Recombinant Candida glabrata Serine/threonine-protein kinase ATG1 (ATG1), partial

What is the primary function of ATG1 in Candida glabrata?

ATG1 is a serine/threonine protein kinase that serves as an essential component in the cytoplasm to vacuole transport (Cvt) pathway and is critical for autophagy induction. The protein is specifically required for autophagosome formation and is involved in clearing protein aggregates that cannot be efficiently processed by the proteasome system . ATG1 functions as a key regulatory kinase that initiates the autophagic process, particularly under stress conditions such as nutrient deprivation or oxidative stress. Experimental data demonstrates that C. glabrata strains lacking ATG1 (Cgatg1Δ) are completely deficient for autophagy, confirming its essential role in this cellular process .

How does ATG1 contribute to C. glabrata virulence?

ATG1 contributes to C. glabrata virulence through multiple mechanisms:

• Stress tolerance: ATG1 enables survival during nitrogen starvation and oxidative stress conditions frequently encountered within host environments .

• Persistence: ATG1-mediated autophagy allows C. glabrata to survive in nutrient-poor environments, as demonstrated by the rapid death of Cgatg1Δ strains in water without nutrients .

• ROS management: ATG1 helps maintain lower intracellular reactive oxygen species (ROS) levels, protecting against oxidative damage .

• Macrophage resistance: Functional ATG1 increases survival following phagocytosis by macrophages, as Cgatg1Δ strains show higher mortality rates when engulfed by mouse peritoneal macrophages .

• In vivo colonization: ATG1 enhances fungal persistence in host tissues, evidenced by significantly decreased CFUs in organs of mouse models infected with Cgatg1Δ strains .

What molecular pathways does ATG1 regulate in C. glabrata?

ATG1 regulates several critical pathways in C. glabrata:

• Autophagosome formation: ATG1 is essential for the initial steps of autophagosome assembly .

• Selective autophagy processes: ATG1 is required for specific types of autophagy including:

  • Nucleophagy (selective degradation of nuclear components)

  • Mitophagy (degradation of mitochondria)

  • ER-specific autophagy (degradation of endoplasmic reticulum components)

• ATG9 trafficking: ATG1 plays a key role in ATG9 and ATG23 cycling through the pre-autophagosomal structure .

• Phosphorylation cascade: ATG1 promotes ATG18 binding to ATG9 through phosphorylation of ATG9, initiating downstream autophagy events .

What experimental approaches can be used to assess ATG1 function in C. glabrata?

Several methodological approaches have been validated for assessing ATG1 function:

• GFP-Atg8 processing assay: This method monitors autophagy induction by tracking the cleavage of GFP-Atg8, which results in free GFP detection by western blotting. In wild-type C. glabrata, increased GFP bands are observed during nitrogen starvation and H₂O₂ exposure, whereas Cgatg1Δ strains show no GFP bands over time, confirming ATG1-dependent autophagy .

• Growth and survival assays:

  • Growth curve analysis in normal and stress conditions (doubling times of wild-type, Cgatg1Δ, and reconstituted strains were 1.173, 1.912, and 1.254 hours respectively in SC-trp medium)

  • Spot assays under nitrogen starvation and H₂O₂ exposure

  • Survival assessment in nutrient-depleted conditions (water)

• ROS measurement: Intracellular ROS levels can be quantified using fluorescent probes to compare wild-type and Cgatg1Δ strains .

• Macrophage interaction studies:

  • Phagocytosis assays using mouse peritoneal macrophages

  • Survival quantification following phagocytosis

• In vivo virulence models:

  • Disseminated candidiasis (DC) mouse model (assessing CFUs in liver, spleen, and kidney)

  • Intra-abdominal candidiasis (IAC) mouse model (examining CFUs in pancreas and other abdominal organs)

How can researchers effectively generate and validate ATG1 deletion mutants in C. glabrata?

The following methodology has been successfully employed for ATG1 deletion and validation:

• Deletion construct generation:

  • Amplify a deletion construct from a suitable plasmid (e.g., pBSK-HIS) using primers tagged with 100-bp sequences homologous to the flanking regions of the ATG1 ORF .

  • Transform C. glabrata parent strains with the deletion construct using the lithium acetate protocol .

  • Select transformants based on appropriate selection markers (e.g., histidine prototrophy) .

• Validation of successful deletion:

  • Verify homologous recombination by diagnostic PCR targeting the deletion junction .

  • Confirm absence of ATG1 mRNA expression using real-time qRT-PCR .

• Complementation for phenotypic verification:

  • Construct a complementation plasmid expressing ATG1 under its native promoter (e.g., pCgACT-CgATG1) .

  • The complementation construct should contain the ATG1 promoter, ORF, and 3'-UTR (approximately 3,781-bp) .

  • Transform the atg1Δ strain with this construct and select transformants based on appropriate markers .

  • Verify ATG1 expression in the complemented strain by qRT-PCR .

  • Compare phenotypes between wild-type, deletion mutant, and complemented strains to confirm that observed defects are specifically due to ATG1 deletion .

What is the relationship between ATG1 activity and oxidative stress response in C. glabrata?

The relationship between ATG1 and oxidative stress response is multifaceted:

• Autophagy induction by oxidative stress:

  • H₂O₂ exposure induces autophagy in C. glabrata in an ATG1-dependent manner .

  • This is evidenced by GFP-Atg8 processing assays, where GFP bands gradually intensify during H₂O₂ exposure in wild-type strains but not in Cgatg1Δ strains .

• Growth under oxidative stress:

  • Cgatg1Δ strains show significant growth defects when exposed to H₂O₂, indicating that ATG1-mediated autophagy is crucial for oxidative stress tolerance .

  • These growth defects are restored to wild-type levels when ATG1 is reintroduced into the mutant .

• ROS management:

  • Cgatg1Δ strains exhibit higher intracellular ROS levels compared to wild-type strains .

  • This suggests that ATG1-mediated autophagy contributes to ROS homeostasis, potentially by:
    a) Removing damaged organelles (particularly mitochondria) that produce excess ROS
    b) Recycling oxidized proteins and lipids
    c) Generating precursors for glutathione synthesis and other antioxidant defenses

• Mitochondrial quality control:

  • ATG1 is required for mitophagy, which regulates mitochondrial quantity and quality .

  • Proper mitochondrial maintenance prevents excess ROS production .

  • The absence of ATG1 likely compromises mitochondrial quality control, contributing to elevated ROS levels.

What infection models are suitable for studying ATG1's role in C. glabrata virulence?

Multiple infection models have been validated for investigating ATG1's contribution to C. glabrata virulence:

• Mouse models of candidiasis:

  • Disseminated candidiasis (DC) model:

    • Assesses C. glabrata spread through blood flow

    • Examines fungal burden in liver, spleen, and kidney

    • Results show Cgatg1Δ strains have significantly decreased CFUs in liver and spleen, with slight non-significant decreases in kidney

  • Intra-abdominal candidiasis (IAC) model:

    • Evaluates intraperitoneal infection

    • Examines fungal burden primarily in pancreas

    • Results show Cgatg1Δ strains have significantly decreased CFUs in all examined organs

• Macrophage interaction assays:

  • Mouse peritoneal macrophages can be used to assess:

    • Phagocytosis efficiency

    • Fungal survival following phagocytosis

    • Results demonstrate higher mortality of Cgatg1Δ strains during phagocytosis by macrophages

• Alternative infection models:

  • Galleria mellonella (wax moth larvae) model:

    • While not specifically mentioned for ATG1 studies, G. mellonella has been validated for C. glabrata virulence studies as seen with other virulence factors

    • This model offers advantages including ease of use, cost-effectiveness, and ethical considerations

    • Could potentially be adapted to study ATG1's role in virulence

What are the technical considerations for working with recombinant C. glabrata ATG1 protein?

When working with recombinant C. glabrata ATG1 protein, researchers should consider:

• Expression and purification:

  • Recombinant C. glabrata ATG1 can be expressed in E. coli expression systems .

  • Protein purity should exceed 90% as determined by SDS-PAGE .

• Storage conditions:

  • Shelf life varies depending on storage conditions and protein stability.

  • For liquid formulations, typical shelf life is 6 months at -20°C/-80°C.

  • For lyophilized formulations, typical shelf life extends to 12 months at -20°C/-80°C .

  • Repeated freezing and thawing should be avoided; working aliquots can be stored at 4°C for short periods .

• Functional assays:

  • Kinase activity assays should be developed to verify the catalytic activity of recombinant ATG1.

  • Substrates including ATG9 should be considered for phosphorylation assays .

  • The recombinant protein can be used for:

    • Antibody production

    • Enzyme kinetics studies

    • Structure-function analyses

    • Inhibitor screening

How can researchers quantitatively assess autophagy induction in C. glabrata?

Several methodologies have been established for quantitative assessment of autophagy in C. glabrata:

• GFP-Atg8 processing assay:

  • Express GFP-CgAtg8 from a transformed plasmid containing the CgATG8 native promoter.

  • Under autophagy-inducing conditions, GFP-Atg8 is cleaved, releasing free GFP.

  • Monitor GFP release via western blotting, where the intensity of the free GFP band correlates with autophagy levels .

  • This assay has been validated for both nitrogen starvation and H₂O₂-induced autophagy in C. glabrata .

• Fluorescence microscopy approaches:

  • GFP-Atg8 can also be used for visualizing autophagosome formation via fluorescence microscopy.

  • Upon autophagy induction, GFP-Atg8 relocates from diffuse cytoplasmic distribution to punctate structures representing autophagosomes.

  • Quantification of GFP-Atg8 puncta per cell provides a measure of autophagy activity.

• Growth and survival assays:

  • Growth curves under normal and autophagy-inducing conditions.

  • Spot assays comparing serial dilutions of wild-type and autophagy-deficient strains.

  • Cell viability assessments in nutrient-deprived conditions .

• ROS measurement:

  • Since autophagy affects ROS levels, measuring intracellular ROS can serve as an indirect assessment of autophagy function.

  • Fluorescent ROS-sensitive probes can be employed to quantify differences between wild-type and autophagy-deficient strains .

• Transmission electron microscopy:

  • Direct visualization of autophagic structures including autophagosomes and autolysosomes.

  • While technically demanding, this approach provides detailed structural information about the autophagic process.

What is the potential of ATG1 as a therapeutic target for C. glabrata infections?

Based on current research, ATG1 shows promise as a potential therapeutic target:

• Rationale for targeting:

  • ATG1 deletion significantly reduces C. glabrata virulence and persistence in multiple infection models .

  • ATG1 is essential for autophagy, which helps C. glabrata survive host defense mechanisms and nutrient-limited environments .

  • Targeting ATG1 could potentially reduce fungal burden in host tissues and increase susceptibility to existing antifungals.

• Potential advantages:

  • Specificity: Targeting fungal-specific aspects of ATG1 structure or regulation could provide selectivity over human homologs.

  • Resistance management: As a novel target class, ATG1 inhibitors might help address emerging antifungal resistance.

  • Combination potential: ATG1 inhibitors could potentially sensitize C. glabrata to conventional antifungals or host immune defenses.

• Challenges to consider:

  • Conservation: ATG1 has mammalian homologs (ULK1/2), necessitating careful inhibitor design to avoid off-target effects.

  • Resistance development: Single-target approaches may face resistance pressure.

  • Delivery: Effective inhibitors would need to penetrate the fungal cell wall and membrane.

• Research directions:

  • Structure-based drug design targeting unique features of fungal ATG1.

  • Screening for compounds that disrupt ATG1 kinase activity or its interactions with other autophagy proteins.

  • Development of combination strategies with existing antifungals.

How does ATG1 function contribute to C. glabrata's resistance against antifungal treatments?

While the search results don't directly address ATG1's role in antifungal resistance, several mechanistic connections can be proposed based on autophagy's known functions:

• Stress adaptation:

  • ATG1-mediated autophagy enhances survival under various stress conditions .

  • This general stress resistance mechanism could potentially contribute to survival during antifungal treatment, which induces cellular stress.

• Cellular remodeling:

  • Autophagy enables cellular component recycling and remodeling .

  • This capability might help C. glabrata adapt to membrane or cell wall disturbances caused by antifungals.

• ROS management:

  • Many antifungals induce oxidative stress as part of their mechanism of action.

  • ATG1-dependent autophagy helps manage ROS levels , potentially providing protection against oxidative damage induced by antifungals.

• Persistence mechanisms:

  • C. glabrata is known for its ability to persist during antifungal therapy.

  • ATG1-mediated autophagy contributes to persistence in host environments , suggesting it might similarly support survival during antifungal exposure.

• Research hypotheses to investigate:

  • Comparative susceptibility testing of wild-type and Cgatg1Δ strains against various antifungal classes.

  • Assessment of autophagy induction following antifungal exposure.

  • Evaluation of combination approaches targeting both ATG1-mediated autophagy and conventional antifungal targets.

Comparative analysis of C. glabrata strains with and without functional ATG1

Data compiled from experimental findings reported in search result .

Functional domains and activities of C. glabrata ATG1 protein

Information compiled from search results and .