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

What is Candida dubliniensis AIM36 protein and how does it differ from other Candida species proteins?

Altered inheritance of mitochondria protein 36 (AIM36) is a mitochondrial protein found in Candida dubliniensis, a pathogenic yeast species closely related to Candida albicans. C. dubliniensis was first distinguished as a separate species from C. albicans in the late 1990s and has been recovered primarily from the oral cavities of immunocompromised individuals, particularly HIV-infected patients and those with AIDS .

AIM36 in C. dubliniensis (UniProt ID: B9WAT8) is also known as FMP39 (Found in mitochondria protein 39) and is encoded by the AIM36 gene (CD36_17300) . The mature protein consists of amino acids 28-293 of the full sequence. While specific functional differences between C. dubliniensis AIM36 and homologous proteins in other Candida species aren't extensively documented in the provided literature, understanding these differences may be crucial given the distinct pathogenicity profiles of Candida species.

How is C. dubliniensis distinguished from C. albicans in laboratory settings?

Given the phenotypic similarities between C. dubliniensis and C. albicans, reliable differentiation requires a combination of methods:

• Growth temperature differentiation: C. dubliniensis fails to grow or grows poorly at 42°C on Potato Dextrose Agar (PDA), while C. albicans typically grows well at this temperature .

• CHROMagar Candida medium: C. dubliniensis produces dark green colonies after 48 hours at 37°C, which can help in initial screening .

• DNA fingerprinting: When genomic DNA is digested with EcoRI and probed with the C. albicans-specific DNA fingerprinting probe 27A, C. dubliniensis produces fewer and fainter bands compared to C. albicans .

• Restriction fragment length polymorphism (RFLP): C. dubliniensis yields distinct RFLP patterns when genomic DNA is digested with HinfI .

• Microsatellite sequence analysis: Using probes homologous to repetitive microsatellite sequences [(GATA)4, (GTG)5, and (GT)8], C. dubliniensis produces fingerprinting patterns distinguishable from conventional C. albicans .

For reliable identification in clinical settings, researchers typically use a combination of these methods, starting with growth on CHROMagar followed by germ tube and chlamydospore tests, and growth at 42°C .

What role does AIM36 play in mitochondrial function and inheritance in Candida species?

While the specific functions of AIM36 in C. dubliniensis aren't explicitly detailed in the search results, the protein name (Altered inheritance of mitochondria protein) suggests involvement in mitochondrial inheritance and potentially in mitochondrial membrane organization. Research into mitochondrial proteins in fungi has revealed their importance in cellular respiration, virulence, and morphogenesis.

Based on our current understanding of mitochondrial proteins in Candida species, AIM36 likely contributes to maintaining mitochondrial integrity during cell division. Further research using recombinant AIM36 could elucidate its precise function through knockout studies, localization experiments, and protein-protein interaction analyses.

The connection between mitochondrial function and filamentation in Candida species suggests that AIM36 might indirectly influence pathogenicity through its effects on mitochondrial dynamics. This is particularly relevant given the differences in filamentation capabilities between C. dubliniensis and C. albicans that contribute to their different pathogenicity profiles .

How does AIM36 expression correlate with filamentation and virulence in C. dubliniensis compared to C. albicans?

While AIM36 isn't specifically mentioned in the filamentation profiles, the research shows that C. dubliniensis and C. albicans differ in the expression of several transcription regulators involved in filamentation. For example:

• NRG1 shows differential expression between the two species, with greater upregulation in C. dubliniensis in serum media .

• Downstream filamentation regulators like UME6 and BRG1 show different patterns of expression between the two species, with more significant downregulation in C. dubliniensis mutants .

Further research could investigate whether AIM36, as a mitochondrial protein, interacts with these regulatory pathways, potentially connecting mitochondrial function to hyphal development and virulence. This is particularly important given that mitochondrial function often influences cell morphogenesis in fungi.

What experimental approaches are most effective for studying AIM36 function in C. dubliniensis?

Based on current research methodologies for studying Candida proteins, the following experimental approaches would be effective for investigating AIM36 function:

• Gene knockout/knockdown studies: Creating AIM36-deficient C. dubliniensis strains to assess changes in mitochondrial inheritance, morphology, and cell viability.

• Localization studies: Using fluorescently tagged AIM36 to confirm mitochondrial localization and observe dynamic changes during cell division and hyphal formation.

• Protein-protein interaction studies: Employing pull-down assays with recombinant His-tagged AIM36 to identify binding partners, potentially revealing functional pathways.

• Comparative expression analysis: Measuring AIM36 expression under various conditions (hyphal induction, stress, antifungal exposure) in both C. dubliniensis and C. albicans.

• Complementation studies: Introducing C. dubliniensis AIM36 into C. albicans AIM36 mutants (or vice versa) to assess functional conservation between species.

The commercial availability of recombinant C. dubliniensis AIM36 protein facilitates antibody generation for these studies and enables in vitro biochemical characterization.

What are the optimal conditions for reconstitution and storage of recombinant C. dubliniensis AIM36 protein?

Based on manufacturer recommendations for the recombinant protein :

Reconstitution Protocol:

• Centrifuge the vial briefly prior to opening to bring contents to the bottom.

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (manufacturer's default is 50%).

• Aliquot for long-term storage.

Storage Conditions:

• Store lyophilized protein at -20°C/-80°C upon receipt.

• Store reconstituted working aliquots at 4°C for up to one week.

• For long-term storage, keep aliquots (with glycerol) at -20°C/-80°C.

• Avoid repeated freeze-thaw cycles as they can compromise protein integrity .

The protein is supplied in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain stability during lyophilization and reconstitution .

What experimental systems are appropriate for functional studies of AIM36 in mitochondrial inheritance?

For investigating AIM36's role in mitochondrial inheritance, several experimental systems can be employed:

In vitro systems:

• Liposome incorporation assays: Reconstituting purified AIM36 into artificial membrane systems to study membrane interactions.

• In vitro protein binding assays: Using the recombinant His-tagged protein to identify interacting partners through pull-down experiments.

Cellular systems:

• C. dubliniensis genetic manipulation: Creating knockout or conditional mutants to assess phenotypic changes.

• Heterologous expression systems: Expressing C. dubliniensis AIM36 in S. cerevisiae to study function in a well-characterized model organism.

• Cell fractionation approaches: Isolating mitochondria to study AIM36 localization and associations with mitochondrial compartments.

Microscopy approaches:

• Fluorescence microscopy: Using fluorescently tagged AIM36 to track mitochondrial dynamics during cell division.

• Electron microscopy: Examining ultrastructural changes in mitochondria in AIM36 mutants.

How can researchers distinguish between the effects of AIM36 on mitochondrial function versus its indirect effects on filamentation and virulence?

Distinguishing direct mitochondrial effects from indirect impacts on filamentation requires careful experimental design:

• Temporal analysis: Monitor mitochondrial changes before filamentation begins using time-lapse microscopy of fluorescently labeled mitochondria in wild-type and AIM36 mutant strains.

• Conditional expression systems: Create strains with controllable AIM36 expression to observe immediate effects of protein depletion/overexpression on mitochondrial function versus delayed effects on morphology.

• Metabolic profiling: Compare respiratory capacity, ATP production, and reactive oxygen species generation between wild-type and AIM36 mutants to assess direct mitochondrial function.

• Epistasis experiments: Combine AIM36 mutations with known filamentation pathway mutations (in genes like NRG1, UME6, or BRG1) to determine genetic interactions .

• Hyphal-inducing conditions that bypass mitochondrial function: Use conditions that induce filamentation through pathways theoretically independent of mitochondrial function to test whether AIM36 effects persist.

• Rescue experiments: Attempt to rescue filamentation defects in AIM36 mutants with compounds that restore mitochondrial function to determine if mitochondrial dysfunction is the primary cause of altered morphogenesis.

How do AIM36 proteins differ between Candida species, and what are the implications for species-specific pathogenicity?

C. dubliniensis is primarily recovered from HIV-infected individuals and AIDS patients, with an incidence of 27-32% in patients with oral candidiasis symptoms and 19-25% in those without symptoms . By contrast, C. albicans has a broader distribution in human populations and causes a wider range of infections.

While not directly linked to AIM36, these differences in pathogenicity correlate with differences in filamentation capabilities. The reduced ability of C. dubliniensis to form hyphae under certain conditions compared to C. albicans may contribute to its more limited pathogenic potential .

A comparative analysis of AIM36 sequences and expression patterns across Candida species could reveal whether variations in this protein contribute to species-specific differences in mitochondrial function, and by extension, to differences in stress resistance, morphogenetic capabilities, and virulence.

What is known about the evolutionary conservation of AIM36 across fungal species?

Future research could address:

• Sequence homology and structural conservation of AIM36 across Candida species and other fungi

• Conservation of functional domains and protein interaction networks

• Whether horizontal gene transfer has played a role in AIM36 evolution

Comparative genomic analyses would help determine whether AIM36 is part of the core genome shared by all Candida species or if it represents a more recently acquired or diverged gene. Such evolutionary insights could inform structure-function relationships and help identify functionally important regions of the protein.

Could AIM36 serve as a diagnostic marker for distinguishing C. dubliniensis from other Candida species in clinical samples?

Based on the challenges in distinguishing C. dubliniensis from C. albicans in clinical settings , AIM36 could potentially serve as a molecular diagnostic marker if species-specific differences in the protein or its expression exist.

Current identification methods rely on phenotypic tests (growth at 42°C, colony morphology on CHROMagar) and genotypic methods (DNA fingerprinting, RFLP analysis) . These methods can be time-consuming and sometimes ambiguous.

If AIM36 contains regions with significant sequence divergence between C. dubliniensis and C. albicans, species-specific antibodies or PCR primers targeting these regions could be developed for rapid diagnostic tests. This approach would be particularly valuable given that approximately 2% of isolates from healthy individuals and 17% of isolates from HIV-infected individuals originally identified as C. albicans were later found to be C. dubliniensis .

What is the relationship between AIM36 function and antifungal drug resistance in Candida species?

While the search results don't directly address AIM36's relationship to antifungal resistance, C. dubliniensis has been noted for its ability to rapidly develop fluconazole resistance in vitro . This is particularly significant given the clinical importance of identifying drug-resistant isolates.

Mitochondrial proteins can contribute to drug resistance through several mechanisms:

• Influencing cellular energetics and stress responses

• Affecting membrane composition and drug permeability

• Participating in signaling pathways that regulate drug efflux pumps

Research into whether AIM36 plays a role in these processes could provide insights into species-specific differences in antifungal susceptibility. This is especially relevant given C. dubliniensis's emergence as an opportunistic pathogen coinciding with epidemiological shifts in Candida infections and increasing antifungal resistance .

What technologies and approaches will advance our understanding of AIM36 function in the next five years?

Emerging technologies likely to advance AIM36 research include:

• CRISPR-Cas9 genome editing: Creating precise mutations in AIM36 to study structure-function relationships with greater efficiency than traditional genetic approaches.

• Single-cell transcriptomics: Examining AIM36 expression patterns across individual cells during morphological transitions and in response to environmental stresses.

• Advanced imaging techniques: Using super-resolution microscopy to visualize AIM36 localization within mitochondrial subcompartments.

• Cryo-electron microscopy: Determining the three-dimensional structure of AIM36 alone and in complexes with interacting proteins.

• Metabolomics and proteomics: Comprehensively analyzing the effects of AIM36 mutations on cellular metabolism and protein expression patterns.

• Host-pathogen interaction models: Investigating how AIM36 function influences interactions with host cells and immune responses using advanced ex vivo and in vivo models.

These approaches, combined with the availability of recombinant AIM36 protein , will enable more detailed functional characterization and potentially reveal therapeutic targets for selective inhibition of pathogenic Candida species.