Recombinant Candida glabrata Autophagy-related protein 20 (ATG20), partial

What is ATG20 in Candida glabrata and what is its function in autophagy?

ATG20 in Candida glabrata is a sorting nexin protein that facilitates autophagy, particularly selective types of autophagy. Based on homology with closely related yeast Saccharomyces cerevisiae, ATG20 likely forms a complex with Snx4/Atg24 and is involved in the cytoplasm-to-vacuole targeting (Cvt) pathway . This protein is part of the broader autophagy machinery that contributes to C. glabrata's ability to survive in nutrient-poor environments and resist host defense mechanisms.

Research methodology approaches:

• Perform sequence alignment between C. glabrata ATG20 and S. cerevisiae ATG20 to identify conserved domains

• Use fluorescent protein tagging to visualize ATG20 localization during autophagy induction

• Employ co-immunoprecipitation to identify binding partners in C. glabrata

How does C. glabrata ATG20 structure relate to its function?

Similar to S. cerevisiae Atg20, C. glabrata ATG20 likely contains a Phox homology (PX) domain that binds to phosphatidylinositol-3-phosphate (PI3P) on membranes, and a Bin/Amphiphysin/Rvs (BAR) domain that can sense and/or induce membrane curvature . These domains enable ATG20 to interact with membranes during autophagosome formation. The protein likely engages both structurally stable domains (PX and BAR) and intrinsically disordered regions for its function .

Methodological approach:

• Perform domain mapping through truncation analyses to identify functional regions

• Use liposome binding assays to assess membrane interaction capabilities

• Employ site-directed mutagenesis to identify critical residues for function

How is ATG20 expression regulated during autophagy in C. glabrata?

While specific regulation of ATG20 in C. glabrata has not been fully characterized, autophagy in C. glabrata is induced by nitrogen starvation and oxidative stress (H₂O₂) . Based on the regulation patterns of other autophagy genes, ATG20 expression is likely upregulated under these stress conditions. The ATG1 kinase complex plays a crucial role in the induction of autophagy in C. glabrata , and may regulate ATG20 activity through direct or indirect mechanisms.

Research approach:

• Perform RT-qPCR to measure ATG20 expression under various stress conditions

• Use ChIP-seq to identify transcription factors that bind to the ATG20 promoter

• Develop reporter assays to monitor ATG20 promoter activity

What are the best methods to produce recombinant C. glabrata ATG20 protein?

Table 1: Comparison of Expression Systems for Recombinant C. glabrata ATG20

Methodological approach:

• Clone the ATG20 gene into an expression vector with an affinity tag

• Express in the chosen system (baculovirus system often provides better folding for membrane-interacting proteins)

• Optimize expression conditions (temperature, induction time)

• Purify using affinity chromatography followed by size exclusion chromatography

• Verify protein integrity by SDS-PAGE and mass spectrometry

• Store with glycerol (final concentration 50%) and aliquot for long-term storage at -20°C/-80°C

How can atg20 knockout strains be generated in C. glabrata?

For generating atg20 knockout strains in C. glabrata, consider the following proven approach:

• Design targeting constructs with homology arms flanking the ATG20 gene

• Include a selectable marker (NAT1, HIS3, LEU2, TRP1) between the homology arms

• Transform C. glabrata cells (strain ATCC2001 or BG2 recommended based on virulence studies)

• Select transformants on appropriate media

• Verify gene deletion by PCR and confirm by sequencing

• Generate complemented strains by reintroducing ATG20 to confirm phenotypes

It's important to note that auxotrophic markers (HIS3, LEU2, TRP1) don't impact C. glabrata virulence, unlike URA3 marker in C. albicans . A recent library of C. glabrata deletion mutants has been constructed using a recyclable NAT1 marker, making it a preferred approach .

What model systems are appropriate for studying C. glabrata ATG20 in vivo?

Table 2: Model Systems for Studying C. glabrata ATG20 Function

Research methodology:

• Compare wild-type, atg20Δ, and complemented strains in each model

• For macrophage assays, measure survival rates and ROS levels

• For G. mellonella, monitor survival daily for 7 days after infection

• For mouse models, measure CFU in target organs (kidney, liver, spleen) after 7 days

• Include atg1Δ strain as a control for general autophagy defects

What is the relationship between ATG20 and C. glabrata virulence?

While the specific role of ATG20 in C. glabrata virulence hasn't been fully characterized, evidence from studies on autophagy in C. glabrata provides insights:

• Autophagy contributes to C. glabrata virulence by conferring resistance to unstable nutrient environments and immune defenses

• Autophagy-deficient strains (e.g., atg1Δ) show:

  • Sensitivity to nitrogen starvation and H₂O₂

  • Rapid death in nutrient-poor conditions

  • Higher intracellular ROS levels

  • Decreased survival after phagocytosis by macrophages

  • Significantly decreased CFUs in organs in mouse models

As ATG20 is part of the autophagy machinery, it likely contributes to these virulence-associated functions, particularly in selective autophagy pathways that may be crucial for adaptation to the host environment.

Research approach:

• Compare virulence of wild-type, atg20Δ, and complemented strains in animal models

• Assess stress resistance profiles (nitrogen starvation, oxidative stress)

• Analyze transcriptomic changes in the atg20Δ mutant under infection-relevant conditions

How does ATG20 interact with other autophagy proteins in C. glabrata?

Based on knowledge from related yeasts, C. glabrata ATG20 likely interacts with:

• SNX4/ATG24 through BAR domains to form a functional complex

• ATG1 complex, which is the key initiator of autophagy

• Potentially ATG9, a transmembrane protein involved in membrane delivery to growing autophagosomes

The ATG1 kinase complex plays a central role in autophagy induction in C. glabrata , and ATG20 may be regulated by this complex either through direct phosphorylation or through other interaction partners.

Methodological approaches:

• Perform co-immunoprecipitation with tagged ATG20 followed by mass spectrometry

• Use yeast two-hybrid screening to identify interaction partners

• Conduct proximity labeling experiments using BioID or APEX2 fused to ATG20

• Analyze the dynamics of protein complex formation using live-cell imaging

How do genetic variations in C. glabrata ATG20 impact autophagy efficiency?

C. glabrata isolates show high genetic diversity with numerous sequence types identified globally . This genetic diversity may extend to autophagy genes including ATG20. Variations in ATG20 sequence could impact:

Studies have shown that C. glabrata isolates from different sequence types can vary in virulence properties , and differences in autophagy efficiency due to ATG20 variants could contribute to this variation.

Research approach:

• Sequence ATG20 from diverse clinical isolates to identify polymorphisms

• Create chimeric proteins or introduce specific mutations to assess functional impact

• Compare autophagy efficiency across different clinical isolates

• Correlate ATG20 sequence variations with virulence phenotypes

How can researchers distinguish between ATG20-specific effects and general autophagy defects?

Table 3: Experimental Approaches to Distinguish ATG20-Specific from General Autophagy Effects

Methodological approach:

• Use multiple independent knockout clones to ensure consistency

• Include complemented strains to confirm phenotype rescue

• Compare with known core autophagy mutants (e.g., atg1Δ) and other selective autophagy mutants

• Employ time-course experiments to distinguish primary from secondary effects

• Use quantitative assays for both bulk and selective autophagy

How should contradictory findings about ATG20 function between different experimental systems be resolved?

When faced with contradictory findings about ATG20 function:

• Consider genetic background differences - C. glabrata shows high genetic diversity with numerous sequence types that may affect autophagy function

• Standardize experimental conditions - nitrogen starvation and oxidative stress are key inducers of autophagy in C. glabrata

• Verify knockout constructs and complementation strains by sequencing

• Use multiple complementary assays to assess autophagy

• Consider strain-specific differences in virulence - different C. glabrata strains show varying levels of virulence in infection models

• Account for experimental model differences - results may vary between macrophage assays, G. mellonella, and different mouse infection models

Research approach:

• Directly compare methods in the same laboratory using identical strains

• Document all experimental parameters in detail

• Conduct collaborative studies between labs with contradictory findings

• Perform meta-analysis of published results with attention to methodological differences

What controls are essential when studying the impact of ATG20 on C. glabrata stress responses?

When studying ATG20's role in stress responses, include these essential controls:

• Wild-type strain (same background as mutants)

• ATG20 complemented strain (atg20Δ + ATG20)

• Known autophagy mutant (e.g., atg1Δ) as a positive control for general autophagy defects

• Related sorting nexin mutant (e.g., snx4/atg24Δ) to assess functional overlap

• Appropriate stress controls:

  • Nitrogen starvation (SC-N medium)

  • Oxidative stress (H₂O₂ at varying concentrations)

  • Nutrient deprivation (water or PBS)

  • Growth under standard conditions (SC-trp or YPD medium)

For infection models, include:

• PBS-injected control animals

• Multiple infectious doses to establish dose-response relationships

• Multiple time points for CFU determination

What novel approaches could advance our understanding of ATG20 function in C. glabrata?

Emerging technologies that could advance ATG20 research include:

• CRISPR-Cas9 genome editing for more efficient generation of mutants and tagged proteins

• Single-cell analysis to examine heterogeneity in autophagy responses within C. glabrata populations

• Cryo-electron microscopy to resolve structural details of ATG20-containing complexes

• Advanced live-cell imaging techniques to monitor ATG20 dynamics during autophagy

• Proximity labeling techniques to identify transient interactions during autophagy induction

• Synthetic genetic array analysis to identify genetic interactions with ATG20

• Quantitative proteomics to analyze post-translational modifications of ATG20 under various conditions

How might understanding ATG20 function contribute to novel antifungal strategies?

The critical role of autophagy in C. glabrata virulence suggests that targeting ATG20 or related autophagy components could be a promising antifungal strategy:

• ATG20 inhibitors could potentially reduce C. glabrata survival under stress conditions encountered during infection

• Combination therapy targeting both ATG20 and conventional antifungal targets might reduce the development of resistance

• Selective inhibition of ATG20-mediated pathways could attenuate virulence without directly killing the fungus, potentially reducing selective pressure for resistance

• Understanding how ATG20 contributes to drug resistance mechanisms could help design more effective treatment strategies for drug-resistant C. glabrata infections

Research approach:

• Screen for small molecule inhibitors of ATG20 function

• Test identified compounds in combination with existing antifungals

• Evaluate effects on C. glabrata virulence in animal models

• Assess potential for resistance development through long-term exposure studies