Recombinant Candida tropicalis Altered inheritance of mitochondria protein 36, mitochondrial (AIM36)

What is Candida tropicalis and why is it significant in research settings?

Candida tropicalis is a diploid dimorphic opportunistic fungal pathogen that has emerged as one of the most important Candida species. It is widely considered the second most virulent Candida species after C. albicans . C. tropicalis is particularly significant because:

• It produces a wide range of virulence factors, including strong biofilm formation (often exceeding C. albicans), adhesion to epithelial and endothelial cells, secretion of lytic enzymes (proteinases, phospholipases, hemolysins), and bud-to-hyphae transition

• It ranks as the second or third most common etiological agent of candidemia in many regions, particularly in Latin American countries and Asia

• It shows distinctive traits including osmotolerance, which allows it to survive in high salt concentration environments

In clinical settings, C. tropicalis infections are associated with mortality rates above 40%, especially in immunocompromised patients . It has been documented as a common agent of candidemia not only in cancer patients but also in adult and child critically ill patients .

What is AIM36 and what is its role in mitochondrial function?

AIM36 (Altered Inheritance of Mitochondria protein 36) is a mitochondrial protein involved in the proper inheritance and distribution of mitochondria during cell division. Based on the information available for Candida dubliniensis AIM36:

• The mature protein spans amino acids 28-293, with a full sequence containing multiple hydrophobic regions suggesting membrane association

• Its amino acid sequence (VRTPIFHQTPIQIIKRNYVIVHKERKKEPVIRYLFYMLVASWVAIYFVANRVDKKKPPKQSFTEREFQSYEEETGLKRRNKLISHHMNSKYKFYVIPYVHDEEELNRVANLLQHKDENAT VKIIDPAQLIEEQKKDEGMKYHYLLEDLDEQGKPYPPGLITAVIKQEIYKILNTREGTFDTNFLIKNYPQTTNEAIKFENDISDIQKCLILHYDMLNELPKNKTDEEQRAIKNVDGYFSS VGKSKTLVEKFDPMDKEFEDIILEDI) suggests potential structural motifs for mitochondrial function

• AIM36 is also known as FMP39 (Found in Mitochondria Protein 39), indicating its localization to this organelle

What are the most effective genetic modification methods for C. tropicalis?

The genetic modification of C. tropicalis requires specialized approaches that differ from those used for C. albicans. Based on current research:

• Homologous recombination with extended homology arms:

  • For successful transformation of C. tropicalis, longer homology arms (~900 bp) flanking the target gene are recommended, as shorter oligonucleotides often result in non-specific integration

  • Target sequence amplification can be done either by digesting with restriction enzymes or by PCR using primers that anneal to the cloned homology arms

• Selection markers and cassettes:

  • The SAT1 marker (conferring nourseothricin resistance) is commonly used, with specialized vectors like pSFS2A or pEM008

  • For C. tropicalis, SAT1 marker recycling is achieved most efficiently using the PCK1 promoter to drive FLP recombinase expression, rather than the MAL2 promoter that works well in C. albicans

• Transformation methods:

  • Electroporation is effective for introducing DNA constructs into C. tropicalis

  • Selection on YEPD plates with 400 μg/ml nourseothricin (NAT) for 48h at 30°C is recommended

For AIM36 specifically, these techniques would need to be adapted to either delete the gene, modify it, or tag it for functional studies.

How can researchers overcome the challenges in marker recycling for sequential genetic modifications in C. tropicalis?

Marker recycling in C. tropicalis presents unique challenges compared to other Candida species. Research has shown that:

• Promoter selection is critical:

  • The MAL2 promoter, which works efficiently in C. albicans, shows low excision rates in C. tropicalis even when the endogenous C. tropicalis promoter is used

  • Replacing the MAL2 promoter with the PCK1 promoter (either from C. tropicalis or C. albicans) leads to efficient excision of the SAT1 marker

• Induction conditions:

  • For PCK1 promoter-driven marker excision, grow cells overnight at 30°C in either:

    • YNB medium with 2% casamino acids, or

    • Synthetic medium containing 2% succinate

  • After induction, screen cells on YEPD supplemented with 400 μg/ml NAT to confirm the loss of NAT resistance

• Verification steps:

  • Confirm successful genetic modifications by:

    • Colony PCR of junction regions

    • PCR amplification of the target gene

    • Phenotypic testing (for auxotrophic markers)

    • Sanger sequencing of modified regions

This approach allows for multiple sequential genetic modifications, essential for complex studies involving proteins like AIM36.

What expression systems are most effective for producing recombinant C. tropicalis AIM36 protein?

Based on existing protocols for similar mitochondrial proteins, the following expression systems are recommended:

• E. coli expression system:

  • Successfully used for C. dubliniensis AIM36 expression

  • Recommended strain: E. coli BL21(DE3) for high-level expression

  • Vector choice: pET-based vectors with N-terminal His-tag for purification

  • Expression conditions: Induction with IPTG at lower temperatures (16-20°C) to enhance proper folding of mitochondrial proteins

• Expression optimization:

  • Codon optimization for E. coli may be necessary for efficient expression

  • Consider expressing only the mature protein (amino acids 28-293) without the mitochondrial targeting sequence to improve solubility

  • Addition of solubility tags (SUMO, MBP, or GST) may enhance protein solubility

• Expression verification:

  • Western blot analysis using anti-His antibodies

  • SDS-PAGE to confirm protein size and purity (expected size approximately 30-35 kDa for the mature protein)

For C. tropicalis AIM36, adaptation of these methods based on the specific amino acid sequence and predicted structural features would be necessary.

What purification strategies yield the highest purity and activity for recombinant AIM36?

For optimal purification of recombinant C. tropicalis AIM36, a multi-step purification strategy is recommended:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective for His-tagged AIM36

  • Lysis buffer: Tris/PBS-based buffer at pH 8.0 with 6% trehalose to maintain protein stability

  • Include protease inhibitors and potentially mild detergents to solubilize membrane-associated regions

• Secondary purification:

  • Ion exchange chromatography based on the predicted isoelectric point of AIM36

  • Size exclusion chromatography to remove aggregates and obtain homogeneous protein

• Quality control:

  • Assess purity by SDS-PAGE (>90% purity recommended)

  • Verify protein identity by mass spectrometry

  • Test functional activity (if applicable assays are available)

• Storage considerations:

  • Store in aliquots at -20°C/-80°C to avoid repeated freeze-thaw cycles

  • For long-term storage, add 5-50% glycerol (final concentration)

  • Stability testing at different temperatures and buffer conditions is recommended

Proper purification is essential for downstream applications such as antibody production, structural studies, or functional characterization.

How can researchers effectively study the role of AIM36 in mitochondrial inheritance in C. tropicalis?

To investigate AIM36 function in mitochondrial inheritance in C. tropicalis, researchers can employ these advanced approaches:

• Gene deletion and complementation:

  • Generate AIM36 knockout strains using homologous recombination with the SAT1 flipper cassette

  • Create complemented strains with wild-type AIM36 to confirm phenotype specificity

  • Develop conditional expression systems using the C. tropicalis PCK1 promoter for regulated expression

• Protein localization and dynamics:

  • Create C-terminal tagged versions of AIM36 using epitope tags like 13× MYC

  • Use vectors like pEM018 or pEM019 that contain the SAT1 cassette with the PCK1 promoter

  • Employ fusion PCR with ~350 bp homology regions for efficient targeting

  • Visualize mitochondrial morphology and inheritance patterns using fluorescence microscopy

• Functional assays:

  • Monitor mitochondrial distribution during cell division

  • Assess mitochondrial membrane potential using fluorescent dyes

  • Evaluate respiratory capacity through oxygen consumption measurements

  • Examine growth under conditions that require mitochondrial function

These approaches would provide comprehensive insights into AIM36's role in mitochondrial dynamics within C. tropicalis.

What techniques are most effective for studying AIM36 protein interactions in C. tropicalis?

To characterize AIM36 protein interactions in C. tropicalis, researchers can employ several cutting-edge approaches:

• Affinity purification coupled with mass spectrometry:

  • Generate strains expressing epitope-tagged AIM36 (e.g., 13× MYC-tagged)

  • Perform co-immunoprecipitation using anti-MYC antibodies

  • Identify interaction partners by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Validate key interactions through reciprocal co-immunoprecipitation

• Proximity-based labeling:

  • Create fusion proteins of AIM36 with BioID or APEX2

  • Identify proteins in close proximity to AIM36 in living cells

  • Map the spatial interactome within mitochondria

• Yeast two-hybrid and split-protein complementation:

  • Screen for direct protein-protein interactions

  • Validate interactions in vivo using bimolecular fluorescence complementation

• Domain mapping and mutational analysis:

  • Generate truncated versions or point mutations of AIM36

  • Identify critical regions/residues for protein interactions and function

  • Correlate molecular interactions with phenotypic outcomes

These methodologies provide complementary approaches to build a comprehensive understanding of AIM36's role within the mitochondrial protein network.

How does genetic variation in AIM36 correlate with phenotypic differences among C. tropicalis isolates?

Analysis of AIM36 genetic variation across C. tropicalis isolates requires integration of genomic and phenotypic data:

• Population genomic analysis:

  • Sequence analysis shows that most C. tropicalis isolates are diploids with approximately 2-6 heterozygous variants per kilobase

  • Specific analysis of AIM36 locus across clinical and environmental isolates could reveal:

    • Selection pressure on specific domains

    • Correlation with mitochondrial function phenotypes

    • Potential adaptations in different ecological niches

• Phenotypic correlation:

  • Growth characteristics in different media and stress conditions vary widely among C. tropicalis isolates

  • Correlate AIM36 variants with:

    • Growth on non-fermentable carbon sources (requiring mitochondrial function)

    • Resistance to mitochondrial stressors

    • Virulence in infection models

• Heterozygosity analysis:

  • Some C. tropicalis isolates show much higher heterozygosity (36-49 variants per kb)

  • Analysis of hybrid isolates (which share one parent with most other isolates) could provide insights into AIM36 evolution

  • Examination of loss of heterozygosity patterns at the AIM36 locus

This integrated approach would reveal how AIM36 variation contributes to the adaptive landscape of C. tropicalis.

What methods can be used to assess the impact of AIM36 modification on C. tropicalis virulence?

To evaluate how AIM36 modifications affect C. tropicalis virulence, researchers can implement these methodologies:

• In vitro virulence factor assessment:

  • Compare wild-type and AIM36-modified strains for:

    • Biofilm formation capacity (C. tropicalis is recognized as a very strong biofilm producer)

    • Hemolytic activity (C. tropicalis produces beta hemolysis)

    • Production of lytic enzymes like proteinases and phospholipases

    • Bud-to-hyphae transition (morphogenesis)

• Host cell interaction models:

  • Adhesion to buccal epithelial and endothelial cells

  • Survival within macrophages

  • Tissue invasion assays

• In vivo infection models:

  • Murine models of disseminated candidiasis to measure:

    • Fungal burden in target organs

    • Survival rates

    • Immune response parameters

  • Compare results to known data for wild-type strains, where fluconazole at 10 mg/kg/day reduces fungal burden

• Comparative analysis with other mitochondrial mutants:

  • Determine if AIM36 modification affects antifungal susceptibility patterns

  • Assess if mitochondrial dysfunction from AIM36 modification relates to virulence attenuation, similar to other strains where acquisition of drug resistance related to loss of virulence

This comprehensive assessment would elucidate the relationship between mitochondrial dynamics, as influenced by AIM36, and pathogenesis.

How can researchers develop specific detection methods for monitoring AIM36 expression in C. tropicalis?

For specific detection of AIM36 expression in C. tropicalis, researchers can utilize several molecular approaches:

• Transcriptional analysis:

  • Develop RT-qPCR assays with primers specific to C. tropicalis AIM36

  • Design primers that distinguish between C. tropicalis AIM36 and homologs in other Candida species

  • Normalize expression to appropriate reference genes for accurate quantification

• Protein detection methods:

  • Generate specific antibodies against C. tropicalis AIM36 using recombinant protein

  • Develop Western blot protocols optimized for mitochondrial proteins

  • Create tagged versions (His-tag, MYC-tag) for detection with commercial antibodies

• Reporter systems:

  • Create AIM36 promoter-reporter constructs to monitor transcriptional regulation

  • Develop fluorescent protein fusions for live-cell imaging of protein localization and abundance

• High-throughput approaches:

  • RNA-seq analysis to monitor AIM36 expression under various conditions

  • Proteomics approaches to detect AIM36 and interacting partners

These methods would provide comprehensive tools for monitoring AIM36 expression and localization under various experimental conditions.

Can AIM36-based approaches be integrated into rapid diagnostic methods for C. tropicalis identification?

While current C. tropicalis diagnostic methods focus on other targets, AIM36-based approaches could potentially be integrated into existing platforms:

• Integration with nucleic acid amplification techniques:

  • Current rapid detection methods for C. tropicalis include:

    • Recombinase Polymerase Amplification combined with Lateral Flow Strip (RPA-LFS)

    • Multiple Cross Displacement Amplification with Lateral Flow Biosensor (MCDA-LFB)

  • AIM36 sequence could be incorporated as an additional target to:

    • Increase specificity for C. tropicalis detection

    • Potentially differentiate between strains with different AIM36 variants

• Specificity considerations:

  • Current nucleic acid-based methods for C. tropicalis detection show high specificity without cross-reactivity with other pathogens, including:

    • C. albicans, C. parapsilosis, C. dubliniensis, and other Candida species

    • Various bacterial pathogens

  • AIM36-based detection would need similar specificity validation

• Performance metrics for clinical implementation:

  • Current RPA-LFS methods can detect 9.94 CFU/μL of C. tropicalis

  • Process time of under 30 minutes (20 minutes for amplification and detection)

  • AIM36-based methods would need to match or exceed these performance characteristics

• Validation with clinical samples:

  • Current methods have been validated with various clinical samples:

    • Blood (93 samples)

    • Sputum (45 samples)

    • Urine (36 samples)

    • Other clinical samples (17 samples)

  • Similar validation would be required for AIM36-based approaches

Integration of AIM36 into diagnostic platforms could potentially enhance the specificity and provide additional strain information in C. tropicalis detection.

How does mitochondrial function, potentially involving AIM36, contribute to antifungal resistance in C. tropicalis?

Mitochondrial proteins like AIM36 may play significant roles in antifungal resistance mechanisms:

• Azole resistance connections:

  • Azole resistance in C. tropicalis has been associated with:

    • Mutations in Cdr1 and Mdr1 efflux pumps

    • Alterations in ergosterol production gene (ERG11)

    • Mitochondrial abnormalities

  • AIM36, as a mitochondrial protein, could influence these pathways through:

    • Effects on membrane composition and drug permeability

    • Energy production for efflux pump activity

    • Stress responses that contribute to resistance

• Echinocandin interactions:

  • C. tropicalis exhibits paradoxical growth in the presence of high caspofungin concentrations:

    • While inhibited at low concentrations, some strains grow at high concentrations

    • Caspofungin becomes fungistatic rather than fungicidal at high concentrations

  • Mitochondrial proteins like AIM36 could contribute to this through:

    • Metabolic adaptations

    • Stress response mechanisms

    • Changes in cell wall composition or maintenance

• Multiple drug resistance mechanisms:

  • Fluconazole-resistant C. tropicalis strains have emerged through:

    • Loss of heterozygosity in genes like FCY2 (purine-cytosine permease)

    • Upregulation of efflux transporters

  • These mechanisms could be influenced by mitochondrial function and inheritance

Understanding AIM36's role in these processes could provide new insights into resistance mechanisms and potential therapeutic targets.

What approaches can be used to study the potential of AIM36 as a novel antifungal target?

Investigating AIM36 as a potential novel antifungal target requires a systematic approach:

• Target validation studies:

  • Generate conditional mutants of AIM36 to confirm essentiality or significant contribution to virulence

  • Perform comparative analyses between mammalian and fungal AIM36 homologs to identify:

    • Structural differences that could be exploited

    • Functional divergence that might allow selective targeting

  • Evaluate effects of AIM36 depletion on:

    • Growth under various conditions

    • Virulence factor production

    • Resistance to existing antifungals

• Structural and biochemical characterization:

  • Determine the three-dimensional structure of AIM36 using:

    • X-ray crystallography

    • Cryo-electron microscopy

    • Nuclear magnetic resonance for specific domains

  • Identify potential binding pockets or active sites

  • Characterize enzymatic activity or protein-protein interactions that could be disrupted

• Compound screening and development:

  • Develop high-throughput screening assays based on:

    • AIM36 function

    • Protein-protein interactions

    • Reporter systems linked to AIM36 activity

  • Screen compound libraries to identify potential inhibitors

  • Test promising compounds for:

    • Antifungal activity against C. tropicalis

    • Selectivity over human homologs

    • Effects on resistant strains

• Combination therapy assessment:

  • Evaluate potential synergy between AIM36 inhibitors and:

    • Existing antifungals (azoles, echinocandins)

    • Other mitochondrial-targeting compounds

  • Similar to the observation that fluconazole eliminates paradoxical growth with caspofungin

These approaches would establish the viability of AIM36 as a novel antifungal target and potentially lead to new therapeutic strategies for C. tropicalis infections.