Recombinant Candida glabrata Spindle pole component BBP1 (BBP1)

What is the function of BBP1 in Candida glabrata spindle pole bodies?

BBP1 is an essential component of the spindle pole body (SPB) in Candida glabrata, functionally similar to its well-studied homolog in Saccharomyces cerevisiae. The protein plays a critical role in SPB duplication and insertion into the nuclear envelope (NE). BBP1 is part of the network of interacting proteins required for the proper insertion of a cytoplasmic SPB precursor into the NE at sites where the inner and outer nuclear membranes fuse. This process is essential for proper chromosome segregation during cell division.

Like related SPB components such as Nbp1, BBP1 likely functions at the inner face of the nuclear envelope to facilitate SPB duplication and insertion. Defects in BBP1 function would be expected to cause monopolar spindles and chromosome segregation errors, leading to genomic instability - a phenotype commonly observed in SPB component mutations .

How should researchers design experiments to localize BBP1 in C. glabrata cells?

To effectively localize BBP1 in C. glabrata cells, researchers should employ the following methodological approaches:

• Fluorescent protein tagging: Generate C. glabrata strains expressing BBP1-GFP or BBP1-mCherry fusion proteins under the control of the native promoter. This approach allows for live-cell imaging of BBP1 localization throughout the cell cycle.

• Immunofluorescence microscopy: Develop specific antibodies against BBP1 for fixed-cell immunofluorescence. Co-stain with antibodies against known SPB markers (such as Spc42 or Mps3) to confirm SPB localization.

• Cell cycle synchronization: Use methods such as α-factor arrest (if applicable to your C. glabrata strain) or nocodazole treatment to obtain synchronized cell populations for examining BBP1 localization at specific cell cycle stages.

• Electron microscopy: Employ immunogold labeling of BBP1 for electron microscopy studies to determine its precise localization within the SPB structure at nanometer resolution.

When conducting these experiments, it's important to include appropriate controls such as co-localization with known SPB markers. Based on studies of related proteins, researchers should expect to observe BBP1 at the SPB throughout the cell cycle, with potential changes in intensity or precise localization during SPB duplication .

What are the challenges in expressing recombinant C. glabrata BBP1 in heterologous systems?

Expressing recombinant C. glabrata BBP1 presents several technical challenges that researchers should anticipate:

• Codon optimization: C. glabrata has distinct codon usage preferences compared to common expression hosts like E. coli. Researchers should optimize the coding sequence for the expression host to ensure efficient translation.

• Protein solubility: SPB components often contain multiple domains including coiled-coil regions that can lead to aggregation when overexpressed. Consider using solubility tags (MBP, SUMO, etc.) and optimizing expression conditions (temperature, induction levels).

• Post-translational modifications: If BBP1 requires specific modifications for proper folding or function, expression in bacterial systems may not provide these modifications. Consider yeast-based expression systems like Pichia pastoris or S. cerevisiae.

• Purification strategy: Design a purification strategy that accounts for BBP1's biochemical properties. If membrane association is expected (as seen with Nbp1 ), include appropriate detergents or extraction methods to maintain protein stability.

• Functional validation: After purification, validate that recombinant BBP1 retains its functional properties through in vitro assays such as binding studies with interaction partners or membrane association assays.

A typical workflow would include cloning BBP1 into an appropriate expression vector with a purification tag, transforming into the expression host, optimizing expression conditions, developing a multi-step purification protocol, and confirming protein identity through mass spectrometry.

How can researchers identify interaction partners of BBP1 in C. glabrata?

To comprehensively identify BBP1 interaction partners, researchers should employ multiple complementary approaches:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged BBP1 (e.g., TAP-tag or FLAG-tag) in C. glabrata

  • Perform gentle cell lysis to preserve protein-protein interactions

  • Isolate BBP1 complexes using affinity chromatography

  • Analyze co-purifying proteins by mass spectrometry

  • Include appropriate controls (untagged strains, tag-only controls)

• Yeast two-hybrid (Y2H) screening:

  • Use BBP1 as bait against a C. glabrata genomic library

  • Screen for positive interactions using reporter gene activation

  • Validate positive hits through secondary assays

• Proximity-based labeling:

  • Fuse BBP1 to a proximity labeling enzyme (BioID or APEX2)

  • Express the fusion protein in C. glabrata

  • Activate labeling to biotinylate proteins in close proximity to BBP1

  • Purify biotinylated proteins and identify by mass spectrometry

• Co-immunoprecipitation with candidate partners:

  • Based on knowledge from related yeasts, test specific candidates

  • Express differentially tagged versions of BBP1 and candidate partners

  • Perform co-immunoprecipitation experiments followed by immunoblotting

Based on studies of SPB components in other yeasts, researchers should expect BBP1 to interact with other SPB components, particularly those involved in SPB duplication and insertion. By analogy to Nbp1, BBP1 might interact with the conserved integral membrane protein Ndc1, which is involved in both SPB insertion and nuclear pore complex assembly .

What phenotypes are expected in C. glabrata BBP1 mutants, and how should they be characterized?

C. glabrata BBP1 mutants would likely exhibit several distinct phenotypes that can be characterized using the following methodologies:

• Cell cycle arrest and morphology:

  • BBP1 mutants would likely arrest as large-budded cells with a G2/M DNA content

  • Quantify cell cycle distribution using flow cytometry

  • Analyze cell morphology using phase contrast and fluorescence microscopy

  • Expected phenotype: large-budded cells with unseparated DNA masses

• Spindle and SPB defects:

  • Visualize spindle morphology using GFP-TUB1 (α-tubulin) to label microtubules

  • Label SPBs using SPC42-mCherry or other SPB markers

  • Expected phenotypes include:

    • Monopolar spindles

    • "Dead poles" that nucleate only cytoplasmic and not nuclear microtubules

    • Failure of SPB duplication or insertion

• Genetic instability:

  • Measure chromosome loss rates using colony sectoring assays

  • Analyze DNA content by flow cytometry to detect aneuploidy or polyploidy

  • Monitor changes in karyotype using pulse-field gel electrophoresis

  • Expected phenotype: increased ploidy as an adaptation mechanism, similar to nbp1-(15-319) cells

• Ultrastructural analysis:

  • Examine SPB structure using transmission electron microscopy

  • Look for specific defects in SPB duplication or insertion

  • Expected phenotypes: SPBs that fail to insert into the nuclear envelope, similar to those observed in Nbp1 mutants

• Temperature sensitivity:

  • Test growth at various temperatures (23°C, 30°C, 37°C)

  • Conditional mutants may be viable at permissive temperature but defective at restrictive temperature

  • Expected phenotype: temperature-sensitive growth defects, particularly at 37°C

These analyses should be performed with appropriate controls, including comparison to wild-type strains and complemented mutants to confirm phenotype specificity.

How does the structure of BBP1 contribute to its function in SPB duplication?

The structure of BBP1 contains several domains that are critical for its function in SPB duplication:

• N-terminal domain: By analogy to other SPB components like Nbp1, the N-terminal region of BBP1 likely contains membrane-binding elements such as an amphipathic α-helix that functions as an in-plane membrane anchor. This domain would be critical for proper localization to the nuclear envelope. Mutations in this region would be expected to disrupt proper SPB insertion into the nuclear envelope .

• Central coiled-coil region: BBP1 likely contains coiled-coil domains that mediate protein-protein interactions with other SPB components. These structural elements are common in SPB proteins and are crucial for the assembly of multi-protein complexes.

• C-terminal domain: This region may be involved in specific interactions with other SPB components.

To experimentally determine the contribution of these domains to BBP1 function, researchers should:

• Generate truncation mutants of BBP1 (e.g., BBP1-(1-173), BBP1-(174-319)) to determine which regions are essential for viability

• Create point mutations in predicted functional motifs (e.g., amphipathic helices, coiled-coil regions)

• Analyze the localization and function of these mutants in vivo

• Perform complementation assays to determine which domains can rescue BBP1 deletion phenotypes

Based on studies of Nbp1, researchers might expect that the N-terminal domain containing a membrane-binding element would be essential for function. Mutations in this region would likely cause conditional lethal growth defects at higher temperatures (37°C) and defects in SPB insertion .

How does the functional role of BBP1 in C. glabrata compare to its homologs in other fungal species?

The functional role of BBP1 in C. glabrata can be compared to its homologs in other fungi through several methodological approaches:

• Comparative genomic analysis:

  • Identify BBP1 homologs across different fungal species using sequence homology searches

  • Analyze conservation of key domains and motifs

  • Construct phylogenetic trees to understand evolutionary relationships

• Complementation studies:

  • Test whether BBP1 homologs from other species (e.g., S. cerevisiae, C. albicans) can complement C. glabrata BBP1 deletion

  • Conversely, test whether C. glabrata BBP1 can complement mutations in homologs from other species

  • These experiments reveal functional conservation or divergence

• Protein localization comparison:

  • Compare localization patterns of BBP1 homologs tagged with the same fluorescent protein across different species

  • Analyze timing of recruitment to the SPB during the cell cycle

• Interaction network comparison:

  • Compare BBP1 protein-protein interaction networks across species using AP-MS or Y2H approaches

  • Identify conserved and species-specific interactions

Based on studies of spindle pole components across fungal species, we would expect both conservation and divergence in BBP1 function. In S. cerevisiae, the SPB is embedded in the nuclear envelope throughout the cell cycle, whereas in other fungi like O. polymorpha, the SPB structure appears to cycle with outer plaque components appearing and disappearing during the cell cycle . C. glabrata, being more closely related to S. cerevisiae than to C. albicans , likely shares more functional similarities with S. cerevisiae BBP1, but may have evolved species-specific adaptations related to its pathogenic lifestyle.

What role might BBP1 play in C. glabrata pathogenesis and virulence?

The potential role of BBP1 in C. glabrata pathogenesis and virulence can be investigated through several experimental approaches:

• Virulence models:

  • Test BBP1 mutants in established C. glabrata infection models:

    • Galleria mellonella larvae model (similar to studies with CgXbp1 and CgDtr1)

    • Mouse models of systemic or mucosal infection

  • Measure survival rates, fungal burden, and host immune responses

  • Compare results with wild-type and complemented strains

• Interaction with host cells:

  • Assess the ability of BBP1 mutants to adhere to and invade host epithelial cells

  • Measure survival within macrophages using CFU assays (similar to CgXbp1 studies)

  • Analyze host cell responses to infection with BBP1 mutants

• Stress response analysis:

  • Test BBP1 mutants for altered responses to stresses encountered during infection:

    • Oxidative stress (H₂O₂, menadione)

    • Nitrosative stress

    • pH stress

    • Nutritional limitation

  • Compare growth, survival, and gene expression profiles under these conditions

• Gene expression studies:

  • Perform RNA-seq to identify genes differentially expressed in BBP1 mutants

  • Look for alterations in known virulence factor expression

  • Conduct ChIP-seq if BBP1 is suspected to have regulatory functions

While BBP1's primary function is in spindle pole body organization, defects in chromosome segregation could indirectly affect pathogenesis through altered gene expression, stress responses, or adaptation to the host environment. The genomic instability resulting from BBP1 dysfunction might potentially contribute to the adaptive capabilities of C. glabrata during infection or antifungal treatment. Additionally, proper cell division is necessary for proliferation within the host, so BBP1 function could indirectly impact virulence by affecting growth rates under infection conditions.

How can researchers develop conditional BBP1 mutants for studying essential functions?

Since BBP1 is likely essential for viability (as are many SPB components), developing conditional mutants is crucial for studying its function. Researchers can employ several strategies:

• Temperature-sensitive alleles:

  • Generate random mutations in BBP1 using error-prone PCR

  • Screen for mutants that grow at permissive temperature (23-25°C) but not at restrictive temperature (37°C)

  • Characterize the mutations by sequencing and identify specific residues critical for function

  • Example workflow:

    • Create a BBP1 deletion strain complemented with a URA3-marked plasmid expressing wild-type BBP1

    • Transform with a library of mutagenized BBP1 on a LEU2-marked plasmid

    • Select for growth on 5-FOA at 25°C (forcing loss of the URA3 plasmid)

    • Screen resulting colonies for temperature sensitivity at 37°C

• Degron-based systems:

  • Create BBP1-td (temperature-sensitive degron) fusions similar to those used for Nbp1

  • Induce degradation of BBP1 by shifting cells to restrictive temperature and inducing the ubiquitin ligase (e.g., Gal1-UBR1)

  • Monitor phenotypes as BBP1 is depleted

• Auxin-inducible degron system:

  • Fuse BBP1 to an auxin-inducible degron (AID) tag

  • Express the TIR1 F-box protein in C. glabrata

  • Induce rapid degradation of BBP1 by adding auxin to the medium

  • Track cellular phenotypes at various time points after induction

• Tetracycline-regulated expression:

  • Place BBP1 under the control of a tetracycline-repressible promoter

  • Repress BBP1 expression by adding doxycycline to the medium

  • Monitor cell cycle progression and SPB duplication as BBP1 levels decrease

When studying conditional BBP1 mutants, researchers should analyze:

• Changes in cell cycle distribution

• SPB duplication and spindle formation defects

• Effects on chromosome segregation

• Alterations in cell morphology and ploidy

These approaches allow for temporal control of BBP1 function, enabling detailed study of its roles at specific cell cycle stages and avoiding the complications of adaptation that can occur in strains with stable mutations in essential genes.

What are the potential applications of BBP1 as a target for antifungal development?

BBP1 represents a potential novel target for antifungal development based on several key factors:

• Essential function: As an SPB component, BBP1 is likely essential for viability, making it an attractive target for developing fungicidal agents.

• Divergence from human proteins: Fungi use SPBs for spindle organization whereas humans use centrosomes, representing a fundamental difference that could be exploited for selective targeting.

• Scientific rationale for targeting BBP1:

  • Disruption would lead to mitotic failure and cell death

  • Sub-lethal inhibition might increase genomic instability, potentially affecting virulence and drug resistance development

  • Structure-based drug design could target specific BBP1 domains or protein-protein interactions

To evaluate BBP1 as an antifungal target, researchers should:

• Target validation approaches:

  • Establish conditional BBP1 mutants in C. glabrata

  • Demonstrate that BBP1 depletion leads to growth inhibition/cell death

  • Evaluate phenotypes in relevant infection models

  • Assess potential for resistance development

• Druggability assessment:

  • Perform computational analysis of BBP1 structure to identify potential binding pockets

  • Develop biochemical or cell-based assays for BBP1 function

  • Conduct fragment-based screening or virtual screening for initial hits

• Novel approaches to BBP1 targeting:

  • G-quadruplex targeting: If the BBP1 gene contains putative G-quadruplex sequences (PQS), these could potentially be targeted similar to approaches suggested for other C. glabrata genes like SDH1

  • Protein-protein interaction inhibitors: Target specific interactions between BBP1 and other SPB components

  • Membrane binding inhibitors: If BBP1 contains an amphipathic helix similar to Nbp1 , this could be targeted to disrupt proper localization

• Combination therapy potential:

  • Evaluate synergy between BBP1 inhibitors and existing antifungals

  • Assess whether BBP1 inhibition affects mechanisms of resistance to current antifungals

The spindle pole body represents a relatively unexplored target for antifungal development, with potential advantages in specificity compared to more commonly targeted pathways like ergosterol biosynthesis (targeted by azoles) or cell wall synthesis (targeted by echinocandins).