Recombinant Candida glabrata Ubiquitin-like modifier-activating enzyme ATG7 (ATG7), partial

What is the functional role of ATG7 in Candida glabrata autophagy?

ATG7 functions as an E1-like activating enzyme involved in two ubiquitin-like systems required for cytoplasm to vacuole transport (Cvt) and autophagy. It specifically activates ATG12 for its conjugation with ATG5 as well as the ATG8 family proteins for their conjugation with phosphatidylethanolamine. Both systems are essential for the ATG8 association to Cvt vesicles and autophagosome membranes, which are critical structures in the autophagy process . This activation function is analogous to the role of ATG7 in other organisms, though with species-specific characteristics that may contribute to C. glabrata's pathogenicity.

How does C. glabrata autophagy differ from that of other Candida species?

While C. albicans is the most common cause of yeast infections, C. glabrata has become increasingly prevalent since the 1990s, often ranking as the second or third most common Candida strain causing infections . Unlike C. albicans, C. glabrata is phylogenetically closer to Saccharomyces cerevisiae . This closer evolutionary relationship suggests that autophagy mechanisms in C. glabrata may be more similar to S. cerevisiae than to C. albicans, though adapted for a pathogenic lifestyle. The high conservation of ATG genes between S. cerevisiae and C. glabrata indicates functional similarities in their autophagy systems .

What expression systems are most effective for producing functional recombinant C. glabrata ATG7?

For recombinant expression of C. glabrata ATG7, researchers should consider:

• Yeast expression systems: Since C. glabrata is phylogenetically close to S. cerevisiae, using S. cerevisiae as an expression host may provide appropriate post-translational modifications and protein folding environment .

• E. coli systems: For higher yield but potentially less native folding, bacterial expression with codon optimization is an alternative approach.

• Expression constructs: Include:

  • A strong inducible promoter (GAL1 for yeast systems)

  • Appropriate purification tags (His-tag, GST-tag)

  • Sequence verification to ensure correct ATG7 sequence integrity

The choice of expression system should be guided by the intended experimental applications, with mammalian or insect cell systems being preferable when studying interactions with host factors.

What are the most reliable methods to assess recombinant C. glabrata ATG7 activity in vitro?

To verify the enzymatic activity of recombinant ATG7, researchers should employ:

• ATP-PPi exchange assay: Measures the first step of the E1 activating enzyme reaction

• Thioester bond formation assay: Detects the covalent intermediate between ATG7 and its substrates

• Conjugation assays: Monitors the formation of ATG12-ATG5 and ATG8-PE conjugates using purified components

• Western blot analysis: Confirms the presence of conjugated products

When establishing these assays, include appropriate controls such as catalytically inactive ATG7 mutants (e.g., cysteine active site mutants) to confirm specificity .

How does ATG7 activity relate to other autophagy proteins in C. glabrata virulence?

ATG7 functions within a network of autophagy proteins that collectively contribute to C. glabrata virulence. While ATG7 is essential for the conjugation systems, ATG1 serves as a key autophagy-inducing factor:

• ATG1: Required for autophagy induction and significantly contributes to virulence. C. glabrata strains with ATG1 deletion (Cgatg1Δ) show:

  • Increased sensitivity to nitrogen starvation and H₂O₂

  • Rapid death in nutrient-depleted environments

  • Higher intracellular ROS levels

  • Decreased survival when phagocytosed by macrophages

  • Significantly reduced CFUs in organs in mouse models of disseminated and intra-abdominal candidiasis

• ATG7: Works downstream of ATG1 in the autophagy pathway, activating the conjugation systems necessary for autophagosome formation. Though specific C. glabrata ATG7 deletion studies are not detailed in the search results, the essential role of ATG7 in autophagy suggests similar virulence attenuation would be observed .

The interaction between these proteins creates a functional autophagy system that enables C. glabrata to adapt to the hostile environment within the host.

What is the relationship between oxidative stress response and ATG7-mediated autophagy in C. glabrata?

Autophagy in C. glabrata can be induced by hydrogen peroxide (H₂O₂), indicating its role in oxidative stress response . The relationship between oxidative stress and ATG7 function involves:

• ROS management: Autophagy deficient strains show elevated intracellular ROS levels compared with wild-type strains, suggesting that functional ATG7 contributes to ROS homeostasis .

• Survival mechanism: ATG7-dependent autophagy likely helps degrade oxidatively damaged organelles and proteins, particularly important during phagocytosis when C. glabrata faces oxidative burst in macrophages.

• Mitochondrial quality control: ATG7 is likely involved in mitophagy, which regulates mitochondrial quantity and quality by eliminating damaged mitochondria that produce excess ROS .

This relationship is particularly relevant in host-pathogen interactions, as phagocytes generate ROS as an antimicrobial strategy, and ATG7-mediated autophagy may help C. glabrata counter this host defense.

What are the critical controls when studying the effects of ATG7 manipulation in C. glabrata?

When designing experiments to study ATG7 function in C. glabrata, researchers should include:

Genetic controls:

• Wild-type C. glabrata strain

• ATG7 deletion mutant (Cgatg7Δ)

• ATG7-reconstituted strain (complementation)

• Catalytically inactive ATG7 mutant (point mutation at active site)

Functional validation:

• Autophagy induction assays using nitrogen starvation and H₂O₂ exposure

• Monitoring autophagy flux with appropriate markers

• Viability assessments under various stress conditions

Experimental conditions:

• Range of physiologically relevant stressors (oxidative, nutrient limitation, pH variation)

• Time-course experiments to capture dynamic responses

• Comparison with other autophagy-deficient strains (e.g., Cgatg1Δ) to position effects within the autophagy pathway

These controls help distinguish ATG7-specific effects from general autophagy deficiency and non-specific stress responses.

How can researchers effectively model host-pathogen interactions to study ATG7's role in C. glabrata virulence?

To investigate ATG7's contribution to C. glabrata virulence, researchers should employ multi-level approaches:

In vitro models:

• Macrophage infection assays: Monitor survival of wild-type versus ATG7-deficient C. glabrata when phagocytosed by macrophages (THP-1 cell line or primary macrophages)

• Neutrophil interaction studies

• Growth in serum or nutrient-limited media

Ex vivo models:

• Mouse peritoneal macrophage infection studies, as described for ATG1 mutants

• Human blood survival assays

In vivo models:

• Mouse models of disseminated candidiasis: Intravenous infection followed by organ CFU determination

• Intra-abdominal candidiasis models: Direct comparison with other autophagy-deficient strains

Virulence assessment parameters:

• Organ fungal burden

• Inflammatory markers

• Host survival rates

• Histopathological changes

Based on findings with ATG1-deficient strains, ATG7-deficient C. glabrata would likely show significantly decreased CFUs in organs in mouse infection models, indicating attenuated virulence .

How does chromatin remodeling interact with ATG7-dependent autophagy during host adaptation?

Recent research has uncovered intriguing connections between chromatin organization and autophagy in C. glabrata:

• Chromatin modification during infection: C. glabrata wild-type cells respond to the intracellular environment of macrophages by modifying their chromatin structure, exhibiting:

  • Altered resistance to micrococcal nuclease digestion

  • Changed epigenetic signatures

  • Decreased protein acetylation

  • Increased cellular lysine deacetylase activity

• Autophagy-chromatin regulatory loop: While direct links between ATG7 and chromatin remodeling aren't explicitly detailed in the search results, chromatin remodeling appears to be a central regulator of survival strategies that facilitates reprogramming of cellular energy metabolism in macrophage-internalized C. glabrata cells .

• Potential mechanism: ATG7-dependent autophagy may influence chromatin structure through:

  • Recycling of histone proteins

  • Regulation of acetyl-CoA levels affecting histone acetylation

  • Selective degradation of chromatin-modifying enzymes

Mutants defective in chromatin organization (Cgrsc3-aΔ, Cgrsc3-bΔ, Cgrsc3-aΔbΔ, Cgrtt109Δ) show attenuated virulence in mouse models, similar to autophagy-deficient strains, suggesting potential functional connections between these processes .

What structural features of C. glabrata ATG7 determine its substrate specificity?

Understanding the structural basis of ATG7 function is critical for developing targeted interventions:

• Functional domains: Based on homology with other ATG7 proteins, C. glabrata ATG7 likely contains:

  • An adenylation domain for ATP binding

  • A catalytic cysteine residue forming thioester bonds with substrates

  • Substrate binding regions specific for ATG12 and ATG8

  • Domains mediating interactions with other autophagy components

• Substrate discrimination: ATG7 must discriminate between two different substrates (ATG12 and ATG8), suggesting the presence of specific recognition motifs or conformational changes.

• Species-specific features: Potential structural differences from human ATG7 could be exploited for selective targeting by antifungal compounds.

Researchers investigating these features should use approaches such as:

• Homology modeling based on solved ATG7 structures

• Site-directed mutagenesis of conserved residues

• Domain swapping experiments

• Cross-linking studies to identify interaction interfaces

How can recombinant C. glabrata ATG7 be utilized for antifungal drug discovery?

Recombinant ATG7 offers several approaches for antifungal development:

• High-throughput screening platform: Developing biochemical assays using recombinant ATG7 to screen compound libraries for:

  • Inhibitors of ATP binding

  • Compounds preventing thioester formation

  • Molecules disrupting ATG7-substrate interactions

• Structure-based drug design: Using structural information about C. glabrata ATG7 to design inhibitors that:

  • Target catalytic sites

  • Disrupt protein-protein interactions essential for autophagy

  • Exploit structural differences between fungal and human ATG7

• Validation workflow:

Given that ATG1-deficient strains show attenuated virulence in mouse models, inhibitors of ATG7 might similarly reduce C. glabrata pathogenicity, particularly in the context of drug-resistant strains .

What are the most promising directions for future research on C. glabrata ATG7?

Future research on C. glabrata ATG7 should focus on:

• Comparative analysis: Detailed characterization of differences between C. glabrata ATG7 and human ATG7 to identify fungal-specific features that could be targeted therapeutically.

• Regulatory mechanisms: Investigation of how ATG7 activity is regulated during different phases of infection and in response to antifungal treatment.

• Combination approaches: Exploring potential synergies between ATG7 inhibition and existing antifungal drugs, particularly for resistant strains.

• Host-pathogen interface: Deeper understanding of how ATG7-dependent autophagy modulates C. glabrata interactions with host immune cells.

• Biomarker development: Exploring whether ATG7 activity or its products could serve as biomarkers for C. glabrata infection progression or treatment response.

• Resistance mechanisms: Investigating how C. glabrata might adapt to or compensate for ATG7 inhibition, which would inform therapeutic strategies.

These research directions hold promise for developing novel approaches to combat C. glabrata infections, especially in immunocompromised patients where this pathogen poses significant clinical challenges .