Recombinant Candida glabrata Mitochondrial inner membrane magnesium transporter mrs2 (MRS2)

What is the role of the MRS2 magnesium transporter in Candida glabrata mitochondrial function?

Mrs2 in Candida glabrata functions as an essential mitochondrial inner membrane magnesium transporter with structural similarity to the bacterial CorA transporter. The protein contains two membrane-spanning domains in its carboxyl-terminal half, with a large amino-terminal and a shorter carboxyl-terminal part exposed to the mitochondrial matrix space .

In yeast systems, MRS2 disruption leads to significant decreases in total mitochondrial magnesium content, which impairs mitochondrial function . Proper magnesium homeostasis is critical for mitochondrial membrane potential maintenance, which research has shown is essential for C. glabrata survival under stress conditions, particularly during host infection .

While most direct experimental evidence comes from Saccharomyces cerevisiae, the high conservation of mitochondrial transporters across fungal species indicates similar functionality in C. glabrata. Researchers investigating MRS2 should consider these functional characteristics:

How does mitochondrial function in C. glabrata relate to its pathogenicity and virulence?

Mitochondrial function is directly linked to C. glabrata pathogenicity through several mechanisms. Under iron-depleted conditions (common during host infection), C. glabrata induces mitophagy to maintain mitochondrial integrity, which contributes to its survival in the host .

Specifically, mitochondrial membrane potential in C. glabrata influences its ability to survive under stress conditions, with research showing that the mitophagy-deficient atg32Δ mutant exhibits decreased mitochondrial membrane potential under iron-depleted conditions. In mouse models of disseminated infection, the atg32Δ strain demonstrates significantly decreased kidney and spleen fungal burdens compared to wild-type strains , indicating that proper mitochondrial function is essential for virulence.

As a mitochondrial magnesium transporter, MRS2 likely contributes to these pathogenicity mechanisms by maintaining proper mitochondrial function during host colonization and infection. Researchers should consider investigating:

• The impact of MRS2 deletion on C. glabrata virulence in animal models

• Changes in MRS2 expression during different stages of infection

• The relationship between mitochondrial magnesium levels and stress adaptation during host colonization

What methods are most effective for expressing and purifying recombinant C. glabrata MRS2 protein?

For successful expression and purification of recombinant C. glabrata MRS2, researchers should consider the following approach based on established protocols for similar mitochondrial membrane proteins:

• Expression System Selection: E. coli expression systems with N-terminal His-tags have proven successful for expressing MRS2 from related fungal species, such as Schizosaccharomyces pombe .

• Protein Construct Design:

  • Include the mature protein sequence (excluding the mitochondrial targeting sequence)

  • Add an N-terminal His-tag for purification

  • Consider using codon optimization for E. coli expression

• Purification Protocol:

  • Lyse cells in Tris/PBS-based buffer

  • Use immobilized metal affinity chromatography (IMAC) for initial purification

  • Add 6% trehalose to the storage buffer at pH 8.0 to enhance stability

  • Store in aliquots at -80°C to prevent repeated freeze-thaw cycles

• Reconstitution: Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL, adding 5-50% glycerol for long-term storage .

For functional studies, researchers should validate the activity of the recombinant protein through magnesium transport assays using reconstituted proteoliposomes.

How can CRISPR-Cas9 be utilized to study MRS2 function in C. glabrata?

CRISPR-Cas9 genome engineering offers a powerful approach for studying MRS2 function in C. glabrata. Researchers can implement this methodology following these key steps:

• System Implementation: Develop a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system. This can be achieved using either S. cerevisiae promoters (pSNR52 or pTEF1) or C. glabrata endogenous promoters (pRNAH1 or pCYC1) .

• Guide RNA Selection: Design efficient guide RNAs targeting the MRS2 gene using online tools like CASTING, which facilitates the selection of the most efficient guide RNAs for a given C. glabrata gene .

• Mutant Identification: Identify mutant strains using the Surveyor technique and sequencing. For MRS2 knockout verification, researchers should assess:

  • Mitochondrial magnesium content

  • Mitochondrial membrane potential using fluorescent dyes like MitoBright LT Red

  • Growth defects under various stress conditions

• Functional Validation: Test the virulence of mutants in vivo using established infection models such as Drosophila melanogaster or mouse models of disseminated infection .

This approach allows for precise genetic manipulation of MRS2 to study its role in mitochondrial function, stress response, and pathogenicity.

What is the relationship between MRS2 and antifungal drug resistance in C. glabrata?

While direct evidence linking MRS2 to antifungal resistance in C. glabrata is limited, several lines of evidence suggest a potential relationship:

• Mitochondrial dysfunction or morphological abnormalities in pathogenic fungi have been shown to contribute to azole resistance . As a critical component of mitochondrial function, MRS2 may influence this resistance mechanism.

• C. glabrata has emerged as a major health threat due to its increasing resistance to multiple drug classes, including triazoles and echinocandins . This multidrug resistance is often associated with genetic diversity and adaptation mechanisms.

• The prevalence of mismatch repair gene (MSH2) mutations in clinical C. glabrata isolates (55% of isolates) correlates with increased frequency of drug-resistant mutants . These mutations often predate the emergence of antifungal resistance .

Researchers investigating the role of MRS2 in drug resistance should examine:

How do iron availability and environmental stress affect MRS2 function in C. glabrata?

Iron availability significantly impacts mitochondrial function in C. glabrata, with iron depletion promoting mitophagy to maintain mitochondrial integrity . While direct data on MRS2 regulation under iron limitation is not available, research suggests the following relationships:

• Under iron-depleted conditions (common during host infection), C. glabrata activates mitophagy through the ATG32 pathway . This process is critical for mitochondrial quality control and cellular longevity.

• The mitochondrial membrane potential in C. glabrata is affected by iron availability, with iron depletion potentially altering the function of mitochondrial membrane proteins including transporters .

• C. glabrata exhibits a complex environmental stress response (ESR) involving transcription factors CgMsn2 and CgMsn4 , which likely regulates mitochondrial function under various stress conditions.

Researchers studying MRS2 under environmental stress should:

• Monitor MRS2 expression and localization under different iron concentrations

• Assess interactions between MRS2 and mitophagy machinery

• Evaluate magnesium transport activity in mitochondria isolated from iron-depleted cells

• Investigate potential regulatory mechanisms involving stress-responsive transcription factors like CgMsn2/4

How does C. glabrata MRS2 compare to homologous proteins in other pathogenic fungi?

Comparative analysis of MRS2 across pathogenic fungi reveals significant conservation with distinct species-specific features:

• Sequence Conservation: MRS2 proteins from C. glabrata, S. cerevisiae, and S. pombe share conserved domains, particularly in the membrane-spanning regions and the magnesium-binding motif .

• Functional Complementation: Bacterial CorA can functionally substitute for the MRS2 gene in yeast, indicating conservation of core transport function across bacterial and fungal kingdoms .

• Species-Specific Adaptations: While core functions are conserved, MRS2 proteins likely have species-specific adaptations related to the particular environments and stresses encountered by different fungal pathogens.

The amino acid sequence alignment of mature MRS2 proteins shows significant conservation in the membrane domains and magnesium-binding sites, with S. pombe MRS2 (373 amino acids, residues 50-422) showing structural similarities to the predicted C. glabrata homolog .

This conservation allows researchers to leverage knowledge from model organisms while investigating the unique properties of C. glabrata MRS2 in pathogenicity and drug resistance contexts.

What techniques can be used to measure MRS2-mediated magnesium transport in C. glabrata mitochondria?

To accurately measure MRS2-mediated magnesium transport in C. glabrata mitochondria, researchers can employ several complementary techniques:

• Isolated Mitochondria Assays:

  • Isolate intact mitochondria from C. glabrata using differential centrifugation

  • Measure magnesium uptake using fluorescent indicators (Mag-Fura-2) or radioactive tracers (²⁸Mg²⁺)

  • Compare transport rates between wild-type and MRS2-deleted strains

• Membrane Potential Monitoring:

  • Use fluorescent dyes like MitoBright LT Red or TMRM to assess mitochondrial membrane potential

  • Monitor changes in membrane potential in response to magnesium availability

  • Determine the effect of MRS2 deletion on membrane potential maintenance

• Recombinant Protein Reconstitution:

  • Express and purify recombinant MRS2 protein

  • Reconstitute into proteoliposomes

  • Measure magnesium flux using stopped-flow spectrofluorometry

• In vivo Magnesium Monitoring:

  • Use genetically encoded magnesium sensors expressed in the mitochondrial matrix

  • Perform real-time imaging of mitochondrial magnesium in living cells

  • Correlate magnesium levels with cellular responses to stress

These methodologies provide complementary data on MRS2 function and can help elucidate its role in mitochondrial physiology and stress adaptation.

How does MRS2 contribute to mitochondrial quality control and cell longevity in C. glabrata?

MRS2's contribution to mitochondrial quality control and cell longevity in C. glabrata likely involves several interconnected mechanisms:

• Maintaining Magnesium Homeostasis: As the principal mitochondrial magnesium transporter, MRS2 ensures optimal magnesium concentrations for mitochondrial enzyme function and ATP production .

• Supporting Mitochondrial Membrane Potential: Proper magnesium levels are essential for maintaining mitochondrial membrane potential, which research has shown is critical for C. glabrata survival under stress conditions .

• Facilitating Stress Adaptation: Under iron-depleted conditions (common during host infection), C. glabrata activates mitophagy to maintain mitochondrial integrity . MRS2-mediated magnesium transport likely supports this adaptation process.

• Influencing Mitochondrial Dynamics: Research on mitochondrial morphology in C. glabrata has shown that proteins like Gem1 control mitochondrial distribution and morphology . MRS2-mediated magnesium transport may interact with these processes to maintain mitochondrial network integrity.

Research has demonstrated that mitophagy-deficient atg32Δ mutants of C. glabrata exhibit decreased longevity under iron-deficient conditions, with significantly lower mitochondrial membrane potential than wild-type cells . As a critical mitochondrial transporter, MRS2 likely plays a role in this longevity mechanism.

What role does MRS2 play in the adaptation of C. glabrata to the host environment during infection?

During infection, C. glabrata faces numerous host-imposed stresses including nutrient limitation, immune attack, and antifungal treatments. MRS2 likely contributes to adaptation through:

• Iron Limitation Response: The host environment is iron-poor due to iron-chelating proteins like transferrin . Under these conditions, C. glabrata induces mitophagy to maintain mitochondrial quality control, a process that likely involves MRS2-mediated magnesium homeostasis.

• Stress Response Coordination: C. glabrata possesses a complex environmental stress response (ESR) system involving transcription factors CgMsn2 and CgMsn4 . MRS2 may be regulated as part of this stress response network to optimize mitochondrial function under challenging conditions.

• Metabolic Adaptation: As a commensal organism that has evolved into a pathogen, C. glabrata must adapt to different nutrient environments during colonization and infection . MRS2's role in maintaining mitochondrial function supports this metabolic flexibility.

• Antifungal Resistance: Mitochondrial dysfunction has been linked to azole resistance in pathogenic fungi . By maintaining proper mitochondrial function, MRS2 may indirectly influence the development of drug resistance, particularly considering that 55% of clinical C. glabrata isolates carry mutations in the mismatch repair gene MSH2 .

In mouse models of disseminated infection, mitochondrial function has been shown to impact C. glabrata virulence , suggesting that MRS2's role in maintaining mitochondrial integrity is clinically relevant.

How can genetic diversity studies in C. glabrata inform research on mitochondrial transporters like MRS2?

Genetic diversity studies in C. glabrata provide valuable context for understanding mitochondrial transporter function and evolution:

• Sequence Type Correlation: Multilocus sequence typing (MLST) has identified over 100 sequence types (STs) in C. glabrata, with ST7 (65.8%) and ST3 (7.6%) being most common in Chinese isolates . These genetic backgrounds may influence MRS2 expression and function.

• Geographical Variation: MSH2 genotypes appear to be geographically dependent, with specific mutations predominating in different regions . This geographical structuring may extend to mitochondrial genes like MRS2.

• Evolutionary Insights: Comparative genomic analysis has identified highly polymorphic loci in C. glabrata , which can help identify regions under selective pressure. Analyzing MRS2 across these diverse genetic backgrounds can reveal evolutionary adaptations.

• Clinical Correlations: Some sequence types are associated with specific drug resistance profiles . Investigating MRS2 variants in these backgrounds may reveal functional differences relevant to pathogenicity and treatment response.

For MRS2 research, genetic diversity data suggests:

• Examining MRS2 sequence variation across different C. glabrata lineages

• Investigating whether specific MRS2 variants correlate with clinical outcomes

• Determining if MRS2 function differs in genetic backgrounds associated with drug resistance

• Exploring co-evolution of MRS2 with other mitochondrial components across C. glabrata diversity