Recombinant Candida glabrata 78 kDa glucose-regulated protein homolog (KAR2), partial

What is KAR2 and what is its primary function in Candida glabrata?

KAR2 (also known as the 78 kDa glucose-regulated protein or GRP78) is an essential endoplasmic reticulum (ER)-resident chaperone protein in Candida glabrata. Its primary function involves maintaining protein homeostasis within the ER and regulating the unfolded protein response (UPR). As an ER chaperone, KAR2 assists in the proper folding of nascent polypeptides entering the ER lumen and prevents protein aggregation during stress conditions.

To study KAR2's primary functions, researchers can employ several methodological approaches:

• Gene expression analysis using quantitative PCR to measure KAR2 mRNA levels under different stress conditions

• Protein localization studies using GFP-tagged KAR2 constructs and fluorescence microscopy

• Immunoprecipitation experiments to identify KAR2-interacting proteins

• In vitro translocation assays using microsomes prepared from C. glabrata to assess KAR2's role in protein translocation

KAR2 is essential for C. glabrata viability, as demonstrated by studies showing that it functions during the translocation of proteins into the ER during the first committed step of the secretory pathway .

How does C. glabrata KAR2 expression change during ER stress?

Unlike in Saccharomyces cerevisiae, transcriptional induction of KAR2 in response to ER stress in C. glabrata appears to be mediated primarily by the calcineurin-Crz1 pathway rather than the canonical Ire1 signaling pathway. When treated with tunicamycin (TM), an ER stress inducer, expression levels of C. glabrata KAR2 increase in wild-type strains and Δslt2 mutants, but not in Δcnb1 and Δcrz1 mutants .

Methodological approach to study KAR2 expression during ER stress:

• Treat C. glabrata cultures with ER stress inducers such as tunicamycin, DTT, or thapsigargin

• Extract total RNA at various time points after treatment

• Perform RT-qPCR or Northern blot analysis to quantify KAR2 mRNA levels

• Use Western blot analysis with anti-KAR2 antibodies to measure protein levels

• Compare expression patterns between wild-type and mutant strains (Δire1, Δcnb1, Δcrz1)

The promoter region of C. glabrata KAR2 contains five copies of the Crz1-binding sequence, including one copy of the full consensus sequence (5′-GNGGCTCA-3′), but lacks the canonical unfolded protein response element (UPRE) found in S. cerevisiae . This provides further evidence for calcineurin-Crz1-dependent regulation of KAR2 in C. glabrata.

What techniques can be used to clone and express recombinant C. glabrata KAR2?

Cloning and expressing recombinant C. glabrata KAR2 requires careful consideration of both molecular biology techniques and expression systems. The following methodological approach has been successfully used:

• PCR amplification of the KAR2 gene:

  • Extract genomic DNA from C. glabrata (strain NIH3172 has been used successfully)

  • Design primers that flank the KAR2 coding sequence with appropriate restriction sites

  • Amplify using high-fidelity DNA polymerase to minimize errors

  • The PCR product should be approximately 2,414 bp for the complete KAR2 gene

• Cloning into an expression vector:

  • Digest both the PCR product and expression vector (e.g., pYEX-BX) with appropriate restriction enzymes (BamHI and SalI have been used)

  • Ligate the digested PCR product into the vector

  • Transform the ligation mixture into E. coli for plasmid amplification

  • Verify the sequence to ensure no mutations were introduced during PCR

• Expression in a heterologous system:

  • Transform the verified plasmid into S. cerevisiae

  • For functional studies, a hemizygous S. cerevisiae strain (e.g., YJ034W BY4743) with one wild-type KAR2 allele and one deleted allele can be used

  • Perform tetrad dissection to obtain haploid cells containing only the C. glabrata KAR2

• Protein purification:

  • Express recombinant KAR2 with an affinity tag (His-tag or GST-tag)

  • Lyse cells and perform affinity chromatography

  • Further purify using size exclusion chromatography if needed

It's important to note that C. glabrata KAR2 lacks CUG codons in the encoded mRNA, which simplifies expression in standard systems without codon optimization .

How does KAR2 function differ between Candida glabrata and Candida albicans?

While both C. glabrata and C. albicans KAR2 proteins serve as essential ER chaperones, there are notable differences in their regulation and potential functions:

• Regulation during ER stress:

  • In C. albicans, KAR2 functions during the translocation of proteins into the ER and is essential for viability

  • In C. glabrata, KAR2 expression during ER stress appears to be regulated through the calcineurin-Crz1 pathway rather than the canonical Ire1-Hac1 pathway used in S. cerevisiae and potentially C. albicans

• Structural conservation:

  • Despite differences in regulation, the KAR2 protein itself is highly conserved functionally

  • C. albicans KAR2 lacks CUG codons, which is notable given the alternative codon usage in Candida species

  • Functional conservation is demonstrated by the ability of C. albicans KAR2 to rescue temperature-sensitive growth defects in S. cerevisiae strains with mutant Kar2 protein

• Role in pathogenesis:

  • C. glabrata is innately resistant to many azole antifungal agents, whereas C. albicans is generally more susceptible

  • The contribution of KAR2 to these differences in drug resistance remains an area of active investigation

  • C. glabrata infections show relatively mild tissue infiltrates of immune cells compared to C. albicans infections, suggesting potential differences in how KAR2 may influence host-pathogen interactions

To study these differences methodologically:

• Perform comparative genome and promoter analyses between the species

• Create chimeric proteins swapping domains between C. glabrata and C. albicans KAR2

• Express each species' KAR2 in the other and assess complementation

• Compare in vitro translocation efficiency using microsomes from each species

These approaches could help elucidate the distinct roles of KAR2 in these related but pathogenically distinct Candida species.

What is the role of KAR2 in the pathogenesis of Candida glabrata infections?

Although C. glabrata was historically considered a relatively nonpathogenic commensal fungal organism, it has emerged as a significant pathogen, particularly in immunocompromised individuals . KAR2, as an essential protein involved in stress response and protein homeostasis, likely contributes to pathogenesis in several ways:

• Stress adaptation:

  • As an ER chaperone, KAR2 helps C. glabrata adapt to various stresses encountered during infection

  • This adaptation is crucial for survival in diverse host microenvironments with varying pH, nutrient availability, and immune pressures

• Cell surface expression:

  • Under stress conditions, KAR2 (GRP78) can translocate to the cell surface

  • Cell surface-associated KAR2 may interact with host proteins and modulate host-pathogen interactions

  • While direct evidence in C. glabrata is limited, studies in other systems suggest surface KAR2 can interact with integrin β1 and affect signaling pathways

• Antifungal resistance:

  • C. glabrata exhibits innate resistance to azole antimycotics, which is a major clinical challenge

  • The ER stress response, potentially involving KAR2, may contribute to this resistance

  • Proper protein folding and quality control mediated by KAR2 may help maintain cellular integrity during drug exposure

Methodological approaches to study KAR2's role in pathogenesis:

• Create conditional KAR2 mutants with reduced expression and assess virulence in animal models

• Perform transcriptomic analysis comparing KAR2 expression in commensal versus invasive isolates

• Study cell surface expression of KAR2 during host cell interactions using biotinylation and flow cytometry techniques similar to those used in other cell types

• Evaluate sensitivity to antifungal drugs and host defense mechanisms in strains with altered KAR2 expression

Understanding KAR2's contribution to pathogenesis could potentially identify new targets for antifungal development.

What is known about cell surface-associated KAR2 (csKAR2) in Candida glabrata and its potential functions?

While cell surface expression of KAR2/GRP78 has not been extensively studied specifically in C. glabrata, research in other cell types provides valuable insights that may be applicable. Studies have shown that under conditions of ER stress, GRP78 can translocate to the cell surface where it plays roles distinct from its ER chaperone function.

Methodology to study cell surface KAR2 in C. glabrata:

• Cell surface protein detection:

  • Use biotinylation/streptavidin pulldown techniques to identify cell surface proteins

  • Confirm using flow cytometry with anti-KAR2 antibodies

  • Employ non-permeabilized immunofluorescence microscopy

• Functional analysis:

  • Investigate potential interactions with host proteins using pulldown assays

  • Examine potential interactions with integrin β1, as observed in other cell types

  • Evaluate downstream signaling pathways potentially activated by csKAR2

In non-fungal cells, HG (high glucose) induces persistent cell surface expression of GRP78, detectable as early as 3 hours after exposure, and this translocation is mediated by ER stress . Studies in kidney mesangial cells have shown that csGRP78 interacts with integrin β1 and activates signaling pathways involving focal adhesion kinase (FAK) and Akt, ultimately affecting matrix protein synthesis .

If similar mechanisms exist in C. glabrata, csKAR2 could potentially:

• Interact with host receptors to influence adhesion and invasion

• Modulate host signaling pathways

• Evade host immune recognition

• Contribute to stress resistance during infection

These possibilities represent important areas for future investigation in C. glabrata pathogenesis research.

How can conditional knockout systems be developed to study the essential KAR2 gene in C. glabrata?

Since KAR2 is essential for viability in C. glabrata, traditional knockout approaches are not feasible. Instead, conditional systems must be employed to study its functions. Based on previous research approaches, the following methodological strategies can be implemented:

• Tetracycline-regulatable expression system:

  • Replace the endogenous KAR2 promoter with a tetracycline-responsive promoter

  • Add tetracycline or doxycycline to repress KAR2 expression

  • Monitor phenotypic changes as KAR2 levels decrease

  • This system allows for temporal control of gene expression

• Conditional promoter replacement:

  • A plasmid-based system (e.g., pCaDIS-KAR2) can be constructed using a 1,000 bp fragment of the 5′ end of the KAR2 gene

  • Replace the endogenous promoter with a conditional promoter such as MAL2 (maltose-inducible) or MET3 (methionine-repressible)

  • Culture cells in repressing conditions to study the effects of KAR2 depletion

• Degron-tagged KAR2:

  • Create a fusion protein with KAR2 and an inducible degron tag

  • The degron tag allows for rapid protein degradation upon a specific trigger

  • This approach enables studying the immediate effects of KAR2 protein loss

• RNA interference (if applicable in C. glabrata):

  • Design shRNA or siRNA targeting KAR2 mRNA

  • Use an inducible promoter to control shRNA expression

  • Monitor KAR2 depletion and resulting phenotypes

• CRISPR interference (CRISPRi):

  • Use a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor

  • Target the KAR2 promoter region to repress transcription

  • This allows for titratable and reversible gene repression

For validation and phenotypic analysis:

• Confirm KAR2 depletion using qPCR and Western blot

• Examine growth rates under various stress conditions

• Assess cell morphology and ultrastructure using microscopy

• Evaluate protein secretion and ER stress markers

• Prepare ER microsomes for in vitro translocation assays to directly measure KAR2 function

These approaches provide powerful tools to study the essential functions of KAR2 while circumventing the lethality of complete gene deletion.

What is the relationship between KAR2 expression and antifungal resistance in C. glabrata?

C. glabrata exhibits intrinsic resistance to azole antifungal agents, which presents a significant clinical challenge . While the direct relationship between KAR2 and antifungal resistance has not been fully elucidated, several lines of evidence suggest potential connections:

• ER stress response and drug resistance:

  • Antifungal drugs often induce ER stress

  • As a key component of the ER stress response, KAR2 may help cells adapt to drug-induced stress

  • The calcineurin-Crz1 pathway, which regulates KAR2 in C. glabrata, has been implicated in antifungal resistance in several fungi

• Protein quality control:

  • KAR2 ensures proper folding of membrane proteins, including drug transporters

  • Enhanced efflux pump expression is a common mechanism of azole resistance

  • KAR2 may indirectly contribute to resistance by ensuring proper folding and trafficking of these transporters

Methodological approaches to investigate this relationship:

• Compare KAR2 expression levels between azole-sensitive and resistant strains using qRT-PCR and Western blotting

• Create strains with tunable KAR2 expression and assess their sensitivity to various antifungals

• Combine KAR2 modulation with inhibitors of different resistance mechanisms (e.g., efflux pump inhibitors)

• Perform genome-wide transcriptional analysis to identify genetic networks connecting KAR2 and resistance genes

• Assess cell surface KAR2 expression in resistant versus sensitive strains

A comprehensive experiment would include:

• Collecting clinical isolates with varying levels of drug resistance

• Measuring baseline and stress-induced KAR2 expression

• Determining minimum inhibitory concentrations (MICs) of different antifungals

• Creating a correlation matrix between KAR2 expression, stress response factors, and drug resistance

• Using pharmacological and genetic approaches to modulate KAR2 and observe effects on resistance

This systematic approach could reveal whether KAR2 represents a potential target for combination therapy to overcome antifungal resistance.

What are the main technical challenges in studying KAR2 protein-protein interactions in C. glabrata?

Studying protein-protein interactions (PPIs) involving KAR2 in C. glabrata presents several technical challenges that require specialized approaches:

• Essential nature of KAR2:

  • Complete deletion is lethal, complicating genetic approaches

  • Solution: Use conditional expression systems or domain-specific mutations that maintain viability but alter specific interactions

  • Employ split complementation systems where KAR2 fragments are expressed separately but can reconstitute function when in proximity

• Membrane association and difficult extraction:

  • KAR2 is primarily localized to the ER membrane, making extraction while preserving interactions challenging

  • Solution: Optimize gentle detergent conditions (digitonin, CHAPS, or DDM) for membrane solubilization

  • Use crosslinking approaches to stabilize transient interactions before extraction

• Multiple cellular pools of KAR2:

  • KAR2 exists in both ER and cell surface pools, each with potentially different interaction partners

  • Solution: Employ subcellular fractionation to separate distinct KAR2 populations

  • Use surface-specific biotinylation to selectively purify cell surface KAR2 complexes

• Distinguishing direct vs. indirect interactions:

  • Co-immunoprecipitation may identify complex components rather than direct binding partners

  • Solution: Use proximity labeling techniques (BioID, APEX) to identify proteins in close proximity

  • Validate direct interactions using purified recombinant proteins and in vitro binding assays

• Temporal dynamics of interactions:

  • KAR2 interactions likely change during different stress conditions and cellular states

  • Solution: Develop time-course experiments with synchronized stress induction

  • Use FRET or BRET systems to monitor interaction dynamics in living cells

Methodological workflow for comprehensive PPI analysis:

• Create C. glabrata strains expressing tagged KAR2 (e.g., TAP-tag, FLAG-tag, or proximity labeling tags)

• Validate that the tag doesn't interfere with KAR2 function

• Perform immunoprecipitation under different conditions (normal growth, ER stress, antifungal exposure)

• Identify interaction partners using mass spectrometry

• Validate key interactions using reciprocal tagging and co-immunoprecipitation

• Assess the biological significance of interactions through functional assays

This comprehensive approach can overcome the technical challenges and provide valuable insights into KAR2's interaction network in C. glabrata.