Recombinant Candida glabrata Topoisomerase I damage affected protein 7 (TDA7)

Basic Research Questions

• What is Candida glabrata TDA7 and what is its role in pathogenesis?

TDA7 is a putative adhesin-like protein in C. glabrata that plays a crucial role in promoting tissue colonization and invasion during infection . As an adhesin, TDA7 mediates the initial interaction between C. glabrata and host surfaces, which can subsequently lead to persistent infections, particularly in immunocompromised patients . Recent proteomic characterization studies have identified TDA7 as part of C. glabrata's virulence arsenal, contributing to its ability to adhere to and invade host tissues.

Methodologically, researchers can investigate TDA7's role in pathogenesis through:

• Gene knockout studies using homologous recombination

• Adhesion assays with epithelial cell lines

• In vivo infection models such as Galleria mellonella larvae

• Comparative proteomic analysis between wild-type and TDA7-deficient strains

• How is TDA7 expression regulated in response to environmental stresses?

C. glabrata adapts to various stress conditions by modifying its gene expression patterns, including those of adhesins like TDA7. Research has shown that C. glabrata responds to the intracellular environment of macrophages by modifying chromatin structure , which affects gene expression patterns. Additionally, azole-resistant C. glabrata strains have been found to possess mutations in the subtelomeric silencer Sir3 that de-represses adhesins, suggesting that antifungal pressure can alter TDA7 expression .

To study TDA7 regulation experimentally:

• Use quantitative RT-PCR to assess expression levels under different stress conditions

• Employ chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the TDA7 promoter

• Analyze epigenetic modifications at the TDA7 locus using techniques like MNase sensitivity assays

• Construct reporter gene fusions to monitor TDA7 promoter activity in real-time

• What methods are recommended for recombinant expression and purification of TDA7?

For successful recombinant expression of C. glabrata proteins like TDA7, researchers can follow methodologies described in recent studies. For example, one approach involves cloning the TDA7 gene into expression vectors with appropriate promoters. The copper-inducible C. glabrata MTI promoter has been successfully used for protein expression in C. glabrata .

A step-by-step approach would include:

• PCR amplification of the TDA7 coding sequence from C. glabrata genomic DNA

• Cloning into an expression vector (e.g., pGREG576 with the MTI promoter)

• Verification of the recombinant plasmid by DNA sequencing

• Transformation into an appropriate expression host

• Induction of protein expression using copper sulfate

• Purification via affinity chromatography (e.g., His-tag purification)

• How does DNA damage affect C. glabrata gene expression and virulence?

C. glabrata responds to DNA damage by activating the DNA damage response (DDR) pathway, which involves a comprehensive defense system to monitor and remove DNA lesions . Research has shown that C. glabrata wild-type cells respond to the intracellular environment of macrophages by modifying their chromatin structure, altering epigenetic signatures, decreasing protein acetylation, and increasing cellular lysine deacetylase activity .

Several genes involved in chromatin organization (Cgrsc3-aΔ, Cgrsc3-bΔ, Cgrsc3-aΔbΔ, Cgrtt109Δ) and DNA damage repair (Cgrtt107Δ, Cgsgs1Δ) have been shown to be critical for C. glabrata virulence in murine models . Genome-wide transcriptional profiling of macrophage-internalized yeasts revealed deregulation of energy metabolism in mutants defective in chromatin remodeling .

Experimental approaches to study DNA damage responses include:

• Exposing C. glabrata to DNA-damaging agents and analyzing expression profiles

• Creating knockouts of DNA repair genes and assessing virulence

• Using fluorescent reporters to monitor DNA damage in real-time during infection

• Performing ChIP-seq to identify genome-wide binding of DNA damage response proteins