Recombinant Candida glabrata 40S ribosomal protein S16 (RPS16)

What is Candida glabrata RPS16 and what is its role in ribosome function?

Candida glabrata 40S ribosomal protein S16 (RPS16) is encoded by the gene CAGL0I00792g and functions as a component of the small 40S ribosomal subunit . Like other ribosomal proteins, it contributes to the structural integrity of the ribosome and participates in protein synthesis. Ribosomal proteins work in concert with ribosomal RNA to facilitate translation of mRNA into proteins. RPS16 belongs to the S9P family of ribosomal proteins and is typically located in the cytoplasm . In the context of Candida glabrata, which demonstrates significant genetic diversity across clinical isolates, ribosomal proteins like RPS16 are essential for pathogen survival and reproduction during infection .

How does C. glabrata RPS16 compare structurally with homologs from other species?

C. glabrata RPS16, like other S16 proteins, contains approximately 100 amino acid residues . While specific structural data for C. glabrata RPS16 is limited in the provided materials, ribosomal protein S16 is known to belong to a family divided into several groups based on sequence similarity: Eubacterial S16, Algal and plant chloroplast S16, Cyanelle S16, and Neurospora crassa mitochondrial S24 (cyt-21) . The sequence and structural conservation of ribosomal proteins across species makes them important for evolutionary studies. Given C. glabrata's high genetic diversity (with nucleotide diversity of 0.00665, higher than the distantly related C. albicans at 0.00298), its ribosomal proteins may exhibit unique features compared to other fungal species .

What expression systems are commonly used for recombinant production of C. glabrata RPS16?

Recombinant C. glabrata RPS16 can be expressed in several host systems including E. coli, yeast, baculovirus-infected insect cells, or mammalian cell expression systems . Each expression system offers distinct advantages: E. coli provides high yield and economic efficiency; yeast systems offer appropriate eukaryotic post-translational modifications; baculovirus systems provide high expression levels for eukaryotic proteins; and mammalian cell systems ensure the most authentic eukaryotic processing. The choice of expression system should align with the research objectives, considering factors such as required yield, post-translational modifications, and downstream applications.

How might genetic diversity in C. glabrata populations affect RPS16 function and structure?

C. glabrata demonstrates remarkable genetic diversity across clinical isolates, with multiple sequence types (STs) identified globally . This diversity may extend to ribosomal proteins including RPS16. Analysis of genome sequences from Scottish hospitals and global isolates revealed greater genetic diversity than previously recognized . This diversity could potentially result in sequence variations in RPS16 that might affect ribosome assembly, translation efficiency, or antibiotic sensitivity. Researchers should consider sequencing RPS16 from multiple isolates to identify potential polymorphisms before conducting functional studies. In microevolution studies, genes involved in cell surface proteins showed enrichment for non-synonymous and frameshift indels during patient infections , suggesting selective pressure that could theoretically extend to components of the protein synthesis machinery under certain conditions.

What is the potential role of RPS16 in antifungal resistance mechanisms in C. glabrata?

While the search results don't directly link RPS16 to antifungal resistance, C. glabrata is known for developing resistance to azole antifungals like fluconazole . Since protein synthesis is crucial for cellular adaptation, including stress responses to antifungal agents, ribosomal components like RPS16 could theoretically play indirect roles in resistance mechanisms. Researchers might investigate whether alterations in ribosomal proteins correlate with resistance patterns. The genome analysis of C. glabrata has identified signatures of positive selection in genes involved in drug resistance, such as ERG4 and FKS1/2 , suggesting that components of essential cellular machinery may be under selective pressure during antifungal therapy. Experimental approaches might include comparing RPS16 expression levels or sequence variations between susceptible and resistant isolates.

How does mitochondrial genome diversity in C. glabrata affect translation and potential interactions with RPS16?

C. glabrata exhibits significant mitochondrial genome diversity, with non-reference ST15 isolates showing reduced mitochondrial genome size and fewer conserved protein-encoding genes . While RPS16 is primarily a cytoplasmic ribosomal protein, mitochondrial translation and cytoplasmic translation are coordinated in eukaryotic cells. Research questions could explore potential cross-talk between these systems in C. glabrata, especially considering the unusual mitochondrial diversity observed. Experimental approaches might include studying translational efficiency in isolates with different mitochondrial genome configurations, or investigating potential moonlighting functions of RPS16 outside the ribosome.

What purification strategies optimize yield and activity of recombinant C. glabrata RPS16?

Recombinant C. glabrata RPS16 is typically purified to ≥85% purity as determined by SDS-PAGE . The optimal purification strategy depends on the expression system and the intended application. A robust purification protocol might include:

• Cell lysis under native or denaturing conditions depending on protein solubility

• Initial capture using affinity chromatography (if a tag is incorporated)

• Intermediate purification via ion exchange chromatography

• Polishing step using size exclusion chromatography

For structural studies requiring higher purity, additional steps may be necessary. The choice between native and denaturing conditions should be guided by protein solubility and the requirement for functional studies. For ribosomal proteins that may participate in multiple interactions, preserving native conformation is often crucial for functional assays.

What are the optimal conditions for functional characterization of recombinant RPS16 in ribosome assembly assays?

Studying RPS16 function in ribosome assembly requires careful consideration of experimental conditions. A systematic approach should include:

• In vitro ribosome reconstitution assays using purified components

• RNA-protein binding assays to assess RPS16 interaction with rRNA

• Translation efficiency assays comparing systems with wild-type versus mutant RPS16

The buffer conditions (pH, ionic strength), temperature, and presence of cofactors can significantly impact results. Researchers should consider:

These conditions should be systematically optimized for each specific assay to ensure reproducible results in C. glabrata ribosomal studies.

How can researchers effectively design knockout or mutation studies to investigate RPS16 function in C. glabrata?

Since ribosomal proteins are generally essential, complete knockout of RPS16 may be lethal. Therefore, researchers should consider:

• Conditional expression systems (e.g., tetracycline-regulated promoters)

• Partial depletion using RNA interference approaches

• Site-directed mutagenesis targeting specific functional residues

• Domain swapping with homologs from other species

For C. glabrata specifically, the genetic manipulation system must account for its haploid nature and different transformation efficiency compared to S. cerevisiae. When designing mutation studies, researchers should consider highly conserved regions identified through multiple sequence alignment of RPS16 from related species. The genetic diversity observed across C. glabrata isolates suggests that strain selection is also an important consideration, as background genetic variation might influence phenotypic outcomes of RPS16 manipulation.

How does RPS16 contribute to virulence and pathogenicity in C. glabrata compared to other Candida species?

C. glabrata is the second most common cause of candidiasis after C. albicans , but with distinct virulence mechanisms. While not directly addressed in the search results, ribosomal proteins like RPS16 could theoretically contribute to virulence through their role in stress adaptation and protein synthesis during infection. Comparative genomic studies have revealed that C. glabrata shows greater genetic diversity than C. albicans (nucleotide diversity of 0.00665 versus 0.00298) , which could extend to ribosomal components.

Researchers investigating this question should consider:

• Comparative expression analysis of RPS16 under infection-relevant conditions

• Heterologous expression experiments swapping RPS16 between Candida species

• Ribosome profiling to identify differentially translated mRNAs under stress conditions

The genetic diversity and evidence of positive selection in C. glabrata virulence factors, particularly Epithelial Adhesins (EPA) , suggests that translation efficiency and accuracy could be important during host-pathogen interactions, potentially implicating ribosomal components like RPS16.

What insights can structural comparisons between prokaryotic and eukaryotic S16 provide for antifungal drug development?

Ribosomal proteins are potential targets for antimicrobials due to their essential nature and structural differences between domains of life. For C. glabrata RPS16, structural comparison with bacterial homologs might reveal fungal-specific features that could be exploited for selective targeting. The S16 protein family encompasses several groups including eubacterial, cyanelle, algal/plant chloroplast, and fungal mitochondrial variants . These evolutionary relationships provide context for structure-based drug design approaches.

A systematic approach would include:

• Structural modeling of C. glabrata RPS16 based on available crystal structures

• Identification of fungal-specific surface pockets or interaction interfaces

• In silico screening for molecules that selectively bind fungal versus human RPS16

• Validation of hits through biochemical and cellular assays

This approach could potentially identify new classes of antifungals targeting protein synthesis in this clinically important pathogen, which shows increasing resistance to conventional antifungal agents .