Recombinant Candida glabrata Altered inheritance of mitochondria protein 23, mitochondrial (AIM23)

What is the function of AIM23 in Candida glabrata?

AIM23 in Candida glabrata functions as a mitochondrial translation initiation factor 3 (mIF3), similar to its homolog in Saccharomyces cerevisiae. It plays a critical role in the initiation of mitochondrial protein synthesis by promoting the dissociation of mitochondrial ribosomes and facilitating the binding of initiator tRNA. Research has demonstrated that AIM23 is essential for proper mitochondrial function, particularly for oxidative phosphorylation and cellular respiration .

In yeast species, deletion of AIM23 results in a respiratory deficient phenotype characterized by:

• Decreased cytochrome c oxidase activity (less than 10% of wild-type levels before adaptation)

• Impaired formation of respiratory chain supercomplexes

• Reduced mitochondrial membrane potential

• Growth defects on non-fermentable carbon sources

How does AIM23 deletion affect mitochondrial function in Candida glabrata?

Key observations in Δaim23 strains include:

What are the differences between AIM23 function in C. glabrata and S. cerevisiae?

While AIM23 serves as mitochondrial translation initiation factor 3 (mIF3) in both species, there are notable differences in how C. glabrata and S. cerevisiae respond to its deletion:

Interestingly, the S. pombe mIF3 can effectively complement a genomic disruption of S. cerevisiae AIM23, verifying that Aim23p functions as a bona fide mitochondrial translation initiation factor .

What are the optimal methods for creating AIM23 deletion mutants in C. glabrata?

To create AIM23 deletion mutants in C. glabrata, researchers should follow a targeted gene deletion approach using homologous recombination. Based on established protocols for genetic manipulation of C. glabrata , the following methodology is recommended:

• Primer Design:

  • Design primers that contain 500-1000 bp gene-specific flanking sequences homologous to regions upstream and downstream of AIM23

  • Include sequences for amplification of a selectable marker cassette (e.g., NAT1 for nourseothricin resistance)

• Deletion Cassette Preparation:

  • Amplify the deletion cassette using PCR with the designed primers

  • Verify the cassette size and integrity by gel electrophoresis

• Transformation:

  • Prepare electrocompetent C. glabrata cells at their logarithmic growth phase

  • Transform cells using electroporation (2.1 kV, 200 Ω, 25 μF) or a modified heat shock method (45°C for 15 min)

  • Plate on selective media containing the appropriate antibiotic

• Verification:

  • Confirm successful deletion by PCR using primers binding outside the integration site

  • Verify the absence of AIM23 transcript by RT-PCR

  • Assess mitochondrial function through respiratory growth tests and cytochrome c oxidase activity measurements

How should researchers assess mitochondrial function in AIM23 mutant strains?

When evaluating mitochondrial function in AIM23 mutant strains, a comprehensive approach involving multiple complementary techniques is advised:

• Respiratory Capacity Assessment:

  • Compare growth rates on fermentable (e.g., glucose) versus non-fermentable (e.g., glycerol, ethanol) carbon sources

  • Monitor adaptation to respiratory growth conditions over time (6h, 24h, 48h, 72h)

• Mitochondrial Membrane Potential Analysis:

  • Use fluorescent dyes such as Rhodamine 123 or DiOC6 followed by flow cytometry

  • Implement fluorescence microscopy to visualize membrane potential changes

• Enzyme Activity Measurements:

  • Quantify cytochrome c oxidase activity in isolated mitochondria

  • Measure ATP synthase function

• Mitochondrial Translation Analysis:

  • Perform pulse-labeling of mitochondrially synthesized proteins using [35S]methionine in the presence of cycloheximide

  • Analyze the translation products by SDS-PAGE and autoradiography

• Respiratory Chain Complex Formation:

  • Assess the formation of respiratory chain supercomplexes using Blue Native PAGE

  • Evaluate complex assembly using specific antibodies

What approaches are effective for complementation studies of AIM23 in C. glabrata?

For complementation studies of AIM23 in C. glabrata, researchers should consider the following strategies:

• Plasmid-Based Complementation:

  • Clone the wild-type C. glabrata AIM23 gene with its native promoter and terminator into a suitable shuttle vector

  • Consider using vectors with different selection markers than those used for the deletion

  • Transform the construct into the Δaim23 strain and select on appropriate media

• Cross-Species Complementation:

  • As demonstrated with S. cerevisiae, test complementation with mIF3 genes from related species (e.g., S. pombe mIF3)

  • This approach can provide insights into functional conservation and specificity

• Controlled Expression Systems:

  • Implement regulatable promoters (e.g., MET3, GAL1) to control the expression level

  • This allows assessment of dosage effects on the complementation efficiency

• Domain Swap Experiments:

  • Create chimeric constructs with domains from mIF3 proteins of different species

  • These constructs can help identify critical functional regions

• Verification of Complementation:

  • Assess restoration of cytochrome c oxidase activity

  • Evaluate growth on non-fermentable carbon sources

  • Measure mitochondrial membrane potential

  • Analyze the formation of respiratory chain supercomplexes

What is the relationship between AIM23 function and antifungal drug resistance in C. glabrata?

The relationship between AIM23 function and antifungal resistance is complex and likely involves several mechanisms:

• Azole Resistance:

  • Mitochondrial dysfunction has been associated with azole resistance in C. glabrata

  • AIM23 deletion may alter ergosterol biosynthesis, which could affect susceptibility to azoles

  • Loss of respiratory function can lead to changes in membrane composition affecting drug uptake and efflux

• Echinocandin Susceptibility:

  • Changes in cell wall composition resulting from metabolic adaptations to mitochondrial dysfunction could potentially affect susceptibility to echinocandins

  • Alterations in FKS1/2 expression patterns might occur as compensatory mechanisms

• Metabolic Adaptations:

  • AIM23 mutants likely undergo metabolic reprogramming to compensate for respiratory deficiencies

  • These adaptations may confer cross-resistance to multiple classes of antifungals

  • Energy-dependent drug efflux systems may be affected by the altered ATP production

• Stress Response Activation:

  • Chronic mitochondrial stress in AIM23 mutants could pre-activate stress response pathways

  • This pre-activation might provide cross-protection against antifungal-induced stress

How does mitochondrial heteroplasmy influence AIM23 function in C. glabrata populations?

Mitochondrial heteroplasmy (the presence of multiple mitochondrial genotypes within a cell or population) could significantly impact AIM23 function in C. glabrata:

• Heteroplasmic Dynamics:

  • C. glabrata shows evidence of mitochondrial DNA heteroplasmy after transformation

  • The ratio of wild-type to mutated mitochondrial DNA can shift under different growth conditions

  • These shifts could result in variable phenotypes related to AIM23 function

• Selection Pressures:

  • In aerobic conditions, certain mitochondrial genotypes may be selected over others

  • The functional state of AIM23 may influence which mitochondrial genomes are maintained

• Mitochondrial DNA Diversity:

  • Recent studies have revealed hypervariability in C. glabrata mitochondrial genomes

  • This diversity could lead to strain-specific interactions with AIM23

  • Population genomic analyses have shown reduced conserved sequences in mitochondrial genomes of certain sequence types

• Evolutionary Implications:

  • Mitochondrial diversity combined with AIM23 variations could contribute to the remarkable adaptability of C. glabrata

  • This might explain how clinical isolates can rapidly adapt to different host environments and antifungal pressures

What are the best approaches for studying AIM23-dependent mitochondrial translation in C. glabrata?

To effectively study AIM23-dependent mitochondrial translation in C. glabrata, researchers should employ a multi-faceted approach:

• In organello Translation Assays:

  • Isolate intact mitochondria from wild-type and Δaim23 strains

  • Perform translation reactions with radiolabeled amino acids

  • Analyze translation products by SDS-PAGE and autoradiography

  • Compare patterns and intensities of newly synthesized mitochondrial proteins

• Ribosome Profiling:

  • Apply ribosome profiling techniques specifically to mitochondrial ribosomes

  • Analyze the distribution of ribosomes on mitochondrial mRNAs

  • Identify specific translation initiation sites affected by AIM23 deletion

• Protein-RNA Interaction Studies:

  • Perform RNA immunoprecipitation to identify mRNAs associated with AIM23

  • Use UV crosslinking to map precise interaction sites

  • Electrophoretic mobility shift assays to characterize binding affinities

• Cryo-EM Structural Analysis:

  • Isolate mitochondrial ribosomes from wild-type and Δaim23 strains

  • Determine structural differences using cryo-electron microscopy

  • Identify how AIM23 interacts with the mitochondrial ribosome and initiation factors

• Live-Cell Imaging:

  • Create fluorescently tagged versions of mitochondrial translation components

  • Track translation dynamics in real-time using high-resolution microscopy

  • Compare translation initiation rates between wild-type and mutant strains

How can researchers effectively measure the impact of AIM23 on mitochondrial function during host infection?

To measure the impact of AIM23 on mitochondrial function during host infection, researchers should implement the following methodological approaches:

• In vivo Infection Models:

  • Use mouse models of disseminated candidiasis to compare organ fungal burden

  • Implement Galleria mellonella larval infection models to assess virulence

  • Recover C. glabrata cells from infected tissues for ex vivo analysis

• Ex vivo Functional Assays:

  • Isolate C. glabrata from infected tissues and immediately assess mitochondrial function

  • Compare respiratory capacity, membrane potential, and ROS production

  • Analyze mitochondrial morphology using electron microscopy

• Gene Expression Analysis:

  • Perform RNA-seq on C. glabrata cells recovered from infection sites

  • Compare expression profiles between wild-type and Δaim23 strains

  • Identify compensatory pathways activated in the absence of AIM23

• Iron Limitation Response:

  • Since host environments are iron-limited, assess the response to iron restriction

  • Compare growth and mitochondrial function under iron-depleted conditions

  • Evaluate the connection between AIM23 function and iron-dependent mitophagy

• Macrophage Interaction Studies:

  • Co-culture C. glabrata with macrophages

  • Measure survival and proliferation within phagocytes

  • Assess mitochondrial function in cells exposed to the macrophage environment

What techniques are most effective for analyzing AIM23-dependent changes in the C. glabrata mitochondrial proteome?

To thoroughly analyze AIM23-dependent changes in the C. glabrata mitochondrial proteome, researchers should consider these methodological approaches:

• Mitochondrial Isolation and Purification:

  • Use differential centrifugation combined with density gradient separation

  • Verify mitochondrial purity using marker proteins for different cellular compartments

  • Ensure minimal contamination from other organelles

• Quantitative Proteomics:

  • Implement stable isotope labeling (SILAC) for precise quantification

  • Use label-free quantification as an alternative approach

  • Apply data-independent acquisition (DIA) for comprehensive proteome coverage

• Targeted Analysis of Respiratory Complexes:

  • Blue Native PAGE to separate intact respiratory complexes

  • Second-dimension SDS-PAGE to identify subunit composition

  • Western blotting with specific antibodies against key components

• Post-translational Modification Analysis:

  • Phosphoproteomics to identify changes in protein phosphorylation

  • Analysis of other modifications (acetylation, succinylation) relevant to mitochondrial function

  • Correlation of modification patterns with functional outcomes

• Spatial Proteomics:

  • Submitochondrial fractionation to determine protein localization

  • Compare distribution between matrix, inner membrane, intermembrane space, and outer membrane

  • Identify proteins that show altered localization in the absence of AIM23

• Integration with Functional Data:

  • Correlate proteomic changes with functional parameters

  • Connect specific protein alterations to observed phenotypes

  • Identify key nodes in the mitochondrial network affected by AIM23 deletion

How does AIM23 function compare across different Candida species?

The function of AIM23 across different Candida species likely shows both conservation and divergence, reflecting their evolutionary relationships and ecological niches:

Key comparative aspects include:

• Sequence conservation and structural features of AIM23 proteins

• Species-specific interactions with mitochondrial ribosomes

• Differential importance in various carbon source environments

• Varying contributions to virulence and stress resistance

What are the differences in AIM23-related phenotypes between laboratory and clinical isolates of C. glabrata?

Laboratory and clinical isolates of C. glabrata likely exhibit significant differences in AIM23-related phenotypes due to adaptation to different environments:

• Genetic Background Variations:

  • Clinical isolates show extensive genetic diversity

  • Different sequence types may interact distinctly with AIM23 function

  • Mitochondrial genome hypervariability in clinical isolates may affect AIM23-dependent translation

• Stress Response Differences:

  • Clinical isolates are adapted to host environments and stressors

  • They may show different compensatory mechanisms for mitochondrial dysfunction

  • Laboratory strains may exhibit more severe phenotypes upon AIM23 deletion

• Drug Resistance Correlations:

  • Clinical isolates with specific mitochondrial haplotypes may show different relationships between AIM23 function and drug resistance

  • Laboratory-evolved resistance may involve different pathways than clinically-evolved resistance

• Mitochondrial Heteroplasmy:

  • Clinical isolates may maintain different levels of mitochondrial heteroplasmy

  • This heteroplasmy could buffer against complete loss of AIM23 function

  • Laboratory strains might show more homogeneous mitochondrial populations

• Experimental Evidence:

  • Studies have shown that clinical isolates exhibit diverse karyotypes and chromosomal rearrangements

  • These genomic variations likely impact nuclear-mitochondrial communication

  • AIM23-dependent phenotypes should be validated across multiple clinical isolates representing different sequence types

How does the role of AIM23 in mitochondrial function compare to other mitochondrial proteins involved in C. glabrata virulence?

AIM23's role in mitochondrial function can be compared with other mitochondrial proteins known to affect C. glabrata virulence:

What are the potential applications of targeting AIM23 for antifungal development?

Targeting AIM23 for antifungal development presents several promising opportunities:

• Selective Inhibition:

  • Design small molecules that specifically inhibit C. glabrata AIM23 function

  • Target unique structural features not present in human mitochondrial translation factors

  • Exploit differences in binding sites between fungal and human mIF3 proteins

• Combination Therapy Approaches:

  • Use AIM23 inhibitors to sensitize C. glabrata to existing antifungals

  • Target both mitochondrial function and established antifungal mechanisms

  • Potentially overcome resistance mechanisms through multi-target approaches

• Biomarker Development:

  • Identify AIM23 variants associated with drug resistance or enhanced virulence

  • Develop diagnostic tests to predict treatment outcomes

  • Guide personalized antifungal therapy based on AIM23 status

• Screening Platforms:

  • Develop high-throughput screening systems using AIM23-dependent reporter strains

  • Screen natural product libraries for compounds that interfere with AIM23 function

  • Implement structure-based virtual screening based on AIM23 protein models

• Challenges to Address:

  • Need for extreme selectivity to avoid human mitochondrial toxicity

  • Requirement for compounds that can access the fungal mitochondrial matrix

  • Potential for development of resistance through compensatory mechanisms

How might CRISPR-Cas9 technologies be optimized for studying AIM23 function in C. glabrata?

CRISPR-Cas9 technologies can be optimized for studying AIM23 function in C. glabrata through several approaches:

• CRISPR-Cas9 Delivery Systems:

  • Develop efficient transformation protocols specific for C. glabrata

  • Optimize RNA polymerase III promoters for sgRNA expression

  • Engineer Cas9 variants with enhanced activity in C. glabrata

• Precise Genetic Modifications:

  • Generate point mutations to study specific domains of AIM23

  • Create conditional knockdown systems using inducible promoters

  • Engineer epitope tags for protein visualization and purification

• Multiplexed Editing:

  • Target AIM23 along with interacting partners

  • Create libraries of AIM23 variants to screen for functional domains

  • Simultaneously edit nuclear and mitochondrial genes affecting the same pathway

• Regulatory Element Modification:

  • Edit AIM23 promoter regions to study expression regulation

  • Modify untranslated regions to analyze post-transcriptional control

  • Engineer stress-responsive elements to study regulation under various conditions

• Technical Considerations:

  • Optimize homology-directed repair templates for maximum efficiency

  • Develop marker-free editing strategies

  • Implement CRISPR interference (CRISPRi) for transient repression studies

What interdisciplinary approaches could advance our understanding of AIM23's role in C. glabrata pathogenesis?

Advancing our understanding of AIM23's role in C. glabrata pathogenesis would benefit from these interdisciplinary approaches:

• Systems Biology Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Build computational models of mitochondrial function in C. glabrata

  • Identify system-wide effects of AIM23 disruption

• Structural Biology and Biophysics:

  • Determine the high-resolution structure of C. glabrata AIM23

  • Characterize interactions with the mitochondrial ribosome

  • Apply single-molecule techniques to study translation initiation dynamics

• Host-Pathogen Interaction Studies:

  • Implement advanced imaging to visualize C. glabrata in host tissues

  • Develop organoid models to study infection in tissue-like environments

  • Analyze host immune responses to wild-type versus Δaim23 strains

• Evolutionary and Comparative Genomics:

  • Analyze AIM23 conservation across pathogenic and non-pathogenic fungi

  • Study correlation between AIM23 variants and ecological niches

  • Examine how mitochondrial genome diversity affects AIM23 function

• Clinical and Translational Research:

  • Correlate AIM23 variants in clinical isolates with patient outcomes

  • Develop diagnostic tools based on AIM23 function

  • Explore potential as a biomarker for drug resistance or virulence

These interdisciplinary approaches would provide a comprehensive understanding of AIM23's role in C. glabrata biology and pathogenesis, potentially leading to new therapeutic strategies against this important fungal pathogen.