Recombinant Candida glabrata tRNA (guanine (9)-N1)-methyltransferase (TRM10)

What is the primary function of TRM10 in Candida glabrata?

TRM10 in C. glabrata functions as a tRNA methyltransferase that catalyzes N-1 methylation of purine residues (primarily guanine) found at position 9 of specific tRNAs. This modification, known as m1G9, is present in multiple tRNA species throughout Eukarya and Archaea . Based on comparative studies with the Saccharomyces cerevisiae homolog, TRM10 likely plays a critical role in ensuring proper tRNA stability and function, which subsequently affects cellular processes dependent on accurate protein translation .

While the exact substrate specificity of C. glabrata TRM10 has not been fully characterized, research on the S. cerevisiae homolog suggests it may modify multiple tRNA substrates but have particularly important biological roles for specific tRNAs, as seen with tRNA^Trp in S. cerevisiae .

How does TRM10 substrate specificity differ between in vivo and in vitro conditions?

This difference suggests that cellular factors not present in purified in vitro systems may regulate TRM10 activity and target selection within the cell. Researchers should be cautious when extrapolating in vitro findings to in vivo scenarios, as the cellular environment likely contains cofactors, inhibitors, or structural elements that influence TRM10's substrate recognition and activity . The specific mechanisms governing this differential specificity remain unelucidated and represent an important area for further research.

What is the significance of the m1G9 modification for tRNA function?

The m1G9 modification appears to have tRNA-specific functional importance. In S. cerevisiae, while TRM10 performs m1G9 modification on 13 different tRNAs, this modification is particularly critical for the biological function of tRNA^Trp .

When TRM10 is deleted (trm10Δ strain), mature tRNA^Trp levels are significantly depleted, while other TRM10 substrates (such as tRNA^Gly) remain largely unaffected. This suggests that the m1G9 modification may be essential for the stability of specific tRNAs but not others, highlighting the complex and substrate-specific nature of tRNA modifications .

The absence of m1G9 modification on tRNA^Trp leads to growth hypersensitivity in the presence of 5-fluorouracil (5FU), which can be rescued specifically by overexpression of tRNA^Trp. This provides strong evidence that the modification plays a biologically important role in maintaining functional levels of particular tRNAs under stress conditions .

How does genetic diversity in C. glabrata TRM10 impact virulence and drug resistance?

The genetic diversity within the C. glabrata population, including variations in genes like TRM10, may significantly impact virulence and drug resistance profiles. Population genetics studies of clinical C. glabrata isolates have identified two major mechanisms generating genetic diversity: microevolution and genetic exchange/recombination .

These variations can affect the function of important cellular components, including tRNA modification enzymes like TRM10. Given that proper tRNA processing and modification are critical for cellular protein synthesis and stress responses, variations in TRM10 function could potentially influence C. glabrata virulence traits and responses to antifungal drugs .

Comparative genomic analysis of clinical C. glabrata isolates has revealed evidence of recombinant sequence types (STs), which could lead to functional differences in genes encoding tRNA modification enzymes. These genetic differences may contribute to the observed variability in virulence and drug resistance profiles among clinical isolates .

What is the relationship between TRM10 activity and antifungal drug resistance mechanisms?

The relationship between TRM10 activity and antifungal resistance is an emerging area of research. Studies in S. cerevisiae have demonstrated that TRM10 deletion (trm10Δ) leads to hypersensitivity to 5-fluorouracil (5FU), an antitumor drug . This suggests that TRM10-mediated tRNA modifications may play a role in cellular responses to certain drugs.

In the clinical context, C. glabrata exhibits marked genetic diversity that impacts drug resistance profiles. For example, studies have documented instances of fluconazole minimum inhibitory concentration changes correlating with genetic alterations . While direct evidence linking TRM10 variations to antifungal resistance in C. glabrata is limited, the enzyme's role in tRNA stability and cellular stress responses suggests it could influence the pathogen's ability to withstand antifungal treatments.

Researchers investigating this relationship should consider examining:

• Correlation between TRM10 sequence variations and minimum inhibitory concentrations (MICs) for common antifungals

• Changes in TRM10 expression levels during exposure to antifungal agents

• The effects of TRM10 deletion or overexpression on antifungal susceptibility profiles

• Potential interactions between TRM10 and known drug resistance mechanisms, such as efflux pumps or drug target alterations

What are the mechanisms of tRNA quality control related to TRM10 activity?

The absence of m1G9 modification catalyzed by TRM10 triggers tRNA quality control mechanisms. In S. cerevisiae, precursor tRNA^Trp species accumulate in trm10Δ strains through at least two distinct mechanisms .

One quality control pathway involves Met22, a component of the methionine biosynthesis pathway that also plays a role in tRNA surveillance. Deletion of MET22 in the trm10Δ background (trm10Δmet22Δ) rescues the levels of mature tRNA^Trp. This is consistent with Met22's known role in tRNA quality control, where its deletion causes inhibition of 5'-3' exonucleases involved in tRNA decay .

Interestingly, none of the known Met22-associated exonucleases appear to be responsible for the decay of hypomodified tRNA^Trp, suggesting the existence of a distinct tRNA quality control pathway specific to tRNA^Trp in S. cerevisiae . This represents an important area for future research to identify the specific nucleases involved in surveilling and degrading hypomodified tRNA^Trp.

What are the optimal conditions for expressing recombinant C. glabrata TRM10?

For successful expression of recombinant C. glabrata TRM10, researchers should consider a heterologous expression system with appropriate promoters. Based on proven methodologies for similar C. glabrata proteins, the following approach is recommended:

• Vector Selection: Utilize a plasmid with a copper-inducible promoter such as the MTI promoter, which has been successfully used for the expression of C. glabrata genes . The pGREG576 vector system can be adapted by replacing the GAL1 promoter with the MTI promoter.

• Promoter Design: The MTI promoter should include approximately 1,000 bp of the promoter region to ensure proper regulation. Primers should be designed with appropriate homology to both the promoter region and the cloning site of the expression vector .

• Expression Conditions: When using the copper-inducible MTI promoter system, expression should be induced with CuSO₄ at concentrations that don't inhibit yeast growth (typically 0.05-0.5 mM) .

• Host Selection: While S. cerevisiae BY4741 has been used successfully for the expression of C. glabrata proteins, expression in C. glabrata itself (such as strain L5U1) may provide more native conditions for proper folding and activity .

• Verification Methods: Confirm successful expression through DNA sequencing of the recombinant plasmid and Western blot analysis of the expressed protein using appropriate antibodies against TRM10 or an added epitope tag.

How can researchers accurately assess TRM10 enzymatic activity in vitro?

To accurately assess TRM10 enzymatic activity in vitro, researchers should implement the following methodological approach:

• Substrate Preparation: Generate purified tRNA substrates, either through in vitro transcription or isolation from appropriate strains. Include known TRM10 substrates (such as tRNA^Trp) as positive controls and non-substrate tRNAs as negative controls .

• Reaction Conditions: Based on studies with related methyltransferases, establish optimal buffer conditions (typically containing Tris-HCl pH 7.5-8.0, MgCl₂, DTT, and sometimes spermidine), temperature (usually 30-37°C), and incubation times (30-60 minutes) .

• Methyl Donor: Include S-adenosylmethionine (SAM) as the methyl donor. Consider using radiolabeled [³H]-SAM or [¹⁴C]-SAM to enable sensitive detection of methylation activity .

• Activity Detection Methods:

  • Radiolabeled methyl group incorporation measured by scintillation counting

  • HPLC analysis of nucleosides after complete digestion of the tRNA

  • Mass spectrometry to detect the mass shift associated with methylation

  • Primer extension assays, which can detect m1G9 modification due to its ability to cause reverse transcriptase stops

• Controls: Include the following essential controls:

  • Enzyme-minus reaction to assess background methylation

  • Heat-inactivated enzyme control

  • Known methyltransferase with different specificity to verify assay functionality

  • Comparative analysis with S. cerevisiae Trm10 as a reference standard

• Substrate Specificity Analysis: Test multiple tRNA substrates to assess the in vitro substrate specificity range, which may differ from in vivo specificity .

What techniques are recommended for analyzing the effects of TRM10 deletion in C. glabrata?

Analysis of TRM10 deletion effects in C. glabrata requires a comprehensive approach combining phenotypic, molecular, and functional assays:

• Generation of Deletion Mutants:

  • Use homologous recombination-based gene replacement techniques with appropriate selection markers

  • Confirm deletion through PCR verification and sequencing of the modified locus

  • Create complemented strains by reintroducing TRM10 under native or controllable promoters to verify phenotype specificity

• Growth and Stress Response Assays:

  • Evaluate growth rates under standard conditions (YEPD, RPMI 1640, BM minimal media)

  • Assess sensitivity to specific stressors including antifungal drugs (particularly 5-fluorouracil), oxidative stress agents (H₂O₂), and pH stress

  • Use growth curve analysis and spot dilution assays to quantify differences in growth capabilities

• tRNA Analysis Techniques:

  • Northern blot analysis to quantify levels of mature and precursor tRNAs

  • High-resolution gel electrophoresis to separate tRNA species

  • Primer extension or reverse transcription stops to detect the presence/absence of m1G9 modification

  • Mass spectrometry to directly assess tRNA modification status

• Virulence Assessment:

  • In vitro macrophage interaction assays to evaluate phagocytosis resistance and intracellular survival

  • Galleria mellonella infection model, which has been validated for C. glabrata virulence studies

  • Monitoring of fungal burden in infection models through CFU counting from recovered tissues/hemolymph

• Data Analysis Table for TRM10 Deletion Effects:

• Molecular Mechanism Investigation:

  • RNA-seq to examine global effects on transcription

  • Ribosome profiling to assess translation efficiency changes

  • Proteomics to identify differentially expressed proteins

  • tRNA microarrays to assess global changes in tRNA abundance and charging status

These methods will provide comprehensive insights into the biological role of TRM10 in C. glabrata, particularly its impacts on tRNA stability, stress responses, and virulence mechanisms.

What role does TRM10-mediated tRNA modification play in stress adaptation and virulence?

TRM10-mediated tRNA modification appears to play crucial roles in stress adaptation and potentially virulence through several mechanisms:

• Stress Response: In S. cerevisiae, TRM10 deletion leads to hypersensitivity to 5-fluorouracil, indicating its importance in responding to certain stress conditions . The m1G9 modification may be particularly important for maintaining functional tRNA levels under stress.

• tRNA Stability: The absence of TRM10-mediated modification leads to depletion of mature tRNA^Trp, suggesting that m1G9 is essential for stability of specific tRNAs . This stability is likely crucial during stress conditions that challenge protein synthesis capabilities.

• Potential Virulence Connection: The genetic diversity observed in clinical C. glabrata isolates, which may include variations in TRM10, has been linked to differences in virulence and drug resistance . While direct evidence for TRM10's role in C. glabrata virulence is limited, insights can be drawn from other virulence factors.

• Comparative Context: Other C. glabrata factors, such as the multidrug transporter CgDtr1, have demonstrated roles in pathogenesis and stress response. CgDtr1 expression increases C. glabrata virulence against G. mellonella, enhances proliferation in hemolymph, and provides protection against stressors encountered during phagocytosis . By analogy, TRM10's role in tRNA modification might similarly contribute to adaptation to host environments.

• Quality Control Mechanisms: The interaction between TRM10 and tRNA quality control pathways suggests that proper tRNA modification is monitored by cellular surveillance systems . These systems may be particularly important during stress conditions encountered during host infection.

How can evolutionary analysis of TRM10 inform therapeutic targeting strategies?

Evolutionary analysis of TRM10 can provide valuable insights for potential therapeutic targeting strategies:

• Conservation Analysis: By examining the conservation of TRM10 across fungal species and comparing it with mammalian homologs, researchers can identify fungal-specific regions or residues that might serve as selective therapeutic targets .

• Structural Evolution: Understanding the evolutionary changes in TRM10 structure can reveal substrate binding pockets or catalytic residues that differ between fungal and human enzymes, allowing for the design of selective inhibitors.

• Functional Divergence: The differential importance of TRM10 for specific tRNAs (e.g., tRNA^Trp in S. cerevisiae) suggests functional specialization that might be exploited therapeutically . If C. glabrata TRM10 has similar substrate-specific critical functions, these could represent vulnerability points.

• Resistance Prediction: Evolutionary analysis across clinical isolates can help predict potential resistance mechanisms that might emerge against TRM10-targeting therapeutics, allowing for preemptive design of combination strategies .

• Sequence Variation Analysis Table:

• Environmental Adaptation: Analysis of TRM10 variants in C. glabrata isolates from different host environments may reveal adaptations to specific stresses or niches, which could inform the development of context-specific therapeutic approaches .

• Epistatic Relationships: Evolutionary analysis can identify genes that have co-evolved with TRM10, potentially revealing synthetic lethal interactions that could be exploited therapeutically through combination approaches targeting TRM10 and its functional partners.

By integrating these evolutionary insights with structural biology and biochemical characterization, researchers can develop targeting strategies that exploit fungal-specific features of TRM10 while minimizing off-target effects on host tRNA modification systems.

What are the key unresolved questions about C. glabrata TRM10 function?

Several critical questions about C. glabrata TRM10 remain unresolved and represent important avenues for future research:

• Substrate Specificity Determinants: The molecular basis for TRM10's substrate selectivity remains poorly understood, particularly the factors that create differences between in vivo and in vitro specificity . Identifying the specific structural features or cofactors that regulate this specificity would provide important insights into tRNA recognition mechanisms.

• tRNA Quality Control Pathways: The specific nucleases responsible for degrading hypomodified tRNAs in TRM10-deficient cells have not been fully identified . The discovery that known Met22-associated exonucleases do not appear responsible for hypomodified tRNA^Trp decay suggests novel quality control pathways that require further characterization.

• Functional Significance in C. glabrata: While studies in S. cerevisiae have identified tRNA^Trp as particularly dependent on TRM10-mediated modification, the critical tRNA substrates in C. glabrata have not been definitively identified . Determining which tRNAs are most affected by TRM10 deficiency in C. glabrata would provide insights into its role in this pathogen.

• Regulatory Mechanisms: The regulation of TRM10 expression and activity during different growth phases, stress conditions, and host environments remains largely unexplored. Understanding these regulatory mechanisms could reveal how C. glabrata modulates tRNA modification in response to environmental challenges.

• Contribution to Pathogenesis: The potential role of TRM10 in C. glabrata virulence, stress resistance, and host interaction requires direct experimental investigation. Comparative studies with other virulence factors like CgDtr1 could illuminate common themes in adaptation to host environments .

What new experimental approaches might advance understanding of TRM10 biology?

Innovative experimental approaches could significantly advance our understanding of TRM10 biology:

• CRISPR-Based Approaches:

  • CRISPR interference (CRISPRi) for tunable repression of TRM10 expression

  • CRISPR activation (CRISPRa) to upregulate TRM10 in specific conditions

  • Base editing to introduce specific mutations in TRM10 without complete gene disruption

• Advanced RNA Analysis:

  • Nanopore direct RNA sequencing for comprehensive detection of tRNA modifications

  • CLIP-seq (crosslinking immunoprecipitation-sequencing) to map TRM10-tRNA interactions in vivo

  • tRNA-seq with specialized library preparation to capture all tRNA species and their modification status

• Structural Biology:

  • Cryo-EM structures of TRM10 bound to substrate tRNAs

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon substrate binding

  • Molecular dynamics simulations to understand substrate recognition mechanisms

• In Vivo Imaging:

  • Live-cell imaging of fluorescently tagged TRM10 to track subcellular localization during infection

  • FRET-based sensors to monitor TRM10 activity in real-time

  • Correlative light and electron microscopy to visualize TRM10 in the context of cellular ultrastructure

• Systems Biology:

  • Multi-omics integration combining transcriptomics, proteomics, and tRNA modification analysis

  • Network analysis to identify functional interactions between TRM10 and other cellular components

  • Computational modeling of tRNA modification dynamics during stress and host interaction

• Host-Pathogen Models:

  • Advanced 3D tissue culture systems to study TRM10's role during host cell interaction

  • Humanized mouse models for studying C. glabrata infection dynamics

  • Ex vivo human tissue infection models to examine TRM10 function in clinical-like settings

How might TRM10 research translate into novel antifungal strategies?

Research on TRM10 has significant potential to inform novel antifungal strategies through several translational pathways:

• Direct Inhibition Strategies:

  • Development of small molecule inhibitors targeting fungal-specific features of TRM10

  • Peptide-based inhibitors designed to interfere with TRM10-tRNA interactions

  • Antisense oligonucleotides targeting TRM10 mRNA to reduce expression

• Combination Approaches:

  • Identification of synthetic lethal interactions between TRM10 and other pathways

  • Sensitization strategies using TRM10 inhibitors to enhance efficacy of existing antifungals

  • Dual-targeting of TRM10 and tRNA quality control pathways to maximize impact on tRNA function

• Stress Enhancement:

  • Compounds that create cellular conditions where TRM10 function becomes essential

  • Targeting pathways that become critical in TRM10-deficient cells

  • Exploiting the hypersensitivity of TRM10-deficient cells to specific stressors like 5FU

• Repurposing Opportunities:

  • Identifying existing drugs that may interact with TRM10 pathways

  • Screening libraries of approved compounds for activity against TRM10

  • Developing novel formulations of existing agents to enhance activity against TRM10-dependent processes

• Diagnostic Applications:

  • TRM10 variant profiling as a biomarker for predicting drug resistance

  • Detection of TRM10 activity as an indicator of metabolically active fungi

  • Monitoring TRM10-dependent tRNA modifications as a measure of treatment efficacy

• Vaccine Development:

  • Exploring TRM10 as a potential antigen for anti-Candida vaccines

  • Using TRM10-deficient strains as potential attenuated vaccine candidates

  • Identifying TRM10-dependent epitopes that might be targeted by the immune system

The translation of TRM10 research into clinical applications will require interdisciplinary collaboration between structural biologists, medicinal chemists, microbiologists, and clinicians to develop and validate these approaches while addressing challenges related to specificity, delivery, and resistance management.