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

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

AIM36 (Altered inheritance of mitochondria protein 36) is a mitochondrial protein in Candida albicans that likely plays a role in mitochondrial inheritance and function. While specific research on AIM36 is limited, studies on mitochondrial proteins in fungi suggest these proteins are crucial for energy metabolism, cellular stress responses, and pathogenicity . Mitochondrial functions are essential for C. albicans virulence and adaptation to host environments.

Based on research of similar mitochondrial proteins, AIM36 may be involved in maintaining mitochondrial DNA stability, regulating mitochondrial division, or facilitating protein import into mitochondria. The "altered inheritance" portion of its name suggests it may influence how mitochondria are distributed during cell division, which is particularly important for a dimorphic fungus like C. albicans that can switch between yeast and hyphal forms .

How do researchers produce and purify recombinant Candida albicans proteins for experimental studies?

Production of recombinant C. albicans proteins typically employs heterologous expression systems. The methodology generally follows these steps:

• Gene identification and isolation: The AIM36 gene sequence is identified from the C. albicans genome and amplified using PCR.

• Cloning: The gene is inserted into an expression vector containing appropriate promoters and selection markers.

• Expression system selection: Common expression hosts include:

  • Escherichia coli: For non-glycosylated proteins

  • Pichia pastoris: For proteins requiring eukaryotic post-translational modifications

  • Saccharomyces cerevisiae: For proteins that may be toxic in bacterial systems

• Protein expression optimization: Parameters like temperature, induction conditions, and media composition are adjusted to maximize yield.

• Purification: Typically achieved through affinity chromatography using tags (His, GST) engineered into the recombinant protein.

For studying AIM36 specifically, researchers may need to optimize conditions to maintain proper folding of this mitochondrial protein. Commercial sources now offer purified recombinant AIM36, which can save researchers significant time and resources in experimental design .

What experimental models are most effective for studying mitochondrial protein function in Candida albicans?

Several experimental models have proven valuable for studying mitochondrial proteins in C. albicans:

• In vitro cellular models:

  • C. albicans mutant strains (knockout, knockdown, or overexpression)

  • Reconstituted human epithelial models for host-pathogen interaction studies

  • Macrophage infection models to assess immune interactions

• Animal models:

  • Mouse systemic infection models, particularly for studying kidney colonization, which is a primary target of C. albicans infection

  • Murine oral candidiasis models for mucosal infection studies

• Molecular and biochemical approaches:

  • Mitochondrial isolation and functional assays

  • Yeast two-hybrid or co-immunoprecipitation for protein interaction studies

  • Fluorescent tagging for subcellular localization studies

For AIM36 specifically, researchers might employ knockout models to assess phenotypic changes in mitochondrial inheritance patterns, morphological transitions, or virulence capabilities. Based on research with other proteins, macrophage interaction models would be particularly valuable since mitochondrial function impacts how C. albicans responds to phagocytosis .

How does Candida albicans mitochondrial function relate to pathogenicity and immune response?

Mitochondrial function in C. albicans is intimately linked to its pathogenicity and interaction with host immune responses:

• Metabolic adaptation: Functional mitochondria allow C. albicans to adapt to different nutrient environments within the host, facilitating infection of diverse tissues.

• Morphogenesis regulation: The yeast-to-hyphal transition, crucial for virulence, requires substantial energy input regulated by mitochondrial activity.

• Stress response: Mitochondria help C. albicans respond to oxidative stress generated by host immune cells like macrophages.

• Innate immune recognition: Pattern recognition receptors like AIM2 (Absent in melanoma 2) can detect fungal DNA, including mitochondrial DNA . While AIM2 and AIM36 are distinct proteins, this illustrates how mitochondrial components can influence immune recognition.

Research shows that AIM2 expression is induced in human and mouse innate immune cells following C. albicans infection . Interestingly, Aim2-deficient mice demonstrate increased resistance to C. albicans infection, with reduced kidney inflammation post-infection. This resistance appears to involve reduced macrophage apoptosis and increased AKT activation . Understanding how mitochondrial proteins like AIM36 might influence these pathways could reveal new therapeutic approaches.

What techniques are recommended for validating the function of recombinant mitochondrial proteins?

To validate recombinant mitochondrial protein function, researchers should employ multiple complementary approaches:

• Structural validation:

  • Circular dichroism spectroscopy to assess secondary structure

  • Mass spectrometry for verification of protein identity and modifications

  • Size-exclusion chromatography to confirm proper folding and oligomeric state

• Functional validation:

  • Enzymatic activity assays (if the protein has known enzymatic function)

  • Binding assays to identify interaction partners

  • Mitochondrial import assays using isolated mitochondria

• Cellular validation:

  • Complementation studies in knockout strains

  • Localization studies using fluorescent tags or immunofluorescence

  • Phenotypic rescue experiments

• In vivo validation:

  • Virulence assays in animal models comparing wild-type, knockout, and complemented strains

  • Host cell interaction studies using primary immune cells or cell lines

For AIM36 specifically, researchers might assess mitochondrial morphology, distribution during cell division, and impact on energy metabolism using techniques like mitochondrial membrane potential measurements and respiratory capacity analysis.

What methodological challenges exist in studying mitochondrial protein inheritance in Candida albicans?

Studying mitochondrial protein inheritance in C. albicans presents several significant challenges:

• Genetic manipulation complexity:

  • C. albicans is diploid with an unconventional codon usage (CUG encodes serine instead of leucine)

  • Requires disruption of both alleles for complete gene knockout

  • Limited selection markers compared to model yeasts

• Mitochondrial targeting issues:

  • Ensuring proper trafficking of tagged proteins to mitochondria

  • Potential interference of tags with protein function or localization

  • Difficulty in distinguishing imported from newly synthesized proteins

• Morphological transition complications:

  • Different mitochondrial dynamics in yeast versus hyphal forms

  • Rapid changes in mitochondrial architecture during morphogenesis

  • Need for continuous live-cell imaging during morphological transitions

• Technical limitations:

  • Challenges in isolating pure, functional mitochondria from C. albicans

  • Limited antibodies available for C. albicans mitochondrial proteins

  • Difficulty in generating mitochondrial DNA mutants

Researchers studying AIM36 might benefit from approaches used in analyzing mitochondrial inheritance in other organisms. For instance, the methodology used to track mitochondrial tRNA transfer in mammalian systems could be adapted to fungal models, using genetic diversity of individual mtDNAs to trace inheritance patterns .

How can researchers optimize heterologous expression systems for difficult-to-express mitochondrial proteins?

Optimizing expression of mitochondrial proteins like AIM36 requires addressing several challenges:

• Expression host selection:

  Expression Host	Advantages	Disadvantages	Best For
  E. coli	Fast growth, high yields, low cost	Lacks eukaryotic PTMs, inclusion body formation	Soluble domains, non-glycosylated proteins
  P. pastoris	Eukaryotic PTMs, high-density cultures	Longer process, more complex media	Full-length glycosylated proteins
  S. cerevisiae	Similar cellular machinery to C. albicans	Lower yields than P. pastoris	Proteins toxic to other systems
  Insect cells	Complex eukaryotic PTMs	Expensive, technically demanding	Highly modified membrane proteins

• Construct design strategies:

  • Use of solubility tags (MBP, SUMO, Trx) at the N-terminus

  • Expression of functional domains rather than full-length protein

  • Codon optimization for the expression host

  • Inclusion of purification tags with cleavage sites

• Expression condition optimization:

  • Lower induction temperatures (16-20°C) to slow folding

  • Reduced inducer concentrations

  • Co-expression with molecular chaperones

  • Addition of specific metal ions or cofactors to the media

• Purification considerations:

  • Mild detergents for membrane-associated mitochondrial proteins

  • Inclusion of stabilizing agents (glycerol, specific lipids)

  • Rapid purification protocols to minimize degradation

  • Size exclusion chromatography as a final polishing step

For mitochondrial proteins like AIM36, researchers might need to express the protein without its mitochondrial targeting sequence, which can cause aggregation in heterologous systems, while ensuring the remaining protein retains functional activity.

What are the current approaches for studying protein-protein interactions involving mitochondrial proteins in Candida albicans?

Investigating protein-protein interactions for mitochondrial proteins requires specialized techniques:

• In vitro methods:

  • Pull-down assays using recombinant proteins

  • Surface plasmon resonance for interaction kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Crosslinking mass spectrometry to identify interaction interfaces

• Cellular techniques:

  • Bimolecular fluorescence complementation (BiFC) adapted for C. albicans

  • Proximity-dependent biotin identification (BioID) for transient interactions

  • Förster resonance energy transfer (FRET) for real-time interaction studies

  • Co-immunoprecipitation from isolated mitochondria

• Systems biology approaches:

  • Interactome mapping using affinity purification-mass spectrometry

  • Genetic interaction screens using CRISPR-based technologies

  • Computational prediction of interactions based on structural modeling

• Specialized mitochondrial approaches:

  • Submitochondrial fractionation to identify compartment-specific interactions

  • Import assays to identify interactions during protein translocation

  • Liposome reconstitution systems with purified components

When studying AIM36, researchers might focus on identifying interaction partners during mitochondrial inheritance events, perhaps using techniques similar to those that revealed the connection between mitochondrial dysfunction and AKT activation, as observed in studies of AIM2 .

How do alterations in mitochondrial proteins affect Candida albicans virulence in different host niches?

Mitochondrial proteins impact C. albicans virulence differently depending on the host environment:

• Bloodstream infections:

  • Mitochondrial proteins support adaptation to glucose-limited environments

  • Enable resistance to oxidative stress from neutrophils

  • Facilitate hyphal transition important for endothelial invasion

• Mucosal infections:

  • Support metabolic flexibility for utilizing alternative carbon sources

  • Enable biofilm formation through energy provision

  • Contribute to epithelial adhesion and invasion

• Kidney colonization:

  • Mitochondrial function is critical for kidney infection, a primary target in systemic candidiasis

  • Supports adaptation to unique nutrient environments

  • Influences interaction with renal macrophages and dendritic cells

• Gastrointestinal colonization:

  • Enables competition with bacterial microbiota

  • Supports adaptation to anaerobic/microaerobic conditions

  • Facilitates response to bile acids and pH changes

Research demonstrates that mitochondrial function influences organ-specific outcomes. For example, studies with AIM2 show that kidney tissues display differential inflammation and apoptosis levels following C. albicans infection, with macrophages playing a critical role . This suggests mitochondrial proteins like AIM36 may have tissue-specific impacts on pathogenesis through their effects on energy metabolism, stress responses, or interaction with host immune cells.

What emerging technologies show promise for advancing our understanding of mitochondrial protein function in fungal pathogens?

Several cutting-edge technologies are poised to transform research on fungal mitochondrial proteins:

• CRISPR-based technologies:

  • CRISPR interference (CRISPRi) for conditional gene repression

  • Base editors for introducing specific mutations without double-strand breaks

  • Prime editing for precise sequence modifications

  • CRISPR-based screening for functional genomics

• Advanced imaging techniques:

  • Super-resolution microscopy for visualizing submitochondrial structures

  • Correlative light and electron microscopy (CLEM) for structural context

  • Live-cell volumetric imaging for 4D mitochondrial dynamics

  • Expansion microscopy for improved spatial resolution

• Single-cell approaches:

  • Single-cell RNA-seq for heterogeneity in mitochondrial gene expression

  • Single-cell proteomics for protein-level analysis

  • Microfluidic devices for real-time single-cell phenotyping

• Synthetic biology tools:

  • Optogenetic control of mitochondrial protein activity

  • Chemically-induced proximity systems for temporal control

  • Synthetic genetic circuits for pathway analysis

  • Engineered proteins with built-in biosensors

• Multi-omics integration:

  • Combined transcriptomics, proteomics, and metabolomics approaches

  • Integration of genetics and functional assays with structural biology

  • Machine learning for pattern recognition across datasets

For proteins like AIM36, emerging approaches similar to those used to track mitochondrial tRNA inheritance in mammalian systems could be particularly valuable . These methods harness genetic diversity of mitochondrial DNA to track specific RNAs and proteins, potentially revealing how mitochondrial components are transferred during cell division or under stress conditions.