Recombinant Candida glabrata Protein PBN1 (PBN1), partial

Basic Research Questions

• What is the functional role of PBN1 in Candida glabrata?

  PBN1 in Candida glabrata functions as the noncatalytic subunit of GPI-MT I (glycosylphosphatidylinositol-mannosyltransferase I) complex, essential for the biosynthesis of GPI anchors. Based on orthology studies, PBN1 is critical for stabilizing the catalytic component of the complex. While not possessing enzymatic activity itself, PBN1 enables proper GPI anchor formation, which tethers essential proteins to the cell surface. This tethering mechanism is crucial for cell wall integrity, host-pathogen interactions, and virulence .

  Methodological approach for investigation: To determine PBN1 function in C. glabrata, researchers should employ gene deletion studies followed by phenotypic characterization of surface proteins. Complementation assays using wild-type PBN1 can confirm functional restoration, while fluorescence microscopy and flow cytometry can visualize alterations in surface protein presentation.

• How conserved is PBN1 across microbial species?

  Sequence analysis reveals remarkable functional conservation despite low sequence identity between species. For example, Leishmania donovani and Trypanosoma cruzi PBN1 share only 35% amino acid identity, while human and yeast orthologs share just 19% identity . Despite this divergence, functional complementation is possible across species. Studies have shown that T. cruzi PBN1 can genetically complement L. donovani PBN1 null mutants, restoring surface presentation of GPI-anchored proteins .

  Conservation analysis approach: Researchers should conduct:

  • Phylogenetic analysis of PBN1 sequences across fungal species

  • Multiple sequence alignment to identify conserved domains

  • Cross-species complementation assays to test functional conservation

  • Structural modeling to identify conserved tertiary structures despite sequence divergence

• What are the optimal storage conditions for recombinant C. glabrata PBN1?

  The recombinant PBN1 protein stability is significantly affected by storage conditions. The protein should be stored at -20°C/-80°C with a liquid form shelf life of approximately 6 months and lyophilized form shelf life of 12 months . For working solutions, aliquot the protein and store at 4°C for up to one week, avoiding repeated freeze-thaw cycles .

  Recommended reconstitution protocol:

  1. Briefly centrifuge the vial before opening

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

  3. Add glycerol to a final concentration of 5-50% (50% is recommended)

  4. Prepare small working aliquots to prevent freeze-thaw damage

• What experimental models are suitable for studying PBN1 function in infection?

  Based on related research with orthologous proteins, several experimental models are appropriate for studying PBN1 function:

  • Cell culture models: THP-1 monocyte-derived macrophages provide an excellent system for studying C. glabrata infection dynamics. These models allow temporal monitoring of transcriptional responses during infection .

  • In vivo models: The Galleria mellonella larval model offers a cost-effective system for virulence studies, as demonstrated with other C. glabrata virulence factors . Murine models of infection have also proven effective for studying orthologs like LdPBN1 .

  • Genetic manipulation approach: Generate PBN1 knockout strains using targeted gene deletion with selectable markers such as hygromycin resistance cassettes, following protocols similar to those used for CgXBP1 deletion .

Advanced Research Questions

• How does PBN1 contribute to antifungal resistance mechanisms in C. glabrata?

  While direct evidence linking PBN1 to antifungal resistance is limited, its role in GPI anchor biosynthesis suggests significant involvement in resistance mechanisms. C. glabrata exhibits innate resistance to azole antifungals and rapidly develops clinical drug resistance . GPI-anchored proteins influenced by PBN1 function likely contribute to cell wall integrity and drug efflux systems.

  Other transcription factors like Pdr1 function as sensors of cellular stresses and regulate ABC transporters, critical elements of pleiotropic defense against azoles . The transcription factors Hap1A and Hap1B regulate ERG genes, with Hap1B deletion resulting in increased azole susceptibility due to decreased ergosterol levels .

  Experimental approach to investigate PBN1's role in resistance:

  1. Generate PBN1 knockout and overexpression strains

  2. Perform antifungal susceptibility testing across multiple drug classes

  3. Analyze expression of known resistance genes (ERG11, CDR1, PDR1) in wild-type vs. PBN1-modified strains

  4. Conduct transcriptomic analysis to identify differentially regulated pathways

• What is the temporal transcriptional response of PBN1 during macrophage infection?

  Understanding PBN1 expression dynamics during infection requires sophisticated temporal transcriptional analysis. While specific PBN1 temporal data is not directly available, methodological approaches can be derived from related C. glabrata infection studies.

  Chromatin Immunoprecipitation followed by Next Generation Sequencing (ChIP-seq) against elongating RNA Polymerase II provides powerful temporal resolution of transcriptional changes during infection . C. glabrata mounts chronological transcriptional responses during macrophage infection, with distinct early and late response patterns .

  Recommended methodology for PBN1 temporal analysis:

  1. Infect THP-1 macrophages with C. glabrata at MOI 5:1

  2. Collect samples at multiple timepoints (0.5h, 2h, 4h, 6h, 8h)

  3. Perform formaldehyde crosslinking (1% for 20 min)

  4. Extract chromatin and conduct ChIP-seq or RNA-seq

  5. Analyze PBN1 expression patterns in context of global transcriptional changes

• How do structural features of PBN1 influence cross-species functional complementation?

  Cross-species functional complementation of PBN1 raises intriguing questions about structural requirements for function. Despite low sequence identity (35% between L. donovani and T. cruzi), functional complementation is possible . The minimal PBN1 sequence requirements for stabilizing GPI14 protein and complementing GPI-MT I function remain unclear .

  Human and yeast gene products (19% identical) are functionally incompatible, while the rat PIG-X homolog (78% identical to human) can support yeast expressing human PIG-M, albeit at reduced rates .

  Structure-function analysis approach:

  1. Generate chimeric proteins with domains from different species

  2. Create alanine scanning mutations of conserved residues

  3. Perform complementation assays in PBN1-deficient strains

  4. Use computational modeling to predict critical interaction interfaces

• How does PBN1 deletion affect C. glabrata survival in different host microenvironments?

  C. glabrata encounters diverse microenvironments within human hosts, each presenting unique challenges. Based on studies of orthologous proteins, PBN1 deletion likely affects survival in these varied niches due to compromised cell surface integrity.

  In L. donovani, PBN1-null mutants show significantly decreased virulence in murine models despite being viable in vitro . Similarly, transcription factor mutants in C. glabrata show altered virulence in G. mellonella infection models .

  C. glabrata can persist within macrophage phagosomes through metabolic flexibility, utilizing alternative carbon sources during nutrient limitation . The glyoxylate cycle gene ICL1 promotes growth during macrophage engulfment .

  Experimental design for microenvironment studies:

  1. Generate PBN1 knockout strains

  2. Test survival under various stress conditions (oxidative stress, pH extremes, nutrient limitation)

  3. Conduct macrophage infection assays measuring fungal survival over time

  4. Perform RNA-seq to identify differentially expressed genes in different microenvironments

• What role does PBN1 play in genetic variation patterns observed in serial clinical isolates?

  Clinical isolates of C. glabrata from the same patient show genetic variation patterns that may reflect adaptation to host environments. Analysis of matched pairs and serial isolates reveals enrichment of non-synonymous changes in genes encoding fungal cell wall proteins .

  While PBN1 has not been specifically identified among these variants, its role in GPI anchor biosynthesis suggests potential involvement in adaptive processes. Clinical strains show low density of SNPs (0.037-0.047 SNPs/Kb), confirming their clonal nature .

  Approach to study PBN1 in clinical adaptation:

  1. Sequence PBN1 locus across serial clinical isolates from individual patients

  2. Identify and characterize any mutations in PBN1 or GPI biosynthesis pathway genes

  3. Assess functional consequences of identified variants through complementation studies

  4. Correlate genetic changes with phenotypic differences in antifungal susceptibility and virulence

• How does PBN1 interact with novel transcription factors like CgXbp1 in regulating C. glabrata virulence?

  Novel transcription factors like CgXbp1 play important roles in C. glabrata's response to macrophage infection and antifungal resistance. CgXbp1 deletion accelerates transcriptional activation of multiple biological processes during macrophage interaction . This transcription factor directly binds to many C. glabrata genes associated with pathogenesis and drug resistance .

  While direct interaction between PBN1 and CgXbp1 has not been established, potential regulatory relationships may exist given their roles in virulence and drug resistance.

  Network analysis methodology:

  1. Perform chromatin immunoprecipitation to determine if CgXbp1 binds the PBN1 promoter

  2. Conduct RNA-seq comparing wild-type, PBN1Δ, and CgXbp1Δ strains to identify shared regulatory networks

  3. Create double knockout strains (PBN1Δ/CgXbp1Δ) to assess genetic interactions

  4. Analyze protein-protein interactions using co-immunoprecipitation or yeast two-hybrid assays