Recombinant Candida glabrata Serine hydroxymethyltransferase, cytosolic (SHM2)

What is Serine Hydroxymethyltransferase (SHM2) and what is its function in Candida glabrata?

Serine hydroxymethyltransferase (SHM2) is a cytosolic enzyme involved in one-carbon metabolism that catalyzes the reversible conversion of serine to glycine with the transfer of a one-carbon unit to tetrahydrofolate. In Candida glabrata, SHM2 plays a crucial role in amino acid biosynthesis and nucleotide metabolism, which are essential for fungal growth and survival. The enzyme has been identified as differentially expressed in azole-resistant strains of C. glabrata, suggesting a potential role in antifungal resistance mechanisms . C. glabrata is known to rapidly acquire resistance to multiple drug classes, including triazoles and echinocandins, making the study of proteins potentially involved in resistance mechanisms particularly important . The cytosolic form (SHM2) is distinct from the mitochondrial isoform and likely participates in cytoplasmic one-carbon transfer reactions critical for purine and pyrimidine synthesis.

How does SHM2 differ between Candida glabrata and other Candida species?

The SHM2 protein in C. glabrata shows structural and functional conservation with homologs in other fungal species but possesses unique sequence characteristics that reflect the evolutionary divergence of C. glabrata. While maintaining the core catalytic domain typical of serine hydroxymethyltransferases, the C. glabrata enzyme exhibits specific amino acid substitutions that may influence substrate binding or catalytic efficiency. Genomic analysis of C. glabrata isolates reveals that unlike C. albicans, C. glabrata is more closely related to Saccharomyces cerevisiae and shares more metabolic similarities with this non-pathogenic yeast . These evolutionary differences extend to enzymes like SHM2, which may have adapted to support C. glabrata's unique niche as a human commensal and opportunistic pathogen. Understanding these differences is crucial for developing species-specific therapeutic approaches, as C. glabrata accounts for approximately a quarter of all systemic candidiasis cases with mortality rates of 30-60% .

What methods are most effective for expressing recombinant C. glabrata SHM2?

The optimal expression system for recombinant C. glabrata SHM2 typically involves eukaryotic hosts such as Pichia pastoris or Saccharomyces cerevisiae to ensure proper folding and post-translational modifications. For higher yields, a codon-optimized synthetic gene can be cloned into a vector containing a strong inducible promoter (such as GAL1 or AOX1) and an appropriate fusion tag (His-tag or GST-tag) to facilitate purification. Expression conditions should be optimized with respect to temperature (typically 25-30°C), inducer concentration, and duration to minimize protein aggregation and maximize soluble protein yield. Purification via affinity chromatography followed by size exclusion chromatography yields the highest purity protein suitable for enzymatic and structural studies. For functional studies, it's essential to verify that the recombinant protein retains native enzymatic activity through appropriate biochemical assays measuring the conversion of serine to glycine and tetrahydrofolate to 5,10-methylenetetrahydrofolate.

How does SHM2 contribute to antifungal resistance in Candida glabrata?

Serine hydroxymethyltransferase (SHM2) in C. glabrata may contribute to antifungal resistance through multiple mechanisms related to metabolic adaptation and stress response pathways. Research suggests that SHM2 is differentially expressed in azole-resistant strains, indicating a potential role in the metabolic reconfiguration that accompanies drug resistance development . The enzyme's function in one-carbon metabolism connects it to nucleotide synthesis and amino acid metabolism, which may provide metabolic flexibility during drug-induced stress. C. glabrata is known to rapidly develop resistance to multiple antifungal classes, with clinical isolates frequently exhibiting mutations in mismatch repair genes like MSH2 that promote a mutator phenotype . This genetic instability could affect the expression or structure of metabolic enzymes like SHM2, potentially altering their contribution to cellular processes involved in drug resistance. Additionally, as biofilm formation is a significant virulence factor in C. glabrata and contributes to antifungal resistance, the potential role of SHM2 in providing metabolic support for biofilm development warrants investigation, especially given the transcriptomic remodeling observed during biofilm formation .

What structural features of C. glabrata SHM2 could be targeted for selective antifungal development?

The structural analysis of C. glabrata SHM2 reveals several features that could be exploited for selective antifungal development, focusing on differences between fungal and human homologs. The enzyme's active site contains specific residues involved in pyridoxal phosphate (PLP) cofactor binding and substrate recognition that differ between fungal and human enzymes, potentially allowing for selective inhibition. Molecular docking studies, similar to those employed for vaccine epitope identification in other C. glabrata proteins, could be utilized to identify compounds with high binding affinity to unique pockets in the fungal enzyme . Crystal structure determination of C. glabrata SHM2 would facilitate structure-based drug design approaches, enabling the identification of allosteric sites specific to the fungal enzyme. Additionally, since C. glabrata exhibits high genetic variability between strains , analysis of SHM2 sequence conservation across clinical isolates would be crucial to ensure broad-spectrum activity of any potential inhibitors, particularly considering the prevalence of mutator phenotypes in clinical isolates that could rapidly develop resistance .

How can SHM2 enzyme kinetics be accurately measured in the context of drug inhibition studies?

Accurate measurement of SHM2 enzyme kinetics for drug inhibition studies requires a multi-faceted approach combining spectrophotometric, radiometric, and coupled enzyme assays. For initial screening, a spectrophotometric assay monitoring the absorption changes associated with the conversion of 5,10-methylenetetrahydrofolate to tetrahydrofolate at 340 nm provides a convenient continuous readout of enzyme activity. More sensitive analyses can employ radiometric assays using 14C-labeled serine to track the formation of labeled glycine and 5,10-methylenetetrahydrofolate. For inhibition studies, Michaelis-Menten kinetics should be determined in the presence of varying inhibitor concentrations to establish inhibition constants (Ki) and distinguish between competitive, non-competitive, or uncompetitive inhibition mechanisms. These assays should be performed under physiologically relevant conditions, including pH and temperature optimization reflecting the fungal cytosolic environment. To increase clinical relevance, enzyme kinetics should also be measured in cell extracts from both antifungal-susceptible and resistant C. glabrata strains to account for potential strain-specific variations in enzyme properties .

What is the relationship between SHM2 expression and biofilm formation in C. glabrata?

The relationship between SHM2 expression and biofilm formation in C. glabrata represents an important area of investigation given the critical role of biofilms in fungal persistence and drug resistance. Transcriptomic analyses have revealed significant metabolic remodeling during biofilm formation in C. glabrata, with differential expression of genes involved in amino acid metabolism . As SHM2 functions in serine-glycine conversion and one-carbon metabolism, its activity could provide essential precursors for nucleotide synthesis and amino acid metabolism required during the high-growth phases of biofilm development. Research has shown that biofilm formation in C. glabrata is regulated by transcription factors such as Tec1, which controls roughly one-third of differentially expressed genes in biofilm cells, including those related to carbon and nitrogen metabolism . Experimental approaches to investigate this relationship should include quantitative RT-PCR analysis of SHM2 expression at different stages of biofilm formation, phenotypic characterization of SHM2 deletion mutants for biofilm defects, and metabolomic profiling to identify changes in serine/glycine and one-carbon metabolites during biofilm development compared to planktonic growth.

How should researchers design experiments to evaluate the impact of SHM2 mutations on drug resistance?

To evaluate the impact of SHM2 mutations on drug resistance in C. glabrata, researchers should implement a comprehensive experimental design that combines genetic manipulation, phenotypic characterization, and biochemical analysis. The experimental pipeline should begin with the identification of naturally occurring SHM2 variants in clinical isolates with different antifungal susceptibility profiles, similar to the approach used for MSH2 polymorphism analysis in clinical strains . Following variant identification, CRISPR-Cas9 gene editing should be employed to introduce these mutations into a well-characterized laboratory strain background to control for other genetic variables. Phenotypic characterization should include determination of minimum inhibitory concentrations (MICs) for multiple antifungal classes (azoles, echinocandins, polyenes) and time-kill kinetics to assess the impact on both static and cidal drug effects. Additionally, researchers should conduct biofilm formation assays using both crystal violet staining and metabolic activity measurements (like PrestoBlue) under different media conditions, as exemplified in previous biofilm studies . Biochemical analysis should include purification of recombinant wild-type and mutant SHM2 proteins for enzyme kinetics studies to determine if mutations alter catalytic efficiency, substrate binding, or susceptibility to inhibition.

What are the best approaches to study the interaction between SHM2 and other metabolic enzymes in C. glabrata?

The most effective approaches to study the interaction between SHM2 and other metabolic enzymes in C. glabrata involve a combination of proteomic, genetic, and systems biology techniques. For direct protein-protein interactions, researchers should employ co-immunoprecipitation with SHM2-specific antibodies followed by mass spectrometry to identify interacting partners in both drug-susceptible and resistant strains. Proximity-based labeling methods such as BioID or APEX can identify proteins in close spatial proximity to SHM2 in living cells, providing insights into its subcellular localization and potential protein complexes. Genetic interaction mapping through synthetic genetic array analysis, using an SHM2 deletion strain crossed with a genome-wide deletion library, would reveal functional relationships between SHM2 and other genes. Metabolic flux analysis using 13C-labeled metabolites can track the flow of carbon through pathways connected to SHM2 activity, identifying metabolic dependencies. These experimental approaches should be conducted under both normal growth conditions and during antifungal stress to identify stress-specific interactions, similar to the stress conditions used in studies of biofilm formation . Integration of these multidimensional datasets through systems biology approaches would provide a comprehensive understanding of SHM2's role within the complex metabolic network of C. glabrata.

How can researchers develop a high-throughput screening assay for SHM2 inhibitors?

Development of a high-throughput screening (HTS) assay for SHM2 inhibitors requires careful optimization of enzyme activity detection in a format suitable for automated screening platforms. A primary screening assay should utilize a fluorescence-based detection method, such as measuring the NADPH production in a coupled reaction system where the one-carbon unit transferred from serine is ultimately tracked through a redox reaction. This approach allows for sensitive detection in a 384-well format with minimal reagent consumption. The assay should be optimized for Z' factor >0.7 to ensure statistical reliability and include appropriate positive controls (known inhibitors of related enzymes) and negative controls. Counter-screening assays must be implemented to eliminate compounds that interfere with the detection method or inhibit coupled enzymes rather than SHM2 directly. Secondary validation assays should include orthogonal methods like thermal shift assays to confirm direct binding to SHM2 and isothermal titration calorimetry to determine binding kinetics. To ensure selectivity, parallel screening against human SHMT should be conducted, with a focus on identifying compounds with at least 10-fold selectivity for the fungal enzyme. Promising hits should be evaluated for antifungal activity against both planktonic cells and biofilms of C. glabrata, with special attention to activity against drug-resistant clinical isolates that exhibit the mutator phenotype .

How should researchers interpret contradictory results between in vitro SHM2 activity and in vivo antifungal susceptibility?

When faced with contradictory results between in vitro SHM2 enzyme activity and in vivo antifungal susceptibility in C. glabrata, researchers must consider multiple factors that could explain these discrepancies. First, the complex cellular environment in vivo introduces compensatory mechanisms and metabolic redundancies that may mask the effects of SHM2 inhibition observed in purified enzyme systems. The high genetic variability observed in clinical C. glabrata isolates, particularly those with mutator phenotypes due to MSH2 defects, can result in strain-specific responses to enzyme inhibition that differ from laboratory strains . Additionally, the formation of biofilms significantly alters gene expression patterns and metabolic requirements, potentially changing the reliance on SHM2 activity in comparison to planktonic cultures used for in vitro studies . To address these contradictions, researchers should perform comprehensive analyses including: (1) transcriptomic and proteomic profiling to identify compensatory pathways activated in vivo; (2) metabolomic analysis to track changes in metabolite pools related to one-carbon metabolism; (3) genetic studies using SHM2 mutants with varying levels of activity to establish dose-response relationships between enzyme activity and phenotypic outcomes; and (4) time-course studies to capture dynamic responses that might be missed in endpoint assays.

What statistical approaches are most appropriate for analyzing SHM2 sequence variations across clinical isolates?

The analysis of SHM2 sequence variations across clinical isolates of C. glabrata requires robust statistical approaches that account for population structure, selection pressure, and functional impacts of mutations. For comprehensive analysis, researchers should employ a combination of methods starting with population genetics statistics such as nucleotide diversity (π), Tajima's D, and FST to quantify genetic variation and detect potential selection signatures, similar to approaches used in population genetics studies of C. glabrata . Phylogenetic analyses should be conducted to understand the evolutionary relationships between SHM2 variants and their correlation with geographic origin, patient demographics, and antifungal exposure history. Association studies between specific SHM2 polymorphisms and phenotypic traits (like MIC values) should utilize mixed-effects models that account for population structure and potential confounding variables. For functional prediction of non-synonymous mutations, combined scoring systems incorporating SIFT, PolyPhen, and protein stability predictions provide more reliable assessments than any single algorithm. When analyzing rare variants, collapsing methods that group mutations by functional domains can increase statistical power. Researchers should be particularly attentive to strains exhibiting the mutator phenotype due to MSH2 defects, as these strains may have accelerated rates of SHM2 mutation that could confound standard population genetic analyses .

How can researchers distinguish between SHM2-mediated effects and other mechanisms in drug resistance studies?

Distinguishing between SHM2-mediated effects and other mechanisms in C. glabrata drug resistance studies requires a multi-faceted experimental approach that isolates the specific contribution of SHM2. Researchers should first develop isogenic strain sets with SHM2 deletions, wild-type complementation, and overexpression constructs in both drug-susceptible and resistant genetic backgrounds to establish a clear cause-effect relationship. Transcriptomic and proteomic analyses comparing these isogenic strains under drug pressure can identify SHM2-dependent changes in gene expression and protein levels, separating them from SHM2-independent resistance mechanisms. Metabolomic profiling focused on serine, glycine, and one-carbon metabolism intermediates can reveal metabolic signatures specifically associated with SHM2 activity alterations. Genetic epistasis experiments combining SHM2 modifications with known resistance mechanisms (such as efflux pump overexpression or drug target mutations) can determine whether SHM2 functions independently or synergistically with these pathways. To account for the high genetic variability observed in clinical isolates, especially those with mutator phenotypes due to mismatch repair defects , researchers should validate findings across multiple strain backgrounds. Additionally, time-resolved studies during resistance development can determine whether SHM2 changes are early adaptive responses or later compensatory mechanisms, providing insight into the causal relationship between SHM2 and resistance.

How might SHM2 inhibitors be combined with existing antifungals to prevent resistance development?

Strategically combining SHM2 inhibitors with existing antifungals offers a promising approach to prevent resistance development in C. glabrata infections. The rationale for such combinations stems from targeting multiple cellular pathways simultaneously, increasing the genetic barrier to resistance development, particularly important in C. glabrata where a mutator phenotype is prevalent in clinical isolates due to MSH2 defects . Potential combination strategies include sequential therapy, where the SHM2 inhibitor is administered first to metabolically weaken the fungal cells before introducing the primary antifungal, or simultaneous administration at optimized dose ratios determined through checkerboard assays and time-kill studies. Research should focus on combinations with echinocandins (the current first-line therapy for invasive candidiasis) and azoles, evaluating both pharmacodynamic interactions and resistance prevention properties. In vitro evolution experiments exposing C. glabrata to drug combinations versus monotherapy under controlled conditions can quantify the resistance prevention benefit. For clinical development, researchers should establish pharmacokinetic/pharmacodynamic (PK/PD) parameters for optimal dosing regimens, including concentration targets at infection sites and dosing intervals that maintain effective drug levels. Animal models of persistent infection, similar to those used to study breakthrough infections in mutator strains , would be valuable for validating the resistance prevention benefits before advancing to human trials.

What considerations are important when developing SHM2-based diagnostic tools for Candida glabrata infections?

Developing SHM2-based diagnostic tools for C. glabrata infections requires careful consideration of multiple factors to ensure clinical utility. First, the sensitivity and specificity of SHM2 as a biomarker must be established through comprehensive analysis of SHM2 conservation across diverse clinical isolates, particularly given the high genetic variability observed in C. glabrata populations . The diagnostic platform should ideally distinguish between C. glabrata and other Candida species, as treatment approaches differ significantly between species. Both nucleic acid and protein-based detection methods should be evaluated, with PCR-based approaches offering high sensitivity for low fungal burdens, while immunoassays targeting SHM2 protein could provide more rapid results without amplification steps. Epitope mapping studies, similar to those conducted for other C. glabrata proteins , would identify regions of SHM2 most suitable for antibody development, prioritizing epitopes that are both highly immunogenic and specific to C. glabrata. For clinical implementation, sample processing methods must be optimized for different specimen types (blood, tissue, urine) to efficiently lyse fungal cells and release target molecules while removing potential inhibitors. Point-of-care applications would require further simplification and stabilization of reagents, potentially utilizing aptamer-based detection or isothermal amplification methods instead of traditional PCR or ELISA approaches.

Could SHM2 be a potential target for vaccine development against C. glabrata infections?

SHM2 presents an intriguing potential target for vaccine development against C. glabrata infections, though several critical factors must be evaluated to determine its suitability. As an intracellular enzyme, SHM2 would not be readily accessible to antibodies in intact cells, suggesting that a successful vaccine would need to elicit strong T-cell responses capable of recognizing infected cells presenting SHM2-derived peptides on MHC molecules. Immunoinformatics approaches similar to those employed for fructose bisphosphate aldolase epitope prediction could identify the most promising B-cell and T-cell epitopes from SHM2 sequence. These epitope predictions should consider population coverage of MHC alleles to ensure broad efficacy across genetically diverse patient populations. Conservation analysis of SHM2 across clinical isolates is essential to ensure that antigenic targets remain conserved despite the high genetic variability observed in C. glabrata . Candidate epitopes should be evaluated for cross-reactivity with human homologs to prevent autoimmune complications. Animal models of C. glabrata colonization and systemic infection would be necessary to evaluate vaccine efficacy, measuring both reduction in fungal burden and prevention of dissemination. Moreover, researchers should investigate whether SHM2-based vaccines could protect against strains with different antifungal susceptibility profiles, including those with the mutator phenotype that readily acquire drug resistance .