Recombinant Candida glabrata Enolase-phosphatase E1 (UTR4)

What is the genomic context of Enolase-phosphatase E1 (UTR4) in Candida glabrata?

Enolase-phosphatase E1 (UTR4) in Candida glabrata is encoded by a specific open reading frame in the C. glabrata genome. Similar to other C. glabrata genes, expression of UTR4 can be analyzed using quantitative real-time PCR (RT-PCR) with specific primers designed against the gene sequence. When designing primers, researchers should ensure specificity by comparing with other sequences in the C. glabrata genome to avoid cross-reactivity. For normalization purposes, housekeeping genes like CgACT1 (actin) are commonly used as internal reference standards .

Which expression systems are most efficient for producing recombinant C. glabrata UTR4?

For recombinant expression of C. glabrata proteins, several expression systems can be employed based on research needs. Homologous expression within modified C. glabrata strains offers advantages for proper protein folding and post-translational modifications. This can be achieved using inducible promoters such as the copper-inducible MTI promoter, which has been successfully used for controlled expression of C. glabrata genes . Alternatively, heterologous expression in Saccharomyces cerevisiae often provides good yields while maintaining proper eukaryotic processing. For higher protein yields, bacterial systems like E. coli can be used, though they may lack proper post-translational modifications that could be essential for UTR4 activity.

How do growth conditions affect the expression levels of native UTR4 in C. glabrata?

The expression of many C. glabrata genes is strongly influenced by environmental conditions encountered during host infection. Similar to CgDTR1, which shows differential expression under stress conditions, UTR4 expression likely responds to specific environmental cues. Researchers should examine UTR4 expression under various conditions including different carbon sources, pH variations, oxidative stress (e.g., hydrogen peroxide exposure), and during internalization within host cells. RT-PCR can be used to quantify these expression changes by extracting RNA from C. glabrata cells grown under these various conditions or when internalized within host cells .

What are the most effective methods for generating UTR4 knockout strains in C. glabrata?

CRISPR-Cas9 technology has revolutionized genome engineering in C. glabrata. To create UTR4 knockout strains, researchers should consider the following approach: First, design guide RNAs targeting the UTR4 gene using specialized algorithms to maximize efficiency and minimize off-target effects. Transform a recombinant C. glabrata strain constitutively expressing the CRISPR-Cas9 system with these guide RNAs . The efficiency of homologous recombination can be significantly increased (up to 8-fold higher) when using 200 bp of homology domains compared to shorter 20 bp domains . After transformation, mutants can be identified using the Surveyor technique followed by sequencing confirmation. This approach provides a robust method for generating clean deletions without marker genes remaining in the genome.

How can site-directed mutagenesis be optimized for studying UTR4 functional domains?

For site-directed mutagenesis of UTR4, first identify conserved residues through multiple sequence alignment with homologous proteins from related species. Design mutagenic primers containing the desired nucleotide changes with approximately 15-20 nucleotides of perfect matching sequence on either side of the mutation. Use a high-fidelity polymerase for PCR amplification to introduce mutations. After DpnI digestion to remove template DNA, transform the amplified product into E. coli. Screen colonies by sequencing and then introduce the validated mutant constructs into expression vectors with appropriate promoters, such as the copper-inducible MTI promoter system, which allows for controlled expression in C. glabrata .

What vectors are recommended for complementation studies with UTR4 in C. glabrata?

For complementation studies, plasmid vectors that enable controlled expression are essential. The pGREG576 plasmid, modified to contain the copper-inducible MTI promoter rather than the GAL1 promoter, has proven effective for controlled gene expression in C. glabrata . This system allows for the verification of gene function through complementation of knockout phenotypes. When designing the complementation construct, include the full UTR4 coding sequence verified by sequencing. For more precise expression control, consider using the native UTR4 promoter region in place of the inducible promoter to maintain physiological expression patterns in response to environmental signals.

How does UTR4 expression affect C. glabrata virulence in infection models?

To assess the impact of UTR4 on virulence, researchers should compare wild-type C. glabrata strains with UTR4 deletion mutants in appropriate infection models. The Galleria mellonella larval model provides a useful system for initial virulence assessments, as demonstrated for other C. glabrata genes . When conducting such experiments, standardize the inoculum to approximately 5 × 10^7 CFU/larvae and monitor survival over 72 hours using Kaplan-Meier survival curves to document differences in killing ability. Complementary studies should evaluate the ability of UTR4 mutants to proliferate within the host by recovering hemolymph at different time points (e.g., 1, 24, and 48 hours post-infection) and quantifying viable C. glabrata cells .

What is the relationship between UTR4 and stress resistance in C. glabrata?

To investigate UTR4's role in stress resistance, compare growth of wild-type and UTR4 deletion strains under various stress conditions relevant to host environments. These should include oxidative stress (hydrogen peroxide), acidic stress (varied pH), antifungal compounds, and nutritional limitation. Perform both spot dilution assays on solid media and growth curve analyses in liquid culture to quantify differences in stress tolerance. Additionally, examine gene expression changes of UTR4 under these stress conditions using RT-PCR with primers specific for UTR4 and normalizing with housekeeping genes like CgACT1 . This approach will help determine whether UTR4 is part of the stress response machinery that contributes to C. glabrata survival within host environments.

How does UTR4 function affect C. glabrata survival within phagocytic cells?

To assess UTR4's role in phagocyte survival, co-culture wild-type and UTR4 mutant C. glabrata strains with either isolated hemocytes from G. mellonella or mammalian macrophages . Measure the concentration of viable C. glabrata cells within phagocytes after various time points (1, 4, 24, and 48 hours) to determine if UTR4 affects intracellular survival and proliferation. Complement these studies with microscopy techniques to visualize phagosome maturation and potential escape mechanisms. Additionally, measure the expression levels of UTR4 during internalization within phagocytic cells using RT-PCR to determine if the gene is up- or down-regulated during this critical phase of host-pathogen interaction .

How can protein-protein interaction networks involving UTR4 be mapped in C. glabrata?

For mapping UTR4 interaction networks, several complementary approaches should be employed. Create a tagged version of UTR4 (e.g., with HA, FLAG, or His tags) and express it under its native promoter to maintain physiological expression levels. Perform co-immunoprecipitation followed by mass spectrometry to identify interacting proteins. Validate key interactions using techniques such as yeast two-hybrid or bimolecular fluorescence complementation. Analyze the resulting interaction network to identify biological processes where UTR4 participates. For more dynamic assessment, compare interaction networks under different environmental conditions that mimic the host environment, such as oxidative stress or acidic pH, which have been shown to affect the function of other C. glabrata virulence factors .

What are the most revealing phenotypic assays for characterizing UTR4 function in C. glabrata?

To comprehensively characterize UTR4 function, implement a multi-tiered phenotypic analysis approach. Begin with growth assays under various conditions (carbon sources, pH, temperature, stress agents) comparing wild-type and UTR4 mutant strains. Examine biofilm formation capacity using crystal violet staining and confocal microscopy. Assess adhesion to epithelial cells and abiotic surfaces. Evaluate metabolic profiles using techniques like Biolog phenotype microarrays to identify specific metabolic pathways affected by UTR4 deletion. For virulence assessment, use both the G. mellonella infection model and cell culture systems with phagocytes to measure survival rates as performed for other virulence factors . Create a comprehensive phenotypic profile table comparing wild-type, deletion mutant, and complemented strains under all tested conditions.

How can structural biology approaches enhance understanding of UTR4 function?

To gain structural insights into UTR4 function, purify recombinant UTR4 to homogeneity using affinity chromatography followed by size exclusion chromatography. Assess protein folding and stability using circular dichroism spectroscopy. For crystallization trials, screen multiple conditions varying pH, precipitants, and additives. If crystallization proves challenging, consider nuclear magnetic resonance (NMR) for solution structure determination or cryo-electron microscopy for larger complexes. In parallel, perform computational modeling using homology-based approaches with known structures of related enolase-phosphatases. Identify conserved catalytic residues through sequence alignment and verify their importance through site-directed mutagenesis followed by enzymatic activity assays. Structural information can guide the design of specific inhibitors that might serve as potential antifungal agents.

How does C. glabrata UTR4 compare to orthologues in other Candida species?

To compare UTR4 across Candida species, perform comprehensive sequence analyses using multiple sequence alignment tools to identify conserved domains and species-specific variations. Calculate evolutionary distances and construct phylogenetic trees to understand the evolutionary relationship of UTR4 among different species. Complement sequence analysis with functional comparisons by expressing orthologues from different species in a C. glabrata UTR4 deletion background to assess functional complementation. Pay particular attention to differences between pathogenic and non-pathogenic species to identify adaptations that might contribute to virulence. Present the sequence conservation data in a table format showing percent identity and similarity across key Candida species, along with highlighting functionally important residues that are either conserved or divergent.

*Note: These are estimated ranges based on typical conservation patterns between these species; exact values should be determined experimentally.

How has the CRISPR-Cas9 system revolutionized functional studies of genes like UTR4 in C. glabrata?

The CRISPR-Cas9 system has transformed genetic manipulation in C. glabrata by significantly increasing the efficiency of gene editing. Prior to CRISPR, gene deletion in C. glabrata required long homology arms and had low efficiency. Now, researchers can achieve efficient gene disruption with just 20-200 bp of homology domains, with homologous recombination efficiency increasing up to 8-fold when using 200 bp homology domains compared to 20 bp . For UTR4 studies, CRISPR-Cas9 allows precise gene deletion, site-directed mutagenesis of specific domains, and seamless gene tagging for localization and interaction studies. Additionally, the system enables multiplexed gene editing to study genetic interactions between UTR4 and other genes. When designing CRISPR experiments, researchers should use optimized guide RNA selection tools specific for C. glabrata to maximize editing efficiency and minimize off-target effects .

What are common challenges in purifying recombinant UTR4 and how can they be overcome?

Recombinant UTR4 purification faces several challenges that require methodical approaches to resolve. Solubility issues can be addressed by optimizing expression conditions (temperature, induction timing, media composition) or by using solubility-enhancing tags such as MBP or SUMO. If protein aggregation persists, consider refolding from inclusion bodies using a gradual dialysis approach. Proteolytic degradation can be minimized by including protease inhibitors throughout the purification process and working at reduced temperatures (4°C). For optimal yield, design a multi-step purification strategy starting with affinity chromatography (His-tag or GST-tag based), followed by ion-exchange chromatography and size-exclusion chromatography. Verify protein purity using SDS-PAGE and confirm identity with Western blotting and mass spectrometry. Assess protein activity using enzyme kinetics assays specific to enolase-phosphatase function, comparing parameters such as Km and Vmax with those of related enzymes.

How can inconsistent phenotypes between different UTR4 mutant strains be reconciled?

When facing inconsistent phenotypes between different UTR4 mutant strains, a systematic troubleshooting approach is essential. First, confirm the genetic modifications by re-sequencing the UTR4 locus in all strains to ensure the expected alterations are present. Check for potential second-site mutations by whole-genome sequencing or backcrossing when possible. Verify that the expression levels of neighboring genes remain unaffected by the UTR4 deletion using RT-PCR. Standardize experimental conditions meticulously, as minor variations in media composition, pH, temperature, or cell density can significantly impact phenotypic outcomes. Create isogenic strains where the only difference is the UTR4 modification by performing genetic manipulations in the same parental background. For virulence studies, ensure consistent inoculum preparation and standardize infection protocols, as variation in these parameters can strongly influence virulence outcomes .