cochaperone Mge1, involved in iron metabolism and protein import into mitochondria, has been identified as a key regulator of fluconazole susceptibility during carbon and pH adaptation .

• Mitochondrial function affects the metabolic flux towards toxic sterol formation, which influences antifungal susceptibility .

• Ribosomal proteins like RPS0 may interact with mitochondrial processes through:

  • Coordination of cytosolic and mitochondrial translation

  • Response to metabolic shifts caused by mitochondrial dysfunction

  • Potential extra-ribosomal functions that might influence mitochondrial processes

Future research could explore whether RPS0 expression or modifications are altered in mitochondrial mutants or under conditions that affect mitochondrial function, potentially revealing new insights into antifungal resistance mechanisms.

How might RPS0 contribute to adaptation to different host microenvironments?

C. glabrata encounters various microenvironments within the host, characterized by different pH levels, carbon sources, and stress conditions. Research indicates that these environments directly influence C. glabrata physiology and its response to antifungal treatment . RPS0 may contribute to these adaptations in several ways:

• Transcriptional Regulation: RPS0 expression might be modulated in response to environmental cues, potentially through mechanisms similar to those observed during heat shock responses .

• pH Adaptation: Acidic pH niches, particularly those associated with acetic acid, modulate gene expression in C. glabrata . RPS0 might contribute to translational adaptations under these conditions.

• Carbon Source Utilization: Fluconazole treatment induces gene expression reprogramming in a carbon source-dependent manner . RPS0 could play a role in this reprogramming by affecting translation of specific mRNAs.

• Biofilm Formation: As C. glabrata transitions to biofilm growth, which enhances antifungal resistance, ribosomal components including RPS0 likely undergo regulatory changes to support this adaptive process .

What PCR protocols are effective for RPS0-based identification of Candida species?

Based on the research by Noumi et al. (2010), specific PCR protocols have been developed for RPS0-based identification of Candida species. The following methodology has proven effective:

• Primer Design: Two pairs of primers targeting the RPS0 gene intron fragment:

  • For C. glabrata: Primers generating a 406 bp amplicon

  • For C. parapsilosis: Primers generating a 150 bp amplicon

• PCR Conditions: While specific cycling parameters are not detailed in the search results, standard PCR protocols for fungal DNA would typically include:

  • Initial denaturation (94-95°C for 3-5 minutes)

  • 30-35 cycles of:

    • Denaturation (94-95°C for 30 seconds)

    • Annealing (temperature optimized for primers, typically 55-60°C for 30 seconds)

    • Extension (72°C for 30-60 seconds)

  • Final extension (72°C for 5-10 minutes)

• Validation: The designed primers were highly specific, amplifying only from their respective species and failing to amplify DNA from other Candida species such as C. albicans .

This PCR-based approach has demonstrated the ability to differentiate between closely related species, even identifying C. orthopsilosis when mistakenly classified as C. parapsilosis .

How can researchers study RPS0 expression changes during stress responses?

To study RPS0 expression changes during stress responses, researchers can adapt methodologies used in related studies of C. glabrata:

• Heat Shock Studies: Following protocols similar to those described by Roetzer et al. (2011), researchers could:

  • Grow C. glabrata cultures at control temperature (22°C)

  • Split cultures and expose them to heat shock temperatures (37°C or 42°C)

  • Harvest cells at specific time points (5, 15, 30, 45, and 60 min after treatment)

  • Quench cells in liquid methanol at -40°C

  • Extract RNA for gene expression analysis

• Antifungal Stress: Based on methodologies from Wächtler et al. (2020):

  • Culture C. glabrata cells to standardized optical density

  • Expose cells to fluconazole or other antifungals

  • Compare gene expression under different carbon source conditions and pH levels

  • Focus on temporal changes in expression

• Macrophage Infection Model: Following approaches like Rai et al. (2021):

  • Differentiate THP-1 monocytes using PMA to form macrophages

  • Infect macrophages with C. glabrata (MOI 5:1)

  • Harvest samples at different time points (0.5, 2, 4 hours post-infection)

  • Extract RNA for transcriptional analysis

• Analysis Methods:

  • Quantitative RT-PCR for targeted analysis of RPS0 expression

  • RNA-seq for genome-wide transcriptional profiling

  • Western blotting to analyze protein levels and potential modifications

What controls should be included in experiments using recombinant RPS0?

For experiments utilizing recombinant RPS0, the following controls should be considered:

• Negative Controls:

  • Buffer-only controls to account for background in binding or activity assays

  • Irrelevant proteins of similar size/structure to test specificity

  • Samples without RPS0 antibodies in immunological experiments

• Positive Controls:

  • Known RPS0-interacting partners in binding studies

  • Confirmed RPS0 substrates in functional assays

  • Commercially available standards for quantification

• Validation Controls:

  • Comparison with native RPS0 purified from C. glabrata (when feasible)

  • Denatured RPS0 to confirm activity is dependent on proper protein folding

  • Tagged versus untagged versions to ensure tag doesn't interfere with function

• Experimental Controls:

  • Different storage conditions to assess stability

  • Varying concentrations to establish dose-dependency

  • Multiple timepoints to determine kinetics of interactions or activities

What are the major challenges in studying RPS0 function in Candida glabrata?

Researchers face several challenges when studying RPS0 function in C. glabrata:

• Essential Nature: As a ribosomal protein, RPS0 is likely essential for viability, making knockout studies difficult. Conditional expression systems or partial knockdowns may be necessary.

• Redundancy: Gene duplication events in Candida species have affected ribosomal protein genes , potentially creating functional redundancy that complicates single-gene studies.

• Extra-ribosomal Functions: RPS0 may have functions beyond its role in ribosomes, which can be difficult to distinguish from its primary role in translation.

• Host-Pathogen Context: Studying RPS0 in the context of host-pathogen interactions requires complex infection models that may not fully recapitulate in vivo conditions.

• Post-translational Modifications: Potential modifications that affect RPS0 function during stress or infection may be technically challenging to identify and characterize.

How might RPS0 research contribute to novel antifungal development?

RPS0 research could contribute to novel antifungal development through several avenues:

• Target Validation: If RPS0 has unique features in Candida compared to human ribosomal proteins, it could potentially serve as a drug target. Research into its structure and function would be essential for this validation.

• Resistance Mechanisms: Understanding how RPS0 expression or function changes during antifungal exposure could reveal resistance mechanisms and potential combination therapies to overcome them .

• Biofilm Targeting: Since biofilms contribute significantly to antifungal resistance, and RPS0 may play a role in biofilm formation or maintenance, targeting these processes could lead to novel anti-biofilm strategies .

• Diagnostic Applications: The species-specific nature of RPS0 gene sequences makes it valuable for rapid identification of Candida species, potentially leading to faster and more targeted antifungal therapy .

• Mitochondrial Connections: Exploring the relationship between RPS0 and mitochondrial function could reveal new targets related to the loss of mitochondrial function associated with fluconazole resistance .

What emerging technologies might enhance RPS0 research?

Several emerging technologies could significantly advance RPS0 research:

• CRISPR-Cas9 Systems: Adapted for fungi, these could enable precise genetic manipulation of RPS0, including conditional knockouts, point mutations, or tagged versions for localization studies.

• Ribosome Profiling: This technique could reveal how RPS0 contributes to translation regulation during stress responses or infection, identifying specifically affected mRNAs.

• Cryo-EM: Structural studies of C. glabrata ribosomes under different conditions could reveal conformational changes in RPS0 and its interactions with other components.

• Single-Cell Technologies: Single-cell RNA-seq or proteomics could identify heterogeneity in RPS0 expression or function within Candida populations during infection or biofilm formation.

• Interactome Analysis: Techniques like BioID or proximity labeling could identify protein interaction networks involving RPS0 during normal growth versus stress conditions.

• Microfluidics: Advanced microfluidic systems could enable real-time monitoring of RPS0 dynamics during host-pathogen interactions or under rapidly changing environmental conditions.