Recombinant Candida glabrata Cytochrome c peroxidase, mitochondrial (CAGL0K08184g)

What is CAGL0K08184g and what is its primary function in Candida glabrata?

CAGL0K08184g encodes a mitochondrial cytochrome c peroxidase (CCP) in Candida glabrata. This enzyme (EC 1.11.1.5) catalyzes the reduction of hydrogen peroxide (H2O2) using reducing equivalents from ferrocytochrome c. Similar to other fungal CCPs, it likely functions as both a peroxidase and a H2O2 sensor protein in respiring yeast when H2O2 levels rise . The protein plays a critical role in mitochondrial reactive oxygen species (ROS) detoxification, helping protect the fungal cell from oxidative damage. The recombinant form is expressed with an amino acid sequence spanning positions 24-357 of the mature protein .

How is the recombinant CAGL0K08184g typically produced and what are its physical properties?

Recombinant CAGL0K08184g is typically produced in E. coli expression systems . The resulting protein (Uniprot: Q6FMG7) has a purity of >85% as determined by SDS-PAGE. The recombinant protein may contain a tag, though the specific tag type is determined during the manufacturing process. The protein can be produced in both lyophilized and liquid forms, with the lyophilized form offering greater stability (shelf life of 12 months at -20°C/-80°C compared to 6 months for the liquid form) .

What are the optimal storage and handling conditions for recombinant CAGL0K08184g?

For optimal storage of recombinant CAGL0K08184g:

Repeated freezing and thawing is not recommended as it can compromise protein activity. For routine experiments, it's advisable to prepare working aliquots stored at 4°C that can be used for up to one week .

How should recombinant CAGL0K08184g be reconstituted for optimal activity?

For reconstitution of lyophilized CAGL0K08184g:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Prepare small aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

The addition of glycerol helps maintain protein stability during freeze-thaw cycles and prevents the formation of ice crystals that can damage protein structure.

What analytical methods are most effective for studying CAGL0K08184g peroxidase activity?

Several methods can be employed to study CAGL0K08184g peroxidase activity:

• Spectrophotometric assays: Monitor the oxidation of ferrocytochrome c at 550 nm in the presence of H2O2 and CAGL0K08184g. This allows real-time measurement of enzyme kinetics.

• High-performance LC-MS/MS: This technique can be used to investigate the oxidation patterns of the protein when exposed to H2O2, similar to studies done with other cytochrome c peroxidases . This method can identify specific oxidized residues and reveal mechanisms such as hole hopping.

• Oxygen electrode measurements: Useful for monitoring oxygen consumption during the catalytic cycle.

• ROS detection assays: Fluorescent probes such as 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) can be used to measure ROS levels in the presence or absence of active CAGL0K08184g.

• EPR spectroscopy: This can be used to detect and characterize transient radical species formed during the catalytic cycle, though it may be biased toward radicals with low O2 reactivity .

How does CAGL0K08184g function as a H2O2 sensor in Candida glabrata?

Based on studies of similar cytochrome c peroxidases in yeast, CAGL0K08184g likely functions as a H2O2 sensor when hydrogen peroxide levels rise during respiration. The mechanism involves:

• H2O2 binding to the heme group of CAGL0K08184g

• The availability of ferrocytochrome c (CycII) determining whether the protein acts as a sensor or peroxidase

• When CycII is limited, H2O2 modifies the protein, generating a signal

• This modification likely involves oxidation of specific amino acid residues (particularly methionines and tryptophans) and formation of dityrosine crosslinks

The sensing function is particularly important during oxidative stress conditions when H2O2 levels rise above normal physiological concentrations. In this context, CAGL0K08184g may trigger downstream adaptive responses that help C. glabrata survive in oxidative environments, such as those encountered during host immune responses.

What is the mechanism of hole hopping in cytochrome c peroxidase and why is it significant?

Hole hopping in cytochrome c peroxidase is a protective mechanism that:

• Involves the transfer of oxidizing equivalents (electron holes) from the heme to specific amino acid residues in the protein

• Occurs in a directed manner toward specific zones rich in redox-active residues

• Prevents irreversible oxidative damage to the heme group

• Results in the oxidation of approximately 20 oxygen atoms, predominantly at methionine and tryptophan residues

• Leads to extensive intramolecular dityrosine crosslinking of neighboring residues

This mechanism is significant because it protects the catalytic center (heme) from irreversible oxidative damage while facilitating controlled heme labilization, which may be important for H2O2 signaling. The oxidation of the proximal heme ligand H175 to oxo-histidine labilizes the heme, but catastrophic heme oxidation is avoided through the hole hopping mechanism until oxidation of the catalytic distal H52 eventually shuts down heterolytic cleavage of H2O2 .

How does mitochondrial function in C. glabrata relate to antifungal resistance mechanisms?

Mitochondrial function in C. glabrata plays a multifactorial role in antifungal resistance, particularly to echinocandins:

• ROS production: Echinocandin exposure induces increased ROS production, though inhibitors of mitochondrial complexes I and IV can reduce both echinocandin-induced ROS formation and cell killing .

• Metabolic adaptation: Mitochondrial metabolism influences the cell's ability to adapt to antifungal stress, with changes in metabolic activity potentially leading to increased ATP production.

• Downstream survival mechanisms: Mitochondrial status influences cell survival downstream of the drug-target interaction, as evidenced by the fact that enzyme sensitivity to echinocandins remains unchanged in mitochondrial mutants despite altered tolerance .

• Genetic diversity: The C. glabrata mitochondrial genome shows remarkable diversity, with reduced conserved sequences and protein-encoding genes in certain sequence types, which may contribute to strain-specific differences in antifungal susceptibility .

This connection between mitochondrial function and antifungal resistance suggests that CAGL0K08184g, as a mitochondrial protein involved in ROS handling, may indirectly contribute to C. glabrata's ability to tolerate certain antifungal drugs.

How conserved is CAGL0K08184g across fungal species and what can ortholog studies reveal?

CAGL0K08184g (Q6FMG7) is part of multiple protein ortholog groups, indicating evolutionary relationships with proteins in other species:

These orthologous relationships suggest that while the core peroxidase function is conserved, the protein has evolved specific adaptations in different organisms. Studying these orthologs can reveal:

• Evolutionarily conserved regions critical for catalytic function

• Species-specific adaptations that may relate to niche-specific stress responses

• Potential functional divergence of peroxidases across evolutionary time

The presence of orthologs in diverse organisms from plants to other fungi highlights the fundamental importance of peroxidase functions across different biological kingdoms.

How has CAGL0K08184g evolved in clinical isolates of C. glabrata?

Clinical isolates of C. glabrata show significant genetic diversity, with at least 19 distinct sequence types identified in global populations and evidence of ancestral recombination . While specific evolution of CAGL0K08184g in clinical isolates hasn't been directly addressed in the available search results, the broader genomic context suggests:

• Microevolution within patients during recurrent infections leads to enrichment for nonsynonymous and frameshift indels in cell surface proteins

• Genes involved in drug resistance, including ergosterol synthesis genes and echinocandin targets, are particularly subject to selective pressure during patient infection

• The mitochondrial genome of C. glabrata shows particular diversity, with reduced conserved sequences and protein-encoding genes in non-reference ST15 isolates

Given that CAGL0K08184g is a mitochondrial protein, it may be subject to selection pressures in the context of this mitochondrial genome diversity. Further research specifically examining variation in CAGL0K08184g across clinical isolates would be valuable for understanding its potential role in adaptation during infection.

How can researchers leverage CAGL0K08184g for developing novel antifungal strategies?

CAGL0K08184g could be leveraged for novel antifungal strategies in several ways:

• Target for combinatorial therapy: Given the connection between mitochondrial function and echinocandin tolerance , inhibitors targeting CAGL0K08184g might sensitize C. glabrata to existing antifungals when used in combination.

• Biomarker development: Changes in CAGL0K08184g expression or activity could serve as biomarkers for monitoring antifungal efficacy or predicting resistance development.

• Structural biology approaches: Determining the three-dimensional structure of CAGL0K08184g could facilitate structure-based drug design to develop specific inhibitors.

• ROS modulation strategies: Since antifungal exposure increases ROS levels and mitochondrial inhibitors can reduce both ROS formation and cell killing , CAGL0K08184g's role in ROS handling makes it a potential target for modulating cellular responses to antifungals.

• Genetic engineering tools: CAGL0K08184g could be used as a sensor component in engineered genetic circuits designed to detect oxidative stress or trigger specific cellular responses.

Methodologically, these applications would require interdisciplinary approaches combining structural biology, medicinal chemistry, genetic engineering, and clinical microbiology.

What experimental challenges exist when studying CAGL0K08184g in the context of host-pathogen interactions?

Studying CAGL0K08184g in host-pathogen interactions presents several methodological challenges:

• Distinguishing host and pathogen responses: In co-culture experiments, separating the oxidative burst produced by host immune cells from the fungal ROS response can be technically challenging.

• Mitochondrial isolation: Obtaining pure, functional mitochondria from C. glabrata during infection models requires specialized techniques to preserve enzyme activity.

• In vivo imaging: Visualizing CAGL0K08184g activity in real-time during infection requires development of specific biosensors or activity-based probes.

• Genetic manipulation: While C. glabrata is genetically tractable, creating targeted mutations in mitochondrial proteins requires specialized approaches to ensure proper targeting and expression.

• Strain variation: The genetic diversity of clinical C. glabrata isolates necessitates testing hypotheses across multiple strain backgrounds to ensure generalizability of findings.

Researchers should consider employing techniques such as:

• Fluorescent protein tagging for localization studies

• Activity-based protein profiling for functional analysis

• Single-cell RNA sequencing to capture heterogeneous responses

• Isotope labeling to track metabolic changes during infection

What are the critical methodological considerations for studying hole hopping mechanisms in CAGL0K08184g?

To effectively study hole hopping mechanisms in CAGL0K08184g, researchers should consider these methodological approaches:

A comprehensive approach would combine these methods to build a detailed map of the electron transfer network within CAGL0K08184g and understand how this network contributes to both peroxidase activity and H2O2 sensing function.

How does CAGL0K08184g activity correlate with virulence in C. glabrata infections?

While direct evidence linking CAGL0K08184g to virulence isn't provided in the search results, its role as a mitochondrial protein involved in oxidative stress response suggests potential connections:

• Oxidative stress resistance: As C. glabrata encounters oxidative bursts from host immune cells during infection, CAGL0K08184g likely contributes to fungal survival by detoxifying ROS.

• Mitochondrial function and fitness: The proper functioning of mitochondrial proteins, including CAGL0K08184g, supports metabolic adaptability during infection. This is particularly important as C. glabrata must adapt to different nutrient conditions in various host niches.

• Evolutionary selection: Clinical isolates show signatures of positive selection in genes that facilitate adhesion to epithelial cells . If CAGL0K08184g indirectly supports these processes through maintaining cellular redox homeostasis, it may contribute to virulence.

• Microevolution during infection: The observation that genes undergo microevolution during recurrent infections suggests that proteins like CAGL0K08184g may adapt to optimize function in the host environment.

Research methodologies to investigate these connections would include comparative analysis of CAGL0K08184g expression and activity across isolates with differing virulence profiles, and construction of gene knockouts or conditional mutants for in vivo virulence studies.

What role might CAGL0K08184g play in C. glabrata's adaptation to antifungal treatment?

CAGL0K08184g may contribute to C. glabrata's adaptation to antifungal treatment through several mechanisms:

• Mitochondrial stress response: Since mitochondrial function influences echinocandin tolerance , CAGL0K08184g's role in maintaining mitochondrial redox balance may indirectly support adaptation to these drugs.

• ROS management: Antifungal exposure increases ROS production . CAGL0K08184g's peroxidase activity could help mitigate this stress, potentially contributing to cell survival.

• Metabolic adaptation: Changes in mitochondrial function during antifungal exposure may require adjustments in the activity of proteins like CAGL0K08184g to maintain cellular homeostasis.

• Genetic diversity: The diverse mitochondrial genome in C. glabrata clinical isolates suggests that mitochondrial proteins may show strain-specific adaptations that affect antifungal responses.

To study these potential roles, researchers could:

• Compare CAGL0K08184g expression before and after antifungal exposure

• Examine protein modifications during drug treatment

• Analyze correlations between CAGL0K08184g sequence variants and antifungal susceptibility profiles

• Create conditional mutants to test the impact of CAGL0K08184g activity levels on drug tolerance

This research could potentially identify CAGL0K08184g as a biomarker for antifungal response or as a target for adjunctive therapies to enhance antifungal efficacy.

What are the most critical unanswered questions regarding CAGL0K08184g function?

Several critical questions about CAGL0K08184g remain unanswered:

• Structure-function relationship: How does the three-dimensional structure of CAGL0K08184g relate to its dual functions in peroxidase activity and H2O2 sensing?

• Regulation mechanisms: What cellular signals regulate CAGL0K08184g expression and activity during different stress conditions?

• Interaction partners: Does CAGL0K08184g form complexes with other proteins in the mitochondria or participate in signaling networks?

• Genetic variation: How do sequence variations in CAGL0K08184g across clinical isolates affect its function and contribution to antifungal responses?

• Post-translational modifications: Beyond oxidative modifications, what other post-translational modifications regulate CAGL0K08184g activity?

Research addressing these questions would significantly advance our understanding of this protein's role in C. glabrata biology and pathogenesis.

How can advanced technologies be applied to study CAGL0K08184g in the context of fungal pathogenesis?

Advanced technologies that could deepen our understanding of CAGL0K08184g include:

• Cryo-electron microscopy: To determine high-resolution structures of CAGL0K08184g in different functional states.

• CRISPR-Cas9 genome editing: For precise genetic manipulation to create point mutations or domain swaps that probe specific aspects of CAGL0K08184g function.

• Single-cell proteomics: To examine CAGL0K08184g expression and modification heterogeneity within fungal populations during infection or drug treatment.

• In vivo imaging with genetically encoded sensors: To visualize CAGL0K08184g activity or ROS levels in real-time during infection processes.

• Metabolomics: To comprehensively analyze how changes in CAGL0K08184g activity affect cellular metabolic profiles during different stress conditions.

• Machine learning approaches: To predict functional consequences of CAGL0K08184g sequence variations observed in clinical isolates.

• Organoid infection models: To study CAGL0K08184g function in more physiologically relevant host-pathogen interaction systems.