Recombinant Candida glabrata Transcription activator of gluconeogenesis ERT1 (ERT1), partial

Introduction to Candida glabrata and Metabolic Adaptation

Candida glabrata (recently renamed as Nakaseomyces glabratus) is an opportunistic fungal pathogen that causes potential life-threatening invasive candidiasis, particularly in immunocompromised populations . Despite its name suggesting a relationship with Candida albicans, C. glabrata is actually more closely related to the non-pathogenic budding yeast Saccharomyces cerevisiae . This phylogenetic relationship provides unique opportunities for comparative genomic studies while presenting challenges in understanding its pathogenicity mechanisms.

A key aspect of C. glabrata's success as a pathogen is its remarkable metabolic adaptability, particularly its ability to thrive in glucose-limited environments such as within host macrophages . During infection, C. glabrata encounters diverse microenvironments within the host, necessitating robust and efficient metabolic adaptation for survival . The reprogramming of carbon metabolism is especially critical for phagocytosed C. glabrata within glucose-deprived conditions during infection .

Molecular Identity and Classification

ERT1 (Ethanol Regulated Transcription factor 1) is a zinc cluster transcription factor in Candida glabrata that plays a crucial role in regulating gluconeogenesis . Zinc cluster proteins form a large family of transcriptional regulators involved in controlling various metabolic processes including the metabolism of sugars, amino acids, fatty acids, as well as contributing to drug resistance mechanisms .

The recombinant form of C. glabrata ERT1 (product code CSB-EP739311CZI) is a partial protein produced in E. coli expression systems with a purity of >85% as determined by SDS-PAGE . It is derived from the Candida glabrata strain ATCC 2001/CBS 138/JCM 3761/NBRC 0622/NRRL Y-65 . The protein corresponds to UniProt accession number Q6FML7 .

Transcriptional Regulation by ERT1

ERT1 functions as a transcription activator that controls the expression of genes involved in gluconeogenesis, a metabolic pathway that generates glucose from non-carbohydrate carbon sources . This regulatory function is particularly important when C. glabrata encounters environments with limited glucose availability, such as within host macrophages during infection .

Target Genes and Metabolic Pathways

While not specifically mentioning ERT1, studies on C. glabrata's metabolic adaptations to alternative carbon sources provide insight into the pathways likely regulated by this transcription factor. Table 1 shows key gluconeogenic genes that are significantly upregulated when C. glabrata shifts from glucose to alternative carbon metabolism:

Table 1: Key Gluconeogenic Genes Upregulated During Carbon Source Shift in C. glabrata

Data derived from transcriptomic analysis of C. glabrata grown in acetate versus glucose .

These genes are likely targets of ERT1 regulation, particularly PCK1, which has been specifically identified as an ERT1 target . The significant upregulation of these genes demonstrates the critical importance of gluconeogenesis for C. glabrata's adaptation to glucose-limited environments.

Expression and Purification

Recombinant Candida glabrata ERT1 is typically produced as a partial protein in E. coli expression systems . The production process generally involves:

1. Cloning the partial ERT1 gene into an appropriate expression vector

2. Transformation into E. coli expression hosts

3. Induction of protein expression

4. Cell lysis and protein extraction

5. Purification using affinity chromatography

6. Quality control assessments including SDS-PAGE to confirm purity (>85%)

Role in Host-Pathogen Interactions

The ability of C. glabrata to adapt to alternative carbon sources is crucial for its survival within host environments, particularly within macrophages where glucose availability is limited . As a key regulator of gluconeogenesis, ERT1 likely plays a significant role in this adaptation process .

Transcriptomic and proteomic studies have revealed that when C. glabrata encounters glucose-deprived conditions, it reprograms its carbon metabolism by upregulating pathways such as the glyoxylate cycle and gluconeogenesis . This metabolic shift allows C. glabrata to synthesize glucose from alternative carbon sources available within the host, thereby enhancing its survival and persistence during infection .

Relationship to Other Transcriptional Regulators

ERT1 is part of a complex regulatory network that controls C. glabrata's metabolic adaptations. It belongs to a family of zinc cluster transcription factors that includes related paralogs such as Gsm1 and Rds2 . These transcription factors exhibit functional redundancy in regulating gluconeogenesis, suggesting a robust system to ensure metabolic adaptability .

BLAST analysis using the full-length Gsm1 polypeptide as a query reveals significant homology with Ert1 (E-value of 8 × 10^-26) and Rds2 (E-value of 2 × 10^-14), indicating their evolutionary relationship and potential functional overlap .

Experimental Applications

Recombinant ERT1 serves as a valuable tool for various research applications, including:

1. Structural studies to elucidate the three-dimensional configuration of the protein

2. Functional assays to determine DNA binding specificity and affinity

3. Protein-protein interaction studies to identify cofactors and regulatory partners

4. Development of antibodies for detection and localization studies

5. Drug discovery efforts targeting transcriptional regulation pathways

Potential Therapeutic Implications

Understanding ERT1's role in C. glabrata's metabolic adaptation may reveal potential therapeutic targets. Since gluconeogenesis is critical for pathogen survival within host environments, inhibiting ERT1 function could potentially compromise C. glabrata's virulence and persistence .

This approach is particularly relevant given the increasing concerns about antifungal resistance in C. glabrata. Studies have shown that C. glabrata can develop resistance to azole antifungals, which are commonly used to treat fungal infections . Targeting metabolic adaptation pathways regulated by ERT1 could provide alternative strategies to combat resistant strains.