Recombinant Neosartorya fumigata Peptidyl-prolyl cis-trans isomerase pin4 (pin4)

What is the taxonomic relationship between Neosartorya fumigata and Aspergillus fumigatus?

Neosartorya fumigata is the teleomorphic (sexual) state of Aspergillus fumigatus, with both names referring to the same organism at different reproductive stages. While A. fumigatus refers to the asexual (anamorphic) state, Neosartorya fumigata represents the sexual form that produces ascospores within cleistothecia . This taxonomic relationship was established through phylogenetic analyses using β-tubulin, calmodulin, and actin gene sequences . The sexual reproduction of A. fumigatus was previously thought to be absent or cryptic until relatively recently, when fertile crosses among geographically restricted environmental isolates were successfully documented . Subsequently, researchers have provided evidence for mating, fruiting body development, and ascosporogenesis accompanied by genetic recombination between unrelated clinical isolates of A. fumigatus .

How can Neosartorya species be distinguished from Aspergillus fumigatus in laboratory settings?

Distinguishing between Neosartorya species and A. fumigatus is crucial in food industry and clinical research settings. PCR-based methods using specific primer sets targeting the β-tubulin and calmodulin genes have been developed for this purpose . These identification methods are rapid, simple, and highly specific, not detecting other fungi involved in food spoilage or environmental contamination . Additionally, morphological characteristics can be used for differentiation, where Neosartorya species produce distinctive ascospores with species-specific surface ornamentations visible under microscopy. For example, N. denticulata produces unique denticulate ascospores with a prominent equatorial furrow, while N. assulata forms ascospores with several large flaps and two distinct equatorial crests .

What is the function of peptidyl-prolyl cis-trans isomerases in fungal biology?

Peptidyl-prolyl cis-trans isomerases (PPIases) are essential enzymes that catalyze the isomerization of peptide bonds preceding proline residues, facilitating protein folding and functioning as molecular chaperones. In fungi like N. fumigata, these enzymes play crucial roles in protein maturation, cellular stress responses, and signal transduction pathways. They contribute to fungal virulence, cell wall integrity, and adaptation to environmental stresses. The pin4 isomerase specifically may participate in maintaining protein conformational stability during stress conditions encountered during host invasion and colonization. Understanding these functions is fundamental to elucidating fungal pathogenicity mechanisms.

What are the optimal expression systems for producing recombinant Neosartorya fumigata pin4 protein?

For recombinant production of N. fumigata pin4, E. coli expression systems typically offer the highest yield and simplicity, similar to the E. coli system used for other N. fumigata proteins like Asp f 2 . The recommended approach employs BL21(DE3) or Rosetta strains with pET vector systems incorporating N-terminal 6xHis-SUMO tags to enhance solubility and facilitate purification . Expression should be induced at OD600 0.6-0.8 with 0.5-1.0 mM IPTG at 18-25°C for 16-20 hours to minimize inclusion body formation.

For researchers requiring post-translational modifications, Pichia pastoris or Aspergillus expression systems may be preferable despite lower yields. Optimization of codon usage according to the expression host is essential, as is careful selection of purification strategies that preserve the enzyme's catalytic activity, typically involving immobilized metal affinity chromatography followed by size exclusion chromatography.

What are the recommended protocols for assessing pin4 enzymatic activity?

The enzymatic activity of pin4 can be assessed using several established protocols:

• Spectrophotometric assays: Measure isomerization of synthetic peptide substrates like Suc-Ala-Ala-Pro-Phe-pNA with spectrophotometric detection at 390 nm.

• Protease-coupled assays: Utilize the observation that proteases like chymotrypsin preferentially cleave after trans-proline, with cleavage rate corresponding to PPIase activity.

• NMR-based methods: For detailed kinetic analysis, NMR spectroscopy can directly monitor cis-trans isomerization in real-time.

The optimal assay conditions typically include:

Controls should include heat-inactivated enzyme and known PPIase inhibitors (cyclosporin A or FK506) to verify specificity.

How can researchers verify the structure and conformational integrity of recombinant pin4?

Verification of pin4 structural integrity should employ multiple complementary techniques:

• Circular Dichroism (CD) spectroscopy: This should be used to assess secondary structure content, with properly folded pin4 typically showing characteristic α-helical and β-sheet signatures.

• Differential Scanning Fluorimetry (DSF): This can determine thermal stability (Tm) and detect the effects of buffer conditions or potential ligands on protein stability.

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): This should be employed to confirm monomeric state and detect any oligomerization or aggregation.

• Limited proteolysis: Time-course digestion with trypsin or chymotrypsin can provide information about flexible regions and domain organization.

A properly folded recombinant pin4 protein should demonstrate enzymatic activity consistent with PPIase function, resistance to aggregation at physiological temperatures, and circular dichroism profiles matching predicted secondary structure elements. Any significant deviations may indicate improper folding requiring optimization of expression or purification protocols.

How does pin4 contribute to Neosartorya fumigata pathogenicity in invasive aspergillosis?

The contribution of pin4 to N. fumigata pathogenicity is multifaceted, functioning at several levels during host invasion:

• Stress adaptation: Pin4 likely contributes to the fungus's ability to withstand host-induced stresses, including thermal stress, oxidative damage, and pH fluctuations encountered during infection. This parallels the known function of other PPIases in stress response pathways.

• Protein homeostasis: By facilitating proper protein folding under stress conditions, pin4 may help maintain functional integrity of virulence factors during host colonization.

• Immunomodulation: Preliminary evidence suggests some fungal PPIases can interact with host immune proteins, potentially manipulating host defense mechanisms.

• Cell wall integrity: Pin4 may participate in ensuring proper maturation and localization of cell wall components, which are critical for evading host recognition.

The protein likely acts in concert with other molecular chaperones in a complex network that maintains cellular functionality under the challenging conditions of the host environment. Researchers investigating this area should employ gene deletion mutants in combination with infection models to quantitatively assess the contribution of pin4 to virulence.

What approaches can be used to identify inhibitors specific to Neosartorya fumigata pin4 for antifungal development?

The development of specific inhibitors targeting N. fumigata pin4 requires a multi-faceted approach:

• Structure-based virtual screening: Using homology models or experimentally determined structures of pin4, computational screening of compound libraries can identify potential binding molecules with inhibitory activity.

• Fragment-based drug discovery: This involves screening small chemical fragments that bind to different regions of pin4, followed by fragment linking or optimization.

• High-throughput enzymatic assays: Adaptation of standard PPIase assays to 384-well formats enables rapid screening of compound libraries:

• Selectivity profiling: Critical assessment of inhibitor selectivity against human PPIases must be conducted to minimize off-target effects and toxicity. This should include testing against human cyclophilins and FKBPs.

• Phenotypic validation: Promising inhibitors should be tested for their effects on fungal growth, morphology, and virulence in infection models.

The ideal inhibitor would demonstrate high affinity (sub-micromolar) binding to N. fumigata pin4, significant selectivity over human orthologs (>100-fold), and antifungal activity in both in vitro and in vivo models.

How can researchers study the interactome of pin4 to understand its role in signaling networks?

Investigating the pin4 interactome requires implementing several complementary approaches:

• Affinity purification coupled with mass spectrometry (AP-MS): Using tagged recombinant pin4 as bait, researchers can identify protein complexes from fungal lysates. This requires:

  • Expression of epitope-tagged pin4 in N. fumigata

  • Gentle cell lysis to preserve protein-protein interactions

  • Affinity purification under native conditions

  • Mass spectrometry identification of co-purified proteins

• Yeast two-hybrid (Y2H) screening: This can identify direct binary interactions between pin4 and fungal proteins, particularly useful for detecting transient interactions that might be missed by AP-MS.

• Proximity-based labeling: BioID or APEX2 fusion proteins can identify proteins in close proximity to pin4 in living cells, offering insight into spatial organization of interactions.

• Co-immunoprecipitation validation: Key interactions should be validated by reciprocal co-IP experiments with antibodies against native proteins.

• Functional validation: RNAi or CRISPR-based knockdown/knockout of identified interaction partners can reveal functional significance of these interactions.

The expected interactome would likely include:

• Other chaperone proteins forming functional complexes

• Substrate proteins requiring isomerization

• Signaling components in stress response pathways

• Potential regulatory proteins that modulate pin4 activity

Analysis should incorporate gene ontology enrichment to identify biological processes over-represented among interacting partners.

What strategies can overcome solubility issues when expressing recombinant Neosartorya fumigata pin4?

Researchers frequently encounter solubility challenges when expressing recombinant fungal proteins like pin4. Effective strategies include:

• Fusion tag optimization: Testing multiple solubility-enhancing tags:

• Expression temperature modification: Lowering induction temperature to 16-18°C significantly slows protein production, allowing more time for proper folding and reducing inclusion body formation.

• Co-expression with chaperones: Co-expressing pin4 with chaperone systems like GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor can improve folding in E. coli.

• Buffer optimization during purification:

  • Including 5-10% glycerol in all buffers

  • Testing various detergents (0.05-0.1% Triton X-100, NP-40, or CHAPS)

  • Adding stabilizing agents like arginine (50-200 mM) or trehalose (5-10%)

• Refolding strategies: If inclusion bodies persist, controlled denaturation followed by step-wise refolding may recover active protein, though typically with lower yields.

The combination of N-terminal 6xHis-SUMO tag, expression at 18°C overnight, and inclusion of glycerol in purification buffers has been particularly successful for similar fungal proteins .

How can researchers address issues of protein stability during purification and storage of recombinant pin4?

Maintaining pin4 stability throughout purification and storage requires careful optimization:

• Buffer composition optimization:

  • Testing pH ranges (typically 7.0-8.0)

  • Varying salt concentrations (100-500 mM NaCl)

  • Including stabilizing agents (10% glycerol, 1-5 mM DTT or TCEP)

  • Testing the effect of divalent cations (Mg²⁺, Ca²⁺)

• Storage condition assessment:

• Cryoprotectant screening: Testing various additives including:

  • Glycerol (10-50%)

  • Sucrose or trehalose (5-10%)

  • BSA as a carrier protein (0.1-1 mg/mL)

• Aggregation prevention:

  • Filtering through 0.22 μm membranes before storage

  • Centrifugation at 100,000×g to remove aggregation nuclei

  • Storage at protein concentrations below aggregation threshold (<1 mg/mL)

• Stability monitoring: Regular quality control using:

  • Enzymatic activity assays

  • Dynamic light scattering to detect aggregation

  • Thermal shift assays to measure stability changes over time

These approaches should be systematically evaluated based on downstream applications, with the optimal protocol balancing maximum stability with practical considerations.

What controls and validations are necessary when studying pin4 interactions with host proteins during infection?

When investigating pin4 interactions with host proteins, researchers should implement rigorous controls and validations:

• Essential experimental controls:

  • Non-specific interaction control: Use an unrelated recombinant fungal protein with the same tag system

  • Tag-only control: Express and purify tag alone to identify tag-mediated interactions

  • Binding specificity control: Include competition assays with unlabeled pin4

  • Host cell negative control: Use cell types not typically infected by N. fumigata

• Validation through multiple methodologies:

  • Initial screening through pull-down or co-immunoprecipitation

  • Confirmation with orthogonal methods (ELISA, SPR, or microscopic co-localization)

  • Functional validation through mutagenesis of key residues

  • In vivo validation in relevant infection models

• Quantitative binding measurements:

  • Determine binding kinetics (kon, koff) through SPR or BLI

  • Calculate binding affinities (KD values) for key interactions

  • Compare affinity values across different experimental conditions

• Biological relevance assessment:

  • Examine interaction under physiologically relevant conditions

  • Determine if interaction occurs at realistic protein concentrations

  • Verify interaction in primary cells or tissues, not just cell lines

  • Correlate interaction strength with pathological outcomes

• Structural validation:

  • If possible, obtain structural data (X-ray crystallography or cryo-EM) of the complex

  • Use hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Employ computational molecular dynamics to assess interaction stability

These rigorous validation steps ensure that identified interactions represent genuine biological phenomena rather than experimental artifacts, providing a solid foundation for therapeutic targeting.

How can functional genomics approaches advance our understanding of pin4 in fungal adaptation?

Functional genomics offers powerful approaches to comprehensively understand pin4's role:

• CRISPR-Cas9 genome editing: Generation of precise pin4 mutants with:

  • Complete gene deletion

  • Point mutations at catalytic sites

  • Domain-specific alterations

  • Promoter modifications for controlled expression

• Transcriptomics analysis: RNA-seq comparisons between wild-type and pin4 mutants under various stress conditions can reveal:

  • Compensatory mechanisms activated in pin4 mutants

  • Co-regulated gene networks

  • Stress-specific transcriptional programs dependent on pin4

• Proteomics integration: Quantitative proteomics to identify:

  • Proteins with altered abundance in pin4 mutants

  • Changes in post-translational modifications

  • Alterations in protein complex formation

• Metabolomics assessment: Analysis of metabolic changes in pin4 mutants to identify:

  • Shifts in primary metabolism

  • Alterations in secondary metabolite production

  • Stress-responsive metabolic adaptations

• Comparative genomics across fungal species: Examination of pin4 conservation and variation to understand:

  • Evolutionary importance and selection pressure

  • Species-specific adaptations

  • Correlation with pathogenicity across the fungal kingdom

Such integrated approaches would provide unprecedented insight into pin4's role in fungal biology and potentially reveal novel therapeutic targets for antifungal development.

What potential exists for developing pin4-based fungal diagnostics for clinical applications?

The development of pin4-based diagnostics presents several promising avenues:

• Antibody-based detection systems:

  • Monoclonal antibodies against unique epitopes of N. fumigata pin4

  • Lateral flow assays for rapid point-of-care testing

  • ELISA-based quantification in clinical samples

• Aptamer technology:

  • Selection of DNA/RNA aptamers with high specificity for pin4

  • Integration into biosensor platforms

  • Potential for increased stability compared to antibodies

• PCR-based detection:

  • Development of highly specific primers targeting the pin4 gene

  • Quantitative PCR for assessing fungal burden

  • Digital PCR for absolute quantification in complex samples

• Mass spectrometry markers:

  • Identification of pin4-specific peptide signatures

  • Integration into clinical proteomics workflows

  • Potential for multiplex detection of multiple fungal biomarkers

The clinical performance parameters for an ideal pin4-based diagnostic would include:

Such diagnostics could significantly improve management of invasive aspergillosis by enabling earlier detection and treatment initiation, potentially reducing mortality rates.