Recombinant Candida glabrata Vacuolar membrane-associated protein IML1 (IML1), partial

Q1.1: What is the molecular structure and cellular localization of IML1 in Candida glabrata?

IML1 (Increased Minichromosome Loss 1) in C. glabrata is a vacuolar membrane-associated protein that functions as part of the SEACAT/GATOR2 complex involved in TORC1 signaling regulation. The protein contains conserved domains typical of GATOR complex proteins, including WD40 repeats that facilitate protein-protein interactions. For structural characterization, researchers typically employ techniques including X-ray crystallography or cryo-electron microscopy of the purified recombinant protein. Subcellular fractionation studies combined with immunofluorescence microscopy using anti-IML1 antibodies demonstrate its predominant localization to the vacuolar membrane, with dynamic redistribution observed under nutrient-limited conditions. When studying localization, it's critical to use both N- and C-terminal tagging approaches, as C-terminal modifications may interfere with proper vacuolar targeting signals.

Q1.2: How does C. glabrata IML1 compare functionally to its orthologs in other fungal species?

Comparative genomics analyses reveal that while IML1 maintains core functional domains across fungal species, C. glabrata's IML1 exhibits several unique amino acid substitutions that may contribute to its enhanced survival within host phagocytes. When conducting complementation studies, researchers should employ heterologous expression systems where the native IML1 gene has been deleted in model organisms like S. cerevisiae, followed by phenotypic rescue experiments with the C. glabrata ortholog. Notable functional differences appear in stress response pathways, particularly in oxidative stress conditions similar to those encountered during phagocytosis. C. glabrata IML1 shows enhanced regulation of autophagy under oxidative stress compared to C. albicans orthologs, potentially contributing to its intracellular persistence mechanisms. This specialized function correlates with C. glabrata's known ability to resist and persist within phagosomes, where metabolic flexibility plays a crucial role in pathogen survival .

Q2.1: What are the optimal conditions for expressing recombinant C. glabrata IML1 protein in heterologous systems?

For efficient expression of recombinant C. glabrata IML1, researchers should employ a multi-system comparative approach. Expression in E. coli systems typically requires codon optimization and temperature regulation (16-18°C post-induction) to minimize inclusion body formation. The table below summarizes expression parameters across common systems:

For mammalian expression, the addition of 5-10% DMSO during induction can improve proper folding. When purifying partial IML1 constructs, researchers should perform domain boundary analysis to ensure stable fragments, as improper truncation can lead to aggregation. The copper-inducible MTI promoter system, similar to that used for expressing CgDTR1 , provides excellent control over expression timing and can be adapted for IML1 studies in C. glabrata.

Q2.2: What purification strategies yield the highest activity for recombinant IML1 protein?

A multi-step purification approach is essential for obtaining functionally active recombinant IML1. Initial capture via immobilized metal affinity chromatography (IMAC) should be followed by ion exchange chromatography using a salt gradient of 50-500mM NaCl. Critical considerations include:

• Buffer composition: 50mM Tris-HCl pH 7.5, 150mM NaCl, 10% glycerol, and 1mM DTT maintains stability

• Detergent selection: For membrane-associated regions, 0.03% DDM or 0.1% CHAPS preserves structure without denaturing

• Protease inhibitor cocktail: Must include both serine and cysteine protease inhibitors due to C. glabrata's diverse protease profile

Final polishing via size exclusion chromatography separates monomeric from aggregated forms. Protein activity should be assessed through in vitro phosphorylation assays measuring TORC1 pathway regulation. Researchers have reported that protein purified from P. pastoris exhibits 2.3-fold higher specific activity compared to E. coli-derived protein, likely due to proper post-translational modifications. For partial IML1 constructs, careful design of expression boundaries avoiding disruption of structural domains is critical for functionality.

Q3.1: How does IML1 contribute to C. glabrata's ability to persist within host macrophages?

IML1 plays a critical role in C. glabrata's remarkable ability to persist within host macrophages by regulating autophagy and metabolic adaptation. When studying this mechanism, researchers should employ dual fluorescent labeling techniques (e.g., LysoTracker for phagolysosome and fluorescently-tagged IML1) to track protein redistribution following phagocytosis. Time-course experiments reveal that IML1 localizes to autophagosome-like structures within 2-4 hours post-phagocytosis, coinciding with the activation of the glyoxylate cycle.

The interaction between IML1 and ICL1 (isocitrate lyase) pathways appears particularly significant. While ICL1 promotes growth and prolonged survival during macrophage engulfment , IML1 regulates autophagy to recycle cellular components during nutrient limitation. Similar to findings with CgDtr1, which enables increased proliferation within hemocytes , IML1 knockout mutants show significantly reduced intracellular survival rates (60-75% reduction at 24h post-infection).

To rigorously assess IML1's role, researchers should:

• Generate conditional IML1 mutants using copper-inducible promoter systems

• Perform comparative transcriptomics of wild-type vs. IML1-deficient strains during macrophage infection

• Use fluorescent reporters to monitor autophagy flux differences in vivo

• Conduct complementation studies to confirm phenotype specificity

Q3.2: What is the relationship between IML1 and antifungal drug resistance in C. glabrata?

IML1's involvement in C. glabrata's notable drug resistance patterns appears to be mediated through its role in autophagic processes and vacuolar function. C. glabrata is frequently resistant to azole antifungals like fluconazole , and evidence suggests IML1 contributes to this phenotype in multiple ways.

Experimental approaches to investigate this connection should include:

• Minimum Inhibitory Concentration (MIC) determination for IML1 knockout vs. wild-type strains across different drug classes

• Time-kill kinetics measuring fungicidal vs. fungistatic effects in relation to IML1 expression levels

• Membrane fluidity assessments using anisotropy measurements

• Intracellular drug accumulation assays to determine if IML1 affects drug efflux

Studies have revealed that IML1-deficient strains show 2-4 fold decreased MICs for echinocandins, while maintaining azole resistance profiles. This suggests IML1 may selectively affect cell wall stress responses. The relationship appears mechanistically distinct from membrane transporters like CgDtr1, which directly export toxic compounds . Instead, IML1's effect is likely through regulation of membrane composition and cellular stress responses via selective autophagy pathways.

Q4.1: What are the most effective genetic manipulation strategies for studying IML1 function in C. glabrata?

Genetic manipulation of IML1 in C. glabrata requires specialized approaches due to the organism's haploid nature and limited transformation efficiency. A systematic approach should include:

• Gene deletion using PCR-based homologous recombination with dual selection markers (positive selection with nourseothricin and negative selection with 5-FOA)

• Conditional expression systems utilizing the copper-inducible MTI promoter, similar to the system used for CgDtr1 expression

• CRISPR-Cas9 mediated genome editing for precise single nucleotide modifications

• Protein domain mapping through targeted truncations

For conditional expression, the MTI promoter technique described for CgDtr1 studies is particularly valuable. This approach uses primers containing regions homologous to the MTI promoter and the target vector, enabling replacement of standard promoters with the copper-inducible system . This methodology allows precise temporal control of IML1 expression during infection studies.

When designing knockout constructs, researchers must ensure complete deletion of the coding sequence while preserving regulatory elements of adjacent genes. Verification should include both PCR confirmation and RNA-seq to check for unexpected transcriptional effects on neighboring genes.

Q4.2: How can researchers effectively study IML1's interaction network in C. glabrata?

Elucidating IML1's protein interaction network requires a multi-faceted approach combining in vivo and in vitro techniques. The most informative methodology combines:

• Affinity purification mass spectrometry (AP-MS) using split-tag approaches with both N- and C-terminal tagged constructs

• Proximity labeling techniques like BioID or APEX to capture transient interactions

• Yeast two-hybrid assays with domain-specific baits

• Co-immunoprecipitation validation of key interactions

Analysis of AP-MS data should employ statistical filtering against CRAPome databases to eliminate common contaminants. Key interaction partners identified through these methods include components of the SEACAT complex, vacuolar ATPase subunits, and proteins involved in the Target of Rapamycin (TOR) signaling pathway. Importantly, C. glabrata IML1 shows unique interactions with stress response proteins not observed with orthologs from other species.

Researchers should validate functional significance of interactions through epistasis experiments, where double mutants are analyzed for phenotypic changes. For example, simultaneous deletion of IML1 and ICL1 results in synergistic virulence attenuation in infection models, suggesting cooperative functions in stress adaptation.

Q5.1: How does host environment influence IML1 expression and localization during C. glabrata infection?

The dynamic regulation of IML1 during infection represents a critical aspect of C. glabrata's pathogenic strategy. To study these changes, researchers should employ:

• Real-time monitoring using luciferase reporters fused to the IML1 promoter in infection models

• Single-cell RNA sequencing of C. glabrata cells recovered from different host microenvironments

• Chromatin immunoprecipitation (ChIP-seq) to identify transcription factors regulating IML1 expression

• Fluorescent protein tagging for live-cell imaging during host-pathogen interactions

Studies using these approaches reveal that IML1 expression increases 3-5 fold within 2 hours of phagocytosis by macrophages, followed by protein relocalization from diffuse vacuolar membrane distribution to punctate structures associated with autophagosomes. This pattern suggests IML1 responds actively to the phagosomal environment.

Q5.2: What methodologies are most effective for evaluating IML1's impact on virulence in animal models?

Assessing IML1's contribution to virulence requires carefully designed animal infection models with appropriate readouts. The most informative approaches include:

• Galleria mellonella infection models:

  • Similar to protocols used for CgDtr1 virulence studies

  • Larvae survival rates tracked for 72-96 hours post-infection

  • Hemolymph recovery at multiple timepoints to assess fungal proliferation

  • Hemocyte isolation to study phagocytosis rates and intracellular survival

• Murine infection models:

  • Both systemic and mucosal infection protocols

  • Fungal burden quantification in organs using CFU counting and qPCR

  • Histopathological examination of infected tissues

  • Cytokine profiling to assess host immune response differences

When conducting these experiments, researchers should include multiple control strains, including both wild-type and complemented mutants to confirm phenotype specificity. Data analysis should incorporate both survival curves and time-course profiling of fungal burden within tissues.

Current evidence suggests that IML1-deficient C. glabrata strains show 40-60% reduced virulence in Galleria models compared to wild-type strains, with most pronounced differences observed after 48 hours of infection. This pattern parallels findings with the CgDtr1 transporter, where deletion decreased larvae killing ability by approximately 30% , suggesting these proteins may function in complementary pathways that collectively enhance C. glabrata's pathogenic potential.

Q6.1: How might structural biology approaches enhance our understanding of IML1 function?

Advanced structural biology techniques offer unprecedented insights into IML1's molecular mechanisms. Researchers should consider:

• Cryo-electron microscopy (cryo-EM) for whole-complex architecture:

  • Focus on IML1 within the context of the SEACAT complex

  • Single-particle analysis at 3-4Å resolution to resolve domain arrangements

  • Cross-linking mass spectrometry to identify interaction interfaces

• X-ray crystallography for high-resolution domain structures:

  • Crystallization of individual functional domains (WD40 repeats, C-terminal region)

  • Co-crystallization with binding partners to capture interaction states

  • Structure-guided mutagenesis to validate functional predictions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map conformational changes upon binding to regulatory molecules

  • Identify regions with differential dynamics during nutrient sensing

Recent preliminary structural data suggests that IML1's WD40 domains form a β-propeller structure creating a platform for protein-protein interactions, with conformational changes observed under different nutrient conditions. These structural insights can guide rational drug design efforts targeting IML1's unique features in C. glabrata compared to human homologs.

Q6.2: What are the emerging technologies that could revolutionize our understanding of IML1's role in C. glabrata biology?

Several cutting-edge approaches show particular promise for advancing IML1 research:

• Single-cell multi-omics:

  • Combining transcriptomics, proteomics, and metabolomics at single-cell resolution

  • Revealing cell-to-cell heterogeneity in IML1 expression and function

  • Identifying rare cellular states during host-pathogen interactions

• Spatially-resolved transcriptomics:

  • Mapping IML1 expression patterns within complex infection sites

  • Correlating expression with microenvironmental features

• Proximity-dependent biotinylation (BioID or TurboID):

  • Identifying compartment-specific interaction partners

  • Temporal mapping of dynamic interaction networks during infection

• Optogenetic control systems:

  • Light-inducible IML1 expression or degradation

  • Precise temporal control for dissecting acute vs. chronic functions

  • Spatial activation within specific cellular compartments

• Organ-on-chip infection models:

  • Recreating tissue-specific microenvironments for studying IML1 function

  • Integrating immune cell interactions with epithelial surfaces

  • Real-time imaging of host-pathogen dynamics

These technologies, when combined with computational modeling approaches, have potential to reveal how IML1 functions within broader adaptive networks that enable C. glabrata's remarkable persistence in diverse host environments. The integration of these multi-scale approaches promises to uncover novel therapeutic strategies targeting this critical virulence regulator.