Recombinant Candida glabrata Sensitivity to high expression protein 10 (SHE10), partial

What is SHE10 and what is its functional role in Candida glabrata pathobiology?

SHE10 (Sensitivity to high expression protein 10) is a protein identified in Candida glabrata with the Uniprot accession number Q6FQR7 . While its precise function remains under investigation, the nomenclature suggests involvement in gene expression regulation, potentially contributing to stress adaptation mechanisms.

Methodological approach to elucidate SHE10 function:

• Comparative genomics with S. cerevisiae homologs, as C. glabrata is phylogenetically closer to S. cerevisiae than to other Candida species

• Creation of SHE10 knockout strains using established gene deletion techniques similar to those used for CgDTR1 deletion

• Phenotypic characterization under various stress conditions, particularly focusing on conditions relevant to the host environment

• Transcriptomic analysis comparing wild-type and SHE10-deficient strains

Given C. glabrata's remarkable capacity for genetic diversity, with at least 19 separate sequence types identified globally , researchers should examine SHE10 across different strains to understand potential functional variations.

What expression systems are most effective for recombinant SHE10 production?

The commercially available recombinant SHE10 is produced using a baculovirus expression system , which offers several advantages for fungal protein expression:

When working with recombinant SHE10, researchers should consider:

• The partial nature of the commercial protein may indicate challenges with full-length expression

• Codon optimization for the expression host may improve yields

• Fusion tags should be selected based on downstream applications

• Expression conditions should be optimized to maximize soluble protein yield

What are the optimal storage conditions for maintaining SHE10 stability and activity?

According to product information , recombinant SHE10 stability varies by formulation:

• Liquid form: 6-month shelf life at -20°C/-80°C

• Lyophilized form: 12-month shelf life at -20°C/-80°C

For optimal handling and reconstitution:

• Centrifuge vials briefly before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage

• Prepare working aliquots to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

Researchers should verify protein integrity after storage using:

• SDS-PAGE to confirm expected molecular weight

• Activity assays specific to hypothesized function

• Circular dichroism to assess secondary structure maintenance

• Dynamic light scattering to evaluate aggregation state

How does C. glabrata genetic diversity impact SHE10 structure-function relationships?

C. glabrata isolates demonstrate remarkable genetic diversity, including microevolution during infection . This diversity may significantly impact SHE10:

• Sequence variation: Clinical isolates may contain SHE10 variants with amino acid differences affecting:

  • Protein stability and half-life

  • Binding affinities to potential partners

  • Subcellular localization patterns

  • Post-translational modification sites

• Expression regulation: Various C. glabrata strains may express SHE10 at different levels or under different conditions, similar to strain-dependent expression patterns observed for other virulence factors .

• Functional adaptation: Evidence of recombination between geographically distinct strains suggests genetic exchange creating new functional variants. If SHE10 contributes to adaptation, it may show positive selection signatures.

Methodology for investigating strain variation:

• Sequence SHE10 across diverse clinical isolates

• Conduct Western blot analysis to compare expression levels

• Perform complementation studies with different SHE10 variants

• Apply selection pressure analysis to identify evolutionary constraints

What experimental models are most suitable for studying SHE10's role in virulence?

Several experimental models can be employed to investigate SHE10's potential role in C. glabrata pathogenicity:

The G. mellonella model has been successfully used to study C. glabrata virulence genes like CgDTR1, where wild-type strains kill significantly more larvae than deletion mutants . A similar approach could evaluate SHE10's contribution to virulence.

For in vitro studies, researchers should note that C. glabrata strains like BG2 replicate to higher numbers in macrophages and are more virulent in G. mellonella infection than CBS138 strains in a dose-dependent manner , highlighting the importance of strain selection.

How can gene editing techniques be optimized for studying SHE10 function?

Several gene editing approaches can be applied to study SHE10 function in C. glabrata:

• Homologous recombination gene deletion:

  • A well-established method in C. glabrata

  • Target gene replacement with selective markers (e.g., CgHIS3)

  • Design primers with ~56 bp homology to flanking regions

  • Verification by PCR using specific primer combinations

• CRISPR-Cas9 modifications:

  • Allows precise gene editing beyond simple knockouts

  • Can create point mutations to test specific domains

  • Enables promoter modifications to alter expression

  • Allows tagging for localization studies

• Conditional expression systems:

  • The copper-inducible MTI promoter has been successfully used in C. glabrata

  • Example from research: replacement of GAL1 promoter with copper-inducible MTI promoter

  • Useful for studying essential genes where knockout may be lethal

C. glabrata's haploid genome simplifies gene editing compared to diploid Candida species, but strain diversity necessitates verifying phenotypes across multiple genetic backgrounds.

What is the relationship between SHE10 and antifungal drug resistance mechanisms?

While specific connections between SHE10 and drug resistance aren't detailed in the search results, researchers investigating this relationship should consider C. glabrata's known resistance mechanisms:

• Azole resistance:

  • Intrinsic tolerance to azoles

  • ~20% of isolates develop fluconazole resistance during therapy

  • Key mechanisms include efflux pump upregulation and ergosterol biosynthesis alterations (ERG4)

  • DNA repair defects (MSH2 mutations) promote adaptive mutations

• Echinocandin resistance:

  • Target gene mutations (FKS1)

  • "Petite" phenotypes show echinocandin tolerance

  • 38 genes identified affecting echinocandin susceptibility

Methodological approach to investigate SHE10's potential role:

• Compare SHE10 expression in drug-susceptible vs. resistant isolates

• Generate SHE10 deletion mutants and determine minimum inhibitory concentrations

• Assess whether overexpression affects drug susceptibility

• Test for potential interactions with known resistance factors

Recent research on CgDTR1 demonstrates how a single protein can significantly affect C. glabrata drug susceptibility, with CgDTR1 conferring resistance to oxidative and acetic acid stress through its function as a plasma membrane acetic acid exporter .

How can proteomics approaches identify SHE10 binding partners and functional networks?

Proteomic approaches offer powerful methods to elucidate SHE10's functional role:

When working with C. glabrata proteins:

• The haploid genome simplifies genetic manipulations for tagging

• Cell lysis conditions need optimization due to the robust cell wall

• Strain selection is critical due to genetic diversity

• Comparisons under different stress conditions may reveal context-dependent interactions

Functional validation should follow proteomic identification, potentially using the large-scale C. glabrata deletion library (619 unique strains) as a resource .

How does SHE10 expression change during host adaptation and microevolution?

C. glabrata undergoes microevolution during infection, with documented changes affecting virulence factors . To investigate SHE10's role in this process:

• Serial clinical isolate analysis:

  • Sequence SHE10 in sequential isolates from the same patient

  • Measure expression levels using RT-PCR

  • Compare with global patterns of genomic change

• Host-mimicking conditions:

  • Monitor SHE10 expression during:

    • Macrophage internalization

    • Exposure to oxidative stress

    • Nutrient limitation

    • Antifungal pressure

  • Use RT-PCR methodology similar to that described for CgDTR1

• Mixed population analysis:

  • Recent evidence shows bloodstream infections contain genetically diverse C. glabrata strains

  • Analyze SHE10 variation within these populations through single-colony isolation and characterization

• Experimental evolution:

  • Laboratory evolution under defined selective pressures

  • Whole genome sequencing to identify mutations

  • Transcriptional profiling to detect expression changes

The high genetic diversity of C. glabrata isolates, with evidence for ancestral recombination and transmission between geographical regions , suggests SHE10 may also show functional diversity worth exploring in adaptation studies.

What computational approaches can predict SHE10 structural features and functional domains?

Computational methods provide valuable insights into potential SHE10 functions:

• Structural analysis:

  • AlphaFold or RoseTTAFold for protein structure prediction

  • Domain identification through InterProScan or SMART

  • Molecular dynamics simulations to assess flexibility and binding sites

  • Structure-based function prediction

• Comparative genomics:

  • Alignment with homologs in related species

  • C. glabrata is phylogenetically closer to S. cerevisiae than other Candida species

  • Identification of conserved motifs and regulatory elements

  • Detection of selective pressure through Ka/Ks analysis

• Network analysis:

  • Guilt-by-association using co-expression data

  • Protein-protein interaction prediction

  • Functional associations through gene ontology analysis

  • Integration with C. glabrata-specific datasets

• Gene expression context:

  • Analysis of co-expressed genes under relevant conditions

  • Identification of shared regulatory elements

  • Correlation with known stress response and virulence pathways

Researchers should validate computational predictions through targeted experimental approaches, considering strain diversity that may affect protein structure and function.

How does SHE10 potentially interact with host immune responses?

While specific information about SHE10's interactions with host immunity isn't provided in the search results, researchers can investigate potential roles based on known C. glabrata pathogenesis mechanisms:

• Phagocyte interactions:

  • C. glabrata can survive within macrophages

  • SHE10 might contribute to:

    • Stress adaptation within phagosomes

    • Neutralization of reactive oxygen species

    • Metabolic adaptation for intracellular survival

• Inflammatory modulation:

  • C. glabrata infections typically elicit less inflammation than C. albicans

  • Women with C. glabrata vaginitis show lower levels of secretory IgA in vaginal secretions

  • SHE10 might modulate pattern recognition receptor signaling

• Immune evasion:

  • Different C. glabrata variants trigger distinct macrophage transcriptional responses

  • SHE10 might contribute to phenotypic variations affecting immune recognition

Methodological approaches:

• In vitro infection assays with immune cells

• Cytokine profiling upon exposure to wild-type vs. SHE10-deficient strains

• Comparison of phagocytosis and killing rates

• Transcriptomic analysis of host cells during infection

The immunological differences between C. glabrata and C. albicans infections suggest unique host-pathogen interactions worth exploring in relation to SHE10 function.

How can microscopy techniques be optimized to study SHE10 localization?

Determining SHE10's subcellular localization provides crucial insights into its function. Several microscopy approaches can be employed:

• Fluorescent protein tagging:

  • GFP/mCherry fusion proteins expressed in C. glabrata

  • Considerations:

    • Tag position may affect protein function

    • Expression control using native or inducible promoters like MTI

    • C. glabrata's haploid nature simplifies genetic manipulations

• Immunofluorescence microscopy:

  • Antibodies against SHE10 or epitope tags

  • Requires optimized cell wall digestion protocols

  • Co-staining with organelle markers confirms specific localization

• Super-resolution microscopy:

  • STED, PALM, or STORM for nanoscale resolution

  • Particularly valuable if SHE10 forms distinct subcellular structures

  • Enables co-localization studies with potential interaction partners

• Live-cell imaging:

  • Dynamic studies of SHE10 localization under different conditions

  • Photoactivatable fluorescent proteins to track protein movement

  • FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

C. glabrata strain selection is important, as different strains like CBS138 and BG2 show significant differences in cell wall properties , which could affect imaging protocols and potentially SHE10 localization.

What methodologies can assess SHE10's potential role in C. glabrata's metabolic adaptation?

C. glabrata demonstrates remarkable metabolic flexibility, which contributes to its survival in diverse host environments. To investigate SHE10's potential role in metabolic adaptation:

• Comparative growth assays:

  • Wild-type vs. SHE10-deficient strains

  • Various carbon and nitrogen sources

  • Different stress conditions (pH, temperature, oxidative stress)

  • BG2 strains utilize a broader range of nitrogen sources than CBS138

• Metabolomic profiling:

  • LC-MS/MS or GC-MS analysis of metabolite pools

  • Flux analysis using isotope-labeled substrates

  • Comparison of intracellular vs. secreted metabolites

• Transcriptomic analysis:

  • RNA-seq under varying nutrient conditions

  • Comparison of wild-type vs. SHE10-deficient responses

  • Time-course analysis during adaptation

• Functional genomics integration:

  • Connection to nutrient sensing pathways

  • Deletion or overexpression of SHE10 in nutrient sensor mutant backgrounds

  • The SNF3 nutrient sensor significantly affects virulence in certain strain backgrounds