Recombinant Candida glabrata Altered inheritance of mitochondria protein 39, mitochondrial (AIM39)

What is Candida glabrata and why is it significant in clinical research?

Candida glabrata is the second most common etiological cause of worldwide systemic candidiasis in adult patients . Clinical isolates display remarkable genetic diversity, with genome analysis revealing at least 19 separate sequence types identified globally, plus newly discovered variants . This pathogen has significant clinical importance due to its ability to develop drug resistance, particularly to azole antifungals, and its capacity to persist within host cells, including macrophages .

Methodological approach: Researchers typically characterize C. glabrata through whole genome sequencing combined with molecular typing methods such as multilocus sequence typing (MLST). Clinical significance is assessed through epidemiological studies, antifungal susceptibility testing, and infection models such as Galleria mellonella larvae .

What is the role of mitochondria in C. glabrata pathogenicity?

Mitochondria play critical roles in C. glabrata pathogenicity through multiple mechanisms:

• Drug resistance: Mitochondrial dysfunction or morphological abnormalities contribute to azole resistance mechanisms

• Genomic diversity: The mitochondrial genome in C. glabrata shows considerable diversity, with reduced conserved sequences and protein-encoding genes in certain sequence types

• Stress response: Mitochondrial function affects cellular responses to environmental stresses encountered during infection

• Virulence factor regulation: Mitochondrial status influences the expression of virulence factors

Methodological approach: Researchers investigate mitochondrial contributions to pathogenicity through gene deletion studies, mitochondrial morphology visualization, and phenotypic characterization of mitochondrial mutants under various stress conditions.

How does AIM39 fit into the broader context of mitochondrial proteins in C. glabrata?

While specific information about AIM39 in C. glabrata is limited in current literature, its name "Altered inheritance of mitochondria protein 39" suggests involvement in mitochondrial inheritance and potentially mitochondrial dynamics . The protein likely functions within the network of mitochondrial proteins that influence morphology, inheritance, and function.

The ERMES (ER-mitochondrial encounter structure) complex, which includes components like GEM1, MDM12, and MDM34, plays important roles in maintaining proper mitochondrial morphology and function in fungi . AIM39 may interact with this complex or function in parallel pathways affecting mitochondrial inheritance.

Methodological approach: Researchers would typically characterize AIM39 function through:

• Gene deletion and complementation studies

• Protein localization using fluorescent tagging

• Protein-protein interaction studies

• Phenotypic analysis of mutants under various growth conditions

What experimental methods are used to produce recombinant mitochondrial proteins from C. glabrata?

Production of recombinant mitochondrial proteins from C. glabrata typically involves:

• Gene cloning: The target gene is amplified from C. glabrata genomic DNA using PCR with specific primers

• Expression vector construction: The gene is inserted into appropriate expression vectors, often using homologous recombination-based cloning strategies

• Heterologous expression: Proteins are commonly expressed in systems such as E. coli, S. cerevisiae, or insect cells

• Purification: Tagged proteins are purified using affinity chromatography followed by additional purification steps

For C. glabrata proteins specifically, expression systems using copper-inducible promoters like MTI have been successfully employed . This approach allows controlled expression of the target protein.

Table 1: Common expression systems for recombinant fungal mitochondrial proteins

How do mutations in mitochondrial proteins affect drug resistance in C. glabrata?

Mutations in mitochondrial proteins significantly impact drug resistance in C. glabrata through several mechanisms:

• Efflux pump regulation: Deletion of GEM1, an ERMES component, increases azole resistance by upregulating drug efflux pumps encoded by CDR1 and CDR2

• Oxidative stress response: Mitochondrial dysfunction leads to increased mitochondrial ROS (mtROS) levels, which can trigger stress response pathways

• Membrane composition: Altered mitochondrial function affects ergosterol biosynthesis, the target of azole antifungals

• Energy metabolism: Changes in energy production can influence cellular responses to antifungal drugs

A key example is the deletion of GEM1, which results in abnormal mitochondrial morphology, increased ROS production, and upregulated expression of drug efflux pumps . Treatment with the antioxidant N-acetylcysteine (NAC) reduces both ROS production and CDR1 expression in Δgem1 mutants, demonstrating the connection between mitochondrial function and drug resistance mechanisms .

What are the structural and functional characteristics of the ERMES complex and how might AIM39 interact with it?

The ER-mitochondrial encounter structure (ERMES) complex forms contact sites between the endoplasmic reticulum and mitochondria, playing crucial roles in mitochondrial morphology and function. Key components include GEM1 (a GTPase that regulates ERMES activity), MDM12, MDM34, and other proteins .

While specific interactions between ERMES and AIM39 are not documented in current literature, potential interactions could include:

• Physical association at ER-mitochondria contact sites

• Functional cooperation in mitochondrial inheritance pathways

• Shared roles in stress response mechanisms

• Involvement in mitochondrial DNA maintenance

Deletion of ERMES components leads to abnormal mitochondrial morphology, with Δgem1 cells displaying shortened or collapsed tubular networks and Δmdm34 cells showing mostly globular mitochondrial morphology . These morphological changes correlate with altered drug resistance profiles.

Methodological approach: Researchers could investigate AIM39-ERMES interactions through techniques such as:

• Co-immunoprecipitation

• Proximity labeling (BioID, APEX)

• Split-fluorescent protein complementation

• Genetic interaction studies (synthetic lethality/sickness screens)

• High-resolution microscopy

How do C. glabrata mitochondrial proteins contribute to survival within host immune cells?

C. glabrata mitochondrial proteins play critical roles in survival within host immune cells through several mechanisms:

• Oxidative stress tolerance: Mitochondrial proteins help counter reactive oxygen species (ROS) produced by phagocytes

• Metabolic adaptation: They enable metabolic flexibility in nutrient-limited phagosomal environments

• Drug resistance: Mitochondrial function influences resistance to host antimicrobial compounds

• Virulence factor regulation: They affect expression of factors that counter immune cell functions

The multidrug transporter CgDtr1 exemplifies these functions, as it was shown to play a role in C. glabrata pathogenesis by protecting cells from stress agents present in macrophagic cells . Deletion of CgDTR1 decreased C. glabrata's ability to proliferate in G. mellonella hemolymph and reduced tolerance to hemocyte action .

Methodological approach: Studies typically employ:

• In vitro macrophage infection models

• G. mellonella hemocyte interaction assays

• ROS measurement in infected cells

• Survival/proliferation quantification within immune cells

• Transcriptomic/proteomic analysis of intracellular fungi

What is the relationship between mitochondrial genome diversity and virulence in different C. glabrata sequence types?

The C. glabrata mitochondrial genome shows remarkable diversity across clinical isolates. Key findings include:

• Reduced conserved sequence and conserved protein-encoding genes in nonreference ST15 isolates

• Evidence for ancestral recombination in several sequence types, suggesting genetic exchange between distinct geographical regions

• Potential correlation between mitochondrial genome diversity and virulence/drug resistance phenotypes

This diversity may impact virulence through:

• Altered energy metabolism affecting growth and stress responses

• Different efficiencies in handling oxidative stress

• Variation in expression of mitochondrially-regulated virulence factors

• Differences in drug resistance mechanisms

Methodological approach: Researchers would typically:

• Compare complete mitochondrial genome sequences across clinical isolates

• Correlate mitochondrial genome features with virulence phenotypes in infection models

• Perform functional characterization of mitochondrial genes unique to specific sequence types

• Conduct population genetics analyses to understand evolutionary relationships

How can high-throughput screening approaches be used to identify novel inhibitors targeting C. glabrata mitochondrial proteins?

High-throughput screening for inhibitors targeting C. glabrata mitochondrial proteins like AIM39 would typically involve:

• Target-based approaches:

  • In vitro enzymatic assays using purified recombinant proteins

  • Thermal shift assays to identify compounds that bind and stabilize proteins

  • Fragment-based screening to identify chemical scaffolds for further development

• Cell-based approaches:

  • Phenotypic screens using C. glabrata wild-type and mitochondrial protein mutants

  • Reporter systems that indicate mitochondrial function (membrane potential, ROS production)

  • Growth inhibition assays under conditions requiring mitochondrial function

• In silico approaches:

  • Structure-based virtual screening if protein structures are available

  • Pharmacophore modeling based on known ligands

  • Machine learning predictions from existing antifungal compound data

Table 2: Comparison of screening approaches for mitochondrial protein inhibitors

What technological challenges exist in studying the role of AIM39 in mitochondrial inheritance and function?

Researchers face several significant challenges when studying mitochondrial proteins like AIM39 in C. glabrata:

• Technical challenges:

  • Limited genetic tools compared to model organisms

  • Difficulties in visualizing mitochondrial dynamics in small fungal cells

  • Challenges in purifying membrane-associated mitochondrial proteins

  • Limited structural information for fungal-specific mitochondrial proteins

• Biological complexities:

  • Genetic redundancy in mitochondrial inheritance pathways

  • Essential functions that complicate knockout studies

  • Strain variation affecting phenotypic outcomes

  • Different phenotypes in laboratory versus host conditions

• Methodological limitations:

  • Need for specialized equipment for mitochondrial imaging

  • Complexity of mitochondrial isolation procedures

  • Difficulties in reconstituting mitochondrial protein functions in vitro

  • Challenges in distinguishing direct from indirect effects on mitochondrial function

To overcome these challenges, integrative approaches combining genetic, biochemical, and imaging techniques are typically employed. Conditional expression systems, partial loss-of-function mutations, and complementary model systems like S. cerevisiae may help address some of these limitations.