Recombinant Candida glabrata Cytochrome c lysine N-methyltransferase 1 (CTM1), partial

What is Cytochrome c lysine N-methyltransferase 1 (CTM1) and what is its function in Candida species?

CTM1 encodes a lysine methyltransferase that facilitates post-translational modification of cytochrome c. Based on studies in related Candida species, CTM1 specifically trimethylates cytochrome c (Cyc1) at the K79 residue, modifying its functionality in cellular processes. This methylation appears critical for regulating morphogenesis in pathogenic Candida species, as demonstrated in C. albicans where inactivating CTM1 blocks hyphal growth, a key virulence trait . The methylation state of cytochrome c influences multiple cellular pathways, including signaling cascades that control growth mode transitions. Unlike some other post-translational modifications, this methylation appears to specifically modulate protein-protein interactions rather than directly affecting catalytic activity .

How conserved is CTM1 across different Candida species?

While comprehensive comparative genomics of CTM1 across all Candida species remains incomplete, available data suggests functional conservation with species-specific adaptations. In C. albicans, CTM1-mediated lysine methylation of cytochrome c regulates hyphal morphogenesis through cAMP-PKA signaling . C. glabrata, despite being more closely related phylogenetically to Saccharomyces cerevisiae than to C. albicans, likely maintains CTM1 function with adaptations reflecting its unique virulence mechanisms. Population genetic studies of C. glabrata have revealed considerable genetic diversity across clinical isolates, with variation in virulence genes and drug targets between sequence types (STs) . This suggests that CTM1 may exhibit strain-specific variations that could affect its activity or regulation, potentially contributing to differences in pathogenicity profiles among clinical isolates.

What are the structural domains of C. glabrata CTM1 and how do they compare to other methyltransferases?

C. glabrata CTM1, like other SET domain-containing lysine methyltransferases, likely possesses a conserved catalytic domain responsible for S-adenosylmethionine (SAM) binding and methyl transfer activity. The enzyme's structure would typically include:

The structural organization allows for specific recognition of cytochrome c and precise methylation at K79. In C. albicans, the specific interaction between CTM1 and cytochrome c enables trimethylation that alters protein-protein interactions relevant to cellular signaling pathways . C. glabrata's CTM1 likely shares core structural elements while potentially exhibiting unique features that reflect its adaptation to different host environments and stress responses compared to other Candida species .

What are the recommended approaches for generating CTM1 knockout mutants in C. glabrata?

For generating CTM1 knockout mutants in C. glabrata, researchers should employ targeted gene disruption strategies using homologous recombination. A recommended protocol involves:

• Design of disruption cassettes containing selectable markers (e.g., NAT1 for nourseothricin resistance) flanked by homologous regions (500-1000 bp) of the CTM1 target locus

• Transformation of C. glabrata cells using lithium acetate/PEG method with optimization for C. glabrata's thick cell wall

• Selection of transformants on appropriate media and PCR verification of successful integration

• Southern blot analysis to confirm single integration at the intended locus

For phenotypic characterization, comparative analyses between wild-type, knockout, and complemented strains should be performed under various conditions to assess:

• Growth kinetics in standard media and under stress conditions

• Virulence factors expression

• Intracellular replication capacity in macrophage infection models

• Biofilm formation capacity

When analyzing CTM1 knockout phenotypes, researchers should be aware that, unlike in C. albicans where hyphal growth is a primary readout , C. glabrata does not form true hyphae, necessitating alternative phenotypic assessments focused on stress responses and virulence traits.

What expression systems are most effective for producing recombinant C. glabrata CTM1?

For optimal expression of recombinant C. glabrata CTM1, researchers should consider multiple expression systems with appropriate optimization:

For E. coli expression, fusion tags such as His6, GST, or MBP can enhance solubility and facilitate purification. When designing expression constructs, researchers should consider:

• Including the full-length CTM1 sequence or focusing on the catalytic domain

• Optimizing temperature (typically lowering to 16-20°C) during induction to improve solubility

• Supplementing growth media with S-adenosylmethionine precursors

• Using lysis buffers containing glycerol and reducing agents to preserve enzymatic activity

Protein functionality should be verified through methyltransferase activity assays using recombinant cytochrome c as substrate and detection of methylation by mass spectrometry or using methyl-lysine specific antibodies, similar to approaches used for C. albicans CTM1 .

How can researchers quantitatively assess CTM1 methyltransferase activity in vitro?

Quantitative assessment of CTM1 methyltransferase activity requires robust in vitro assays that measure the enzyme's capacity to transfer methyl groups to cytochrome c. Recommended methodological approaches include:

• Radiometric assay: Using S-[methyl-³H]-adenosylmethionine as methyl donor, followed by scintillation counting to detect transferred radioactivity to purified cytochrome c

• Coupled enzymatic assay: Monitoring S-adenosylhomocysteine (SAH) production through coupled reactions with SAH hydrolase and adenosine deaminase

• Mass spectrometry-based approaches: Using LC-MS/MS to detect and quantify methylated lysine residues on cytochrome c, with particular focus on K79 trimethylation

A standard reaction buffer typically contains:

• 50 mM Tris-HCl (pH 8.0)

• 5 mM MgCl₂

• 1 mM DTT

• 0.01% Triton X-100

• 50 μM S-adenosylmethionine

• Purified recombinant CTM1 (10-100 nM)

• Cytochrome c substrate (1-5 μM)

Kinetic parameters (Km, kcat) should be determined under varying substrate and enzyme concentrations. Site-directed mutagenesis of potential catalytic residues in CTM1 can provide valuable insights into its mechanism, while mutation of the K79 residue in cytochrome c to alanine (K79A) serves as a negative control, similar to approaches used in C. albicans studies .

How does CTM1 function differ between C. glabrata and C. albicans?

The functional divergence of CTM1 between C. glabrata and C. albicans reflects their distinct evolutionary trajectories and pathogenic strategies. In C. albicans, CTM1-mediated trimethylation of cytochrome c at K79 plays a crucial role in regulating hyphal morphogenesis, a well-recognized virulence trait . When CTM1 is inactivated or cytochrome c carries a K79A mutation in C. albicans, cells remain in yeast form even under hyphae-inducing conditions, demonstrating that unmethylated cytochrome c suppresses hyphal growth .

In contrast, C. glabrata does not form true hyphae and employs different virulence mechanisms. C. glabrata exhibits:

• Greater genetic diversity among clinical isolates

• Distinct stress response mechanisms, as evidenced by its unique UPR pathway that operates independently of HAC1 mRNA splicing, unlike S. cerevisiae and C. albicans

• Different intracellular survival strategies within macrophages

These differences suggest that while CTM1 may retain its basic methyltransferase function in C. glabrata, its role in pathogenicity likely diverges from the morphogenesis-focused function observed in C. albicans. Instead, CTM1 in C. glabrata may be more involved in stress responses, metabolism regulation, or other aspects of pathogen-host interaction that don't involve hyphal transition but remain critical for virulence.

What role does cytochrome c methylation play in stress responses across different Candida species?

Cytochrome c methylation appears to function as a regulatory mechanism in stress response pathways across Candida species, though with species-specific adaptations. In C. albicans, methylated cytochrome c influences cyclic AMP (cAMP)-protein kinase A (PKA) signaling, with unmethylated Cyc1 (in ctm1Δ/Δ or cyc1 K79A mutants) inhibiting PKA kinase activity . This signaling pathway is critical for cellular responses to various stressors and for virulence traits.

C. glabrata possesses unique stress response mechanisms compared to other Candida species, particularly in the unfolded protein response (UPR). Unlike S. cerevisiae and C. albicans, C. glabrata's Ire1 functions independently of HAC1 mRNA splicing . This fundamental difference in stress response architecture suggests that:

• Cytochrome c methylation may interface with C. glabrata's unique UPR pathway

• CTM1 might play roles in oxidative stress responses, particularly relevant during phagocytosis

• Methylation status could influence C. glabrata's exceptional echinocandin tolerance, especially in petite variants

The connection between methylation status and stress responses is particularly relevant considering C. glabrata's capacity to persist within macrophages and exhibit varied intracellular replication rates depending on genetic background . Further research is needed to elucidate how CTM1-mediated methylation integrates with these species-specific stress response mechanisms.

How can researchers conduct meaningful cross-species complementation studies with CTM1?

Cross-species complementation studies with CTM1 provide valuable insights into functional conservation and divergence among Candida species. A systematic approach should include:

Experimental Design Framework:

• Generate CTM1 deletion mutants in both species (e.g., C. glabrata and C. albicans)

• Create expression constructs containing:

  • Native CTM1 from the same species (positive control)

  • CTM1 from the complementary species under native or constitutive promoters

  • Chimeric CTM1 proteins with swapped domains to identify functional regions

• Transform constructs into respective deletion mutants and assess phenotypic restoration

Analytical Approaches:

When interpreting results, researchers should consider that functional complementation may be incomplete due to species-specific interaction partners and regulatory networks. Similar to observations with Ire1 and HAC1 complementation between S. cerevisiae and C. glabrata , CTM1 function may be influenced by species-specific substrate recognition or downstream effectors. Sequencing the methylated regions of cytochrome c from both species after complementation provides molecular evidence of functional conservation or divergence of the methyltransferase activity.

How does CTM1 activity affect C. glabrata virulence in animal models?

The impact of CTM1 activity on C. glabrata virulence in animal models is likely complex and multifaceted. Based on related research in Candida species, researchers should consider several key aspects when designing animal studies:

• Infection Model Selection:

  • Systemic infection models: Assess fungal burden in kidneys, liver, and spleen

  • Colonization models: Evaluate gastrointestinal persistence

  • Biofilm-associated infection models: Examine catheter-associated infections

• Virulence Parameters to Monitor:

  • Survival rates of infected animals

  • Fungal burden in target organs

  • Inflammatory responses (cytokine profiles)

  • Histopathological evidence of tissue damage

Unlike C. albicans, where CTM1 deletion affects hyphal morphogenesis but unexpectedly maintains virulence due to accelerated proliferation , C. glabrata CTM1 mutants might show different phenotypes. In C. glabrata, virulence is more dependent on factors like adherence, biofilm formation , stress resistance, and intracellular survival within macrophages .

Researchers should specifically examine whether CTM1 deletion affects C. glabrata's exceptional capability to persist within macrophages, as this represents a key virulence mechanism. Interestingly, C. glabrata petite variants (with mitochondrial defects) show distinct macrophage interaction patterns and pronounced type-I interferon responses , suggesting potential connections between mitochondrial function (where cytochrome c operates) and immune evasion that might involve CTM1-mediated regulation.

What is the relationship between CTM1 activity and antifungal drug resistance in C. glabrata?

The potential relationship between CTM1 activity and antifungal drug resistance in C. glabrata represents an important research direction, especially given C. glabrata's intrinsic and acquired resistance to multiple antifungals. Several mechanistic connections should be investigated:

• Echinocandin Tolerance: Experimental evidence indicates that C. glabrata petite variants (with mitochondrial defects) exhibit increased tolerance to echinocandins . Since cytochrome c is a mitochondrial protein subject to CTM1-mediated methylation, researchers should investigate whether altered CTM1 activity affects echinocandin susceptibility through changes in:

  • Cell wall composition and β-glucan exposure

  • Stress response activation during drug exposure

  • Metabolic adaptations that promote survival

• Azole Resistance: C. glabrata frequently develops resistance to azole antifungals. The potential role of CTM1 could involve:

  • Influence on membrane composition through metabolic effects

  • Alterations in efflux pump expression

  • Adaptations in ergosterol biosynthesis pathways

• Biofilm-Associated Resistance: Since biofilms contribute significantly to C. glabrata's drug resistance , researchers should examine whether CTM1 affects biofilm formation and structure.

Experimental approaches should include:

Particular attention should be paid to gene expression changes in CTM1 mutants during drug exposure, potentially revealing regulatory connections between methylation status and resistance mechanism activation.

How does CTM1 function influence C. glabrata interaction with host immune cells?

The influence of CTM1 function on C. glabrata interactions with host immune cells, particularly macrophages, represents a critical aspect of virulence. Research approaches should focus on several key aspects:

• Phagocytosis and Intracellular Survival:
  C. glabrata exhibits remarkable ability to survive within macrophages. Research indicates that certain variants, like petites, show different phagocytosis rates and intracellular replication patterns . Experiments should assess whether CTM1 deletion affects:

  • Rate of phagocytosis by macrophages

  • Intracellular replication capacity

  • Phagolysosome maturation and acidification

  • Macrophage survival during infection

• Immunomodulatory Effects:
  Host immune responses to C. glabrata infection show distinctive patterns, with specific variants eliciting different cytokine profiles. For example, petite-infected macrophages exhibit pronounced type-I interferon and pro-inflammatory cytokine responses at later infection stages compared to non-petite strains . Researchers should examine whether CTM1 activity influences:

  • Cytokine production profiles (TNF-α, IL-1β, IL-6, IFN-β)

  • Pattern recognition receptor activation

  • Inflammasome assembly and activation

  • Neutrophil recruitment and activation

• Methodological Approaches:

  • Ex vivo infection models using THP-1 macrophages, primary human macrophages, or murine bone marrow-derived macrophages

  • Flow cytometry to assess phagocytosis rates and macrophage activation markers

  • Confocal microscopy to visualize intracellular trafficking

  • RT-qPCR and ELISA to measure cytokine expression and secretion

  • RNAseq to comprehensively profile host transcriptional responses

Researchers should specifically compare wild-type, CTM1 deletion mutants, and complemented strains in these assays to establish direct links between CTM1 function and immune interaction phenotypes. Additionally, creating cytochrome c K79A mutants (analogous to those studied in C. albicans ) would help determine whether the immune interaction phenotypes are specifically related to the methyltransferase activity of CTM1.

What techniques are most effective for analyzing the methylation state of cytochrome c in C. glabrata?

The accurate analysis of cytochrome c methylation states in C. glabrata requires sophisticated analytical techniques that can detect specific post-translational modifications with high precision. The most effective approaches include:

• Mass Spectrometry-Based Methods:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) following tryptic digestion

  • Targeted Selected Reaction Monitoring (SRM) for quantification of specific methylated peptides

  • Top-down proteomics for intact protein analysis to distinguish mono-, di-, and trimethylation states

• Antibody-Based Detection:

  • Western blotting using methyl-lysine specific antibodies

  • Immunoprecipitation followed by mass spectrometry (IP-MS)

  • Enzyme-linked immunosorbent assay (ELISA) for quantitative assessment

• Genetic and Biochemical Approaches:

  • Site-directed mutagenesis of the K79 residue to confirm methylation sites

  • In vitro methyltransferase assays with recombinant CTM1 and cytochrome c

  • Protein-protein interaction studies to assess how methylation affects binding partners

Sample Preparation Protocol:

• Isolate mitochondria from C. glabrata cells using differential centrifugation

• Extract cytochrome c using mild detergents or osmotic shock

• Purify using ion exchange or affinity chromatography

• For MS analysis, perform tryptic digestion followed by enrichment of methylated peptides

• For antibody detection, use validated anti-methyl-lysine antibodies with appropriate controls

When interpreting results, researchers should consider that methylation patterns may vary under different growth conditions or stress exposures. Comparative analysis between wild-type and CTM1 mutant strains provides essential validation of CTM1-dependent methylation events. This approach has successfully identified the specific K79 trimethylation site in C. albicans cytochrome c and can be adapted for C. glabrata studies.

How can researchers identify other potential substrates of CTM1 beyond cytochrome c?

Identifying novel CTM1 substrates beyond cytochrome c requires comprehensive proteomic approaches combined with biochemical validation. A systematic workflow should include:

• Global Methylome Profiling:

  • Quantitative proteomics comparing wild-type and CTM1 deletion strains

  • Stable isotope labeling by amino acids in cell culture (SILAC) for accurate quantification

  • Enrichment of methylated peptides using anti-methyl-lysine antibodies prior to MS analysis

  • Bioinformatic analysis identifying proteins with altered methylation patterns

• Substrate Prediction and Validation:

  • Motif analysis around known methylation sites to generate consensus sequences

  • In silico prediction of proteins containing potential CTM1 recognition motifs

  • In vitro methyltransferase assays with recombinant CTM1 and candidate substrates

  • Site-directed mutagenesis of predicted methylation sites to confirm specificity

• Protein Interaction Studies:

  • Affinity purification-mass spectrometry (AP-MS) using CTM1 as bait

  • Yeast two-hybrid screening to identify physical interactors

  • Biolayer interferometry or isothermal titration calorimetry to measure binding affinities

Decision Matrix for Candidate Validation:

Researchers should prioritize candidates with evidence across multiple categories. When analyzing results, consider that methylation may be context-dependent and influenced by cellular compartmentalization, protein complex formation, or stress conditions. This approach has been successful in identifying novel substrates for other methyltransferases and can be adapted for CTM1 studies in C. glabrata.

What are the challenges in designing inhibitors specific to C. glabrata CTM1?

Designing specific inhibitors of C. glabrata CTM1 presents several unique challenges that researchers must address through integrated structural, biochemical, and computational approaches:

• Structural Considerations:

  • Limited structural information about C. glabrata CTM1

  • Conserved SET domain architecture across many methyltransferases

  • Need to identify unique structural features for selectivity

  • Challenge of targeting the S-adenosylmethionine binding pocket without affecting other methyltransferases

• Specificity Requirements:

  • Selectivity against human methyltransferases to minimize off-target effects

  • Discrimination between CTM1 and other fungal methyltransferases

  • Need to maintain activity against potential variants of CTM1 across clinical isolates, given the genetic diversity observed in C. glabrata populations

• Drug Development Challenges:

  • Designing compounds with appropriate physicochemical properties for cellular penetration

  • Addressing potential efflux through C. glabrata's robust drug export systems

  • Achieving sufficient stability in biological environments

  • Demonstrating efficacy in relevant infection models

Rational Design Strategy:

• Initial Steps:

  • Homology modeling of C. glabrata CTM1 based on related methyltransferase structures

  • Molecular dynamics simulations to identify potential binding pockets

  • Fragment-based screening to identify starting chemical scaffolds

  • Virtual screening of compound libraries against modeled structures

• Optimization Process:

  • Structure-activity relationship studies to improve potency and selectivity

  • Biochemical assays to assess inhibition of methyltransferase activity

  • Cellular assays in C. glabrata to confirm target engagement

  • Counter-screening against human methyltransferases to assess selectivity

• Validation Methods:

  • X-ray crystallography or cryo-EM to confirm binding modes

  • Thermal shift assays to evaluate compound binding

  • Cellular thermal shift assays to assess target engagement in cells

  • Assessment of phenotypic effects matching CTM1 genetic deletion

The unique stress response mechanisms in C. glabrata, particularly its distinct unfolded protein response pathway that operates independently of HAC1 mRNA splicing , suggest that targeting CTM1 might produce species-specific effects on stress adaptation and virulence. This presents both a challenge for inhibitor design and an opportunity for developing narrow-spectrum antifungals with potentially reduced resistance development.

What are the most promising research directions for understanding CTM1 function in C. glabrata pathogenesis?

The study of CTM1 function in C. glabrata pathogenesis offers several high-priority research directions that could significantly advance our understanding of fungal virulence mechanisms. The most promising areas include:

• Comparative Functional Genomics:
  Systematic comparison of CTM1 function across Candida species, particularly examining how C. glabrata CTM1 differs from its C. albicans counterpart in regulation of cellular processes. While C. albicans CTM1 influences hyphal morphogenesis , C. glabrata lacks true hyphal formation, suggesting CTM1 may have evolved different functions aligned with C. glabrata's unique pathogenicity mechanisms. This approach should incorporate RNA-seq analysis of CTM1 deletion mutants under various stress conditions to identify species-specific regulatory networks.

• Host-Pathogen Interaction Dynamics:
  Investigation of how CTM1-mediated cytochrome c methylation affects C. glabrata interactions with host immune cells, particularly focusing on:

  • Survival within macrophages, as C. glabrata shows distinctive intracellular persistence patterns

  • Modulation of host immune responses, including cytokine production profiles

  • Potential effects on mitochondrial function and metabolic adaptation during infection

• Integration with Stress Response Pathways:
  Exploration of how CTM1 functions intersect with C. glabrata's unique stress response mechanisms, particularly its distinctive unfolded protein response that operates independently of HAC1 mRNA splicing . This could reveal novel connections between post-translational modifications and stress adaptation that contribute to C. glabrata's exceptional resilience in diverse host environments.

• Clinical Correlations:
  Analysis of CTM1 sequence variants and expression patterns across diverse clinical isolates from the expanding collection of C. glabrata genomic data . This population-level approach could identify potential correlations between CTM1 variants and virulence traits, antifungal susceptibility, or clinical outcomes.

The intersection of these research directions promises to yield comprehensive insights into how post-translational modifications like CTM1-mediated methylation contribute to C. glabrata's success as an opportunistic pathogen and may reveal novel targets for therapeutic intervention.

How might advances in structural biology techniques facilitate CTM1 research?

Advanced structural biology techniques offer unprecedented opportunities to elucidate CTM1 function at the molecular level, providing insights crucial for understanding its role in C. glabrata pathogenicity:

• Cryo-Electron Microscopy (Cryo-EM):
  Recent advances in cryo-EM resolution now enable detailed structural analysis of proteins previously resistant to crystallization. For CTM1 research, cryo-EM could:

  • Reveal the complete three-dimensional structure of CTM1 alone and in complex with cytochrome c

  • Visualize conformational changes during the catalytic cycle

  • Identify structural determinants of substrate specificity

  • Provide templates for structure-based drug design

• Integrative Structural Biology Approaches:
  Combining multiple techniques creates comprehensive structural models:

  • X-ray crystallography for high-resolution active site details

  • Small-angle X-ray scattering (SAXS) for solution-state conformational dynamics

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map protein-protein interaction surfaces

  • Nuclear magnetic resonance (NMR) for dynamic regions and ligand binding studies

• Computational Methods:
  Advances in computational structural biology enhance experimental approaches:

  • AlphaFold2 and related AI methods for accurate structure prediction

  • Molecular dynamics simulations to study conformational flexibility

  • Enhanced sampling techniques to identify cryptic binding sites

  • Quantum mechanics/molecular mechanics (QM/MM) calculations to understand catalytic mechanisms

These structural insights would address fundamental questions about CTM1 function:

• How does CTM1 achieve specificity for the K79 residue in cytochrome c?

• What structural changes occur in cytochrome c upon methylation?

• How do these structural changes alter interactions with downstream effectors like PKA?

• What features might be exploited for selective inhibitor design?

With these advanced approaches, researchers could overcome current limitations in understanding the molecular basis of CTM1 function and potentially identify novel intervention strategies targeting this key regulatory enzyme in C. glabrata.

What interdisciplinary approaches might yield new insights into CTM1 biology and function?

Interdisciplinary research approaches offer powerful strategies to uncover previously unrecognized aspects of CTM1 biology in C. glabrata. The integration of diverse methodologies across fields provides complementary perspectives that can reveal emergent properties of this regulatory system:

• Systems Biology Integration:
  Combining global analytical techniques to build comprehensive models of CTM1 function:

  • Multi-omics integration (genomics, transcriptomics, proteomics, metabolomics)

  • Network modeling to identify regulatory hubs connected to CTM1 activity

  • Flux balance analysis to quantify metabolic impacts of CTM1 deletion

  • Mathematical modeling of signaling dynamics affected by cytochrome c methylation

• Chemical Biology Approaches:
  Using small molecule probes and chemical genetic strategies:

  • Development of CTM1-specific activity-based probes

  • Click chemistry for in situ labeling of methylation targets

  • Chemogenomic profiling to identify synthetic lethal interactions

  • Small molecule perturbation screens to map CTM1-dependent pathways

• Advanced Imaging Technologies:
  Visualizing CTM1 function in cellular contexts:

  • Super-resolution microscopy to track CTM1 localization during infection

  • Förster resonance energy transfer (FRET) to measure protein-protein interactions

  • Live-cell imaging during host-pathogen interactions

  • Correlative light and electron microscopy to connect molecular events with ultrastructural changes

• Host-Pathogen Interface Studies:
  Examining CTM1 in the context of host interactions:

  • Organoid infection models to recapitulate tissue-specific interactions

  • Single-cell RNA-seq of infected host cells to capture heterogeneous responses

  • Interspecies metabolic modeling to identify critical exchange points

  • CRISPR screening in both pathogen and host to identify interaction networks

These interdisciplinary approaches could reveal unexpected connections between CTM1-mediated methylation and diverse cellular processes, potentially uncovering novel therapeutic targets. The integration of data across scales—from atomic structures to organismal phenotypes—provides a holistic understanding of how this post-translational modification contributes to C. glabrata's success as a pathogen, particularly in its unique ecological niches within the human host.