Recombinant Candida glabrata Autophagy-related protein 31 (CIS1)

How does Atg31/CIS1 relate to virulence in Candida glabrata infections?

Autophagy proteins, including Atg31, likely contribute to C. glabrata virulence by enhancing survival during phagocytosis and nutrient limitation within host tissues. Research on the autophagy-inducing factor Atg1 has demonstrated that autophagy-deficient strains (Cgatg1Δ) show significantly decreased survival when phagocytosed by macrophages and reduced colony-forming units (CFUs) in mouse models of disseminated and intra-abdominal candidiasis .

The specific contribution of Atg31 to virulence remains to be fully characterized, but based on its role in the autophagy pathway, it likely enhances resistance to oxidative stress generated by immune cells and adaptation to nutrient-limited environments in the host. Researchers should design comparative virulence studies between wild-type, Atg31-deficient, and complemented strains in both macrophage infection models and animal models of candidiasis.

What methodologies are recommended for detecting Atg31/CIS1 expression in C. glabrata?

For detecting Atg31 expression in C. glabrata, researchers should consider multiple complementary approaches:

• RT-qPCR for transcript analysis under different stress conditions

• Western blotting using epitope-tagged Atg31 (e.g., GFP, HA, or FLAG tags)

• Fluorescence microscopy with GFP-tagged Atg31 to observe localization patterns

When analyzing expression patterns, it's important to examine Atg31 under conditions known to induce autophagy in C. glabrata, including nitrogen starvation and hydrogen peroxide exposure . A time-course experiment would be valuable to determine the kinetics of Atg31 expression in response to these stressors.

What expression systems are optimal for producing recombinant C. glabrata Atg31/CIS1?

For successful expression of recombinant C. glabrata Atg31/CIS1, consider these expression systems with their respective advantages:

For initial characterization studies, an E. coli system with a fusion tag (His6 or GST) may be most practical. For functional studies, yeast expression systems would better preserve native protein conformation and activity. When using SC-trp or YPD agar media for C. glabrata cultivation, maintain consistent growth conditions (30°C) as used in autophagy research protocols .

What are the recommended protocols for purifying recombinant Atg31/CIS1?

A systematic purification strategy for recombinant C. glabrata Atg31/CIS1 should include:

• Affinity Chromatography:

  • For His-tagged proteins: Ni-NTA resin with imidazole gradient elution (20-250 mM)

  • For GST-tagged proteins: Glutathione Sepharose with reduced glutathione elution

• Ion Exchange Chromatography:

  • Based on Atg31's predicted isoelectric point

  • Use anion exchange (Q Sepharose) if pI < 7.0

  • Use cation exchange (SP Sepharose) if pI > 7.0

• Size Exclusion Chromatography:

  • Final polishing step using Superdex 75/200 columns

  • Assess oligomeric state and homogeneity

Buffer optimization is critical, particularly considering that Atg31 functions in the context of autophagy protein complexes. Include reducing agents (1-5 mM DTT or 1-2 mM β-mercaptoethanol) to prevent oxidation, and protease inhibitors to minimize degradation. For functional studies, ensure purification under non-denaturing conditions to maintain native protein conformation.

How can researchers effectively verify the functionality of purified recombinant Atg31/CIS1?

To verify functionality of purified recombinant Atg31/CIS1, implement these complementary approaches:

• Protein-Protein Interaction Assays:

  • Co-immunoprecipitation with known binding partners (Atg17, Atg29)

  • Pull-down assays with GST-tagged Atg31 to identify interacting proteins

  • Surface plasmon resonance to quantify binding affinities

• In vitro Reconstitution Assays:

  • Assemble minimal PAS components with purified proteins

  • Monitor complex formation via size-exclusion chromatography or light scattering

• Complementation of Atg31-deficient C. glabrata:

  • Transform atg31Δ strains with recombinant Atg31

  • Assess restoration of autophagy using:

    • GFP-Atg8 processing assays

    • Electron microscopy for autophagosome formation

    • Survival under nitrogen starvation and H₂O₂ stress

Functionality verification should include positive controls (wild-type Atg31) and negative controls (mutated Atg31 lacking key functional domains). Research on autophagy-deficient C. glabrata strains demonstrates that functional autophagy proteins confer resistance to nitrogen starvation and oxidative stress, which can serve as phenotypic indicators of protein functionality .

How does Atg31/CIS1 interact with the ROS response pathway in C. glabrata during infection?

Research on C. glabrata autophagy proteins indicates a significant interplay between autophagy and reactive oxygen species (ROS) management. Autophagy-deficient strains (Cgatg1Δ) exhibit elevated intracellular ROS levels even under normal growth conditions, suggesting that functional autophagy is essential for ROS homeostasis . For Atg31/CIS1 research, investigators should examine:

• ROS Level Assessment:

  • Measure baseline and stress-induced ROS in wild-type vs. atg31Δ strains using fluorescent probes (DCFH-DA, DHE)

  • Compare ROS production during macrophage phagocytosis between wild-type and atg31Δ strains

• Oxidative Stress Response Gene Expression:

  • Analyze expression of catalase genes (CTA1) and other antioxidant enzymes in atg31Δ strains

  • Perform RNA-seq comparing wild-type and atg31Δ strains under H₂O₂ challenge

• Interaction with Antioxidant Systems:

  • Investigate potential direct interactions between Atg31 and components of antioxidant defense

  • Determine if Atg31-mediated autophagy selectively degrades damaged mitochondria (mitophagy) to prevent ROS accumulation

When examining the relationship between Atg31 and oxidative stress, researchers should note that autophagy-deficient C. glabrata strains show sensitivity to H₂O₂ and display growth defects that can be partially rescued by overexpression of catalase (CTA1) , suggesting complex interactions between autophagy and oxidative stress response.

What structural and biochemical differences exist between C. glabrata Atg31/CIS1 and its orthologs in non-pathogenic yeasts?

Investigating structural and biochemical differences between C. glabrata Atg31/CIS1 and orthologs in non-pathogenic yeasts requires:

• Comparative Sequence Analysis:

  • Perform multiple sequence alignment of Atg31 sequences from C. glabrata, S. cerevisiae, and other Candida species

  • Identify conserved domains and pathogen-specific sequence variations

• Structural Characterization:

  • Determine secondary structure elements using circular dichroism spectroscopy

  • Pursue X-ray crystallography or cryo-EM of Atg31 alone and in complex with binding partners

  • Use homology modeling based on solved structures if available

• Functional Domain Analysis:

  • Generate chimeric proteins exchanging domains between C. glabrata and S. cerevisiae Atg31

  • Assess functionality of chimeras in complementation assays

  • Map interaction domains through truncation and site-directed mutagenesis

Research should focus on identifying adaptations specific to pathogenic contexts, as autophagy in C. glabrata contributes to virulence by enhancing survival during macrophage phagocytosis and in nutrient-limited host environments . Examining differences in protein-protein interaction networks between pathogenic and non-pathogenic species may reveal virulence-associated adaptations of the autophagy machinery.

How can researchers design effective C. glabrata Atg31/CIS1 knockout and complementation systems for in vivo studies?

For effective genetic manipulation of C. glabrata Atg31/CIS1, researchers should implement:

• Knockout Strategy:

  • Use homologous recombination with selection markers (e.g., TRP1) flanked by sequences upstream and downstream of ATG31

  • Verify deletion by PCR, Southern blotting, and RT-PCR

  • Confirm autophagy deficiency using GFP-Atg8 processing assays

• Complementation System Design:

  • Create centromeric or integrative plasmids containing ATG31 under its native promoter

  • Include epitope tags (HA, FLAG) or fluorescent protein fusions for tracking

  • Use selection markers different from those used for knockout generation

• Conditional Expression Systems:

  • Implement tetracycline-regulated or methionine-repressible promoters for controlled expression

  • Engineer auxin-inducible degron systems for rapid protein depletion

For in vivo studies, considerations should include:

• Generate multiple independent knockout and complemented strains to control for off-target effects

• Include wild-type revertants to confirm phenotype restoration

• Design point mutations affecting specific functions rather than complete knockouts for nuanced analysis

Based on techniques used for studying Atg1 in C. glabrata , similar genetic approaches should be applicable for Atg31/CIS1 research, with careful attention to strain verification and controlled expression systems.

What are common challenges in detecting autophagy-related phenotypes in Atg31/CIS1 mutant C. glabrata strains?

Researchers investigating Atg31/CIS1 function in C. glabrata may encounter these challenges:

• Phenotypic Redundancy:

  • Multiple autophagy pathways may compensate for specific protein deficiencies

  • Solution: Combine Atg31 deletion with other autophagy protein mutations to reveal masked phenotypes

• Variable Stress Responses:

  • Inconsistent autophagy induction under different stress conditions

  • Solution: Standardize stress application protocols and measure multiple stress markers simultaneously

• Strain Background Effects:

  • Different C. glabrata clinical isolates may show variable autophagy responses

  • Solution: Use multiple strain backgrounds and include appropriate isogenic controls

• Detection Sensitivity Limitations:

  • Subtle autophagy defects may be difficult to quantify

  • Solution: Combine multiple detection methods (biochemical, microscopic, and genetic)

For autophagy induction evaluation, researchers should examine nitrogen starvation responses using SD-N media and oxidative stress responses using H₂O₂ treatment at standardized concentrations. When assessing viability, complement CFU counts with metabolic activity assays and membrane integrity tests for comprehensive evaluation.

How can researchers address solubility and stability issues with recombinant C. glabrata Atg31/CIS1?

To overcome common solubility and stability challenges with recombinant Atg31/CIS1:

When designing expression constructs, consider domain boundaries based on structural predictions to avoid disrupting functional domains. Including stabilizing binding partners may significantly improve solubility, as Atg31 naturally functions in complex with other autophagy proteins like Atg17 and Atg29.

What experimental controls are essential when investigating C. glabrata Atg31/CIS1 function during host-pathogen interactions?

When studying C. glabrata Atg31/CIS1 in host-pathogen interactions, include these essential controls:

• Strain Controls:

  • Wild-type parental strain

  • Atg31 deletion mutant

  • Complemented strain with reintroduced ATG31

  • Multiple independent isolates of each strain

• Experimental Controls for Macrophage Infection Models:

  • Uninfected macrophages

  • Heat-killed C. glabrata strains

  • Known autophagy-deficient strains (e.g., atg1Δ) as positive controls

  • Phagocytosis inhibitors to distinguish between attached and internalized fungi

• Environmental Condition Controls:

  • Standard growth media (SC-trp, YPD)

  • Nitrogen starvation conditions (SD-N)

  • Various H₂O₂ concentrations to assess oxidative stress resistance

  • Time-course measurements to capture dynamic responses

• Molecular Controls for Protein Interaction Studies:

  • Empty vector controls

  • Irrelevant protein controls

  • Known interaction partners as positive controls

  • Mutated binding sites as specificity controls

Based on research with C. glabrata Atg1 , macrophage co-culture experiments should include viability assessments at multiple time points (24, 48, 72, and 96 hours) to capture the full spectrum of autophagy-dependent survival effects in the context of host immune cells.

What are promising approaches for targeting C. glabrata Atg31/CIS1 in antifungal research?

Building on evidence that autophagy contributes to C. glabrata virulence , researchers exploring Atg31/CIS1 as an antifungal target should consider:

• Structure-Based Drug Design:

  • Determine high-resolution structures of Atg31 and its complexes

  • Identify druggable pockets, particularly at protein-protein interaction interfaces

  • Use fragment-based screening to identify starting compounds

• Functional Inhibition Strategies:

  • Develop peptide inhibitors mimicking key interaction domains

  • Screen for small molecules disrupting Atg31-Atg29-Atg17 complex formation

  • Design interfering RNA or antisense oligonucleotides for gene silencing

• Combination Therapy Approaches:

  • Test autophagy inhibitors with conventional antifungals (azoles, echinocandins)

  • Combine with oxidative stress inducers to exploit vulnerability of autophagy-deficient strains

  • Pair with immunomodulators to enhance host immune response

• Selective Targeting Strategies:

  • Focus on unique structural features of C. glabrata Atg31 not present in human homologs

  • Exploit pathogen-specific regulation of autophagy

  • Target fungal-specific post-translational modifications

These approaches should be evaluated in both in vitro systems and animal models of candidiasis to assess efficacy, specificity, and potential for resistance development. The high resistance of C. glabrata to some antifungal medications underscores the importance of novel therapeutic targets like autophagy proteins.

How might C. glabrata Atg31/CIS1 function differ in biofilm formation versus planktonic growth?

Investigating Atg31/CIS1 function in biofilm formation presents an important research direction:

• Comparative Expression Analysis:

  • Measure Atg31 expression in planktonic versus biofilm conditions

  • Perform temporal analysis during biofilm development stages

  • Use RNA-seq to identify co-regulated genes in both growth modes

• Biofilm Formation Assessment:

  • Compare wild-type and atg31Δ strains for:

    • Adhesion capacity to various surfaces

    • Biofilm architecture using confocal microscopy

    • Extracellular matrix composition

    • Resistance to antifungal agents

• Stress Response in Biofilms:

  • Evaluate oxidative stress markers within biofilms

  • Assess nutrient gradient effects on autophagy activation

  • Determine spatial distribution of autophagy activity in mature biofilms

• Host-Biofilm Interactions:

  • Examine immune cell interactions with wild-type versus atg31Δ biofilms

  • Assess inflammatory responses to different biofilm compositions

  • Determine persistence in mixed species biofilms

Understanding Atg31's role in biofilm formation could reveal new targets for disrupting this virulence mechanism of C. glabrata. Since autophagy contributes to stress resistance , it may play crucial roles in the adaptation to the nutrient-limited and oxidatively stressed environment within mature biofilms.