Recombinant Neosartorya fumigata Peptidyl-prolyl cis-trans isomerase B (cpr2)

What is Neosartorya fumigata and how is it related to Aspergillus fumigatus?

Neosartorya fumigata is the teleomorph (sexual form) name of the organism commonly known as Aspergillus fumigatus. This filamentous fungus is the most common cause of invasive aspergillosis, a frequently fatal lung disease primarily affecting immunocompromised individuals . In scientific literature, both names are used, though recent taxonomy efforts have consolidated many species under the Aspergillus genus. The organism produces several specialized metabolites that contribute to its pathogenicity, including ergot alkaloids and various proteins that interact with host immune systems .

How is recombinant cpr2 typically produced for research applications?

Recombinant cpr2 is typically produced using E. coli expression systems. Commercial preparations, such as that mentioned in the search results, indicate the protein is available in E. coli-derived formulations . The production process generally involves:

• Cloning the cpr2 gene into an appropriate expression vector

• Transforming the construct into E. coli strains optimized for protein expression

• Inducing protein expression under controlled conditions

• Purifying the recombinant protein using affinity chromatography techniques

• Verifying protein identity and activity through biochemical assays

The resulting purified protein is then used for various research applications, including structural studies, functional assays, and immunological investigations .

How can recombinant A. fumigatus antigens like cpr2 be used in diagnostic applications?

Recombinant A. fumigatus antigens have shown significant potential for improving diagnostic accuracy in Aspergillus-related respiratory conditions. While the search results don't specifically mention cpr2 in diagnostics, studies with other recombinant A. fumigatus antigens demonstrate their utility in differentiating true fungal sensitization from cross-reactivity.

In a study evaluating recombinant A. fumigatus (rAsp) antigens for diagnosing Aspergillus-sensitized asthma (ASA) and allergic bronchopulmonary aspergillosis (ABPA), researchers found that specific recombinant antigens (f1 and f2) could potentially detect true Aspergillus sensitization with greater specificity than crude antigen preparations . The methodology involved:

• Measuring IgE antibodies against both crude A. fumigatus (cAsp) and recombinant antigens

• Comparing responses in patients with A. fumigatus-unsensitized asthma (n=51), ASA (n=71), and ABPA (n=123)

• Evaluating cross-reactivity with other fungal antigens

Results showed that 19 subjects diagnosed with ASA and one with ABPA using crude antigen testing were negative for rAsp f1 and f2, suggesting possible misclassification due to cross-reactivity with other fungi . Similar methodological approaches could be applied to evaluate cpr2's potential as a diagnostic marker.

What experimental methods are most effective for studying gene expression of factors like cpr2 during infection?

Real-time reverse transcription-PCR (RT-PCR) has proven effective for analyzing A. fumigatus gene expression during infection. A study examining developmental gene expression used this method to track expression patterns in both in vitro cultures and in vivo mouse infection models .

For in vivo analysis, researchers employed:

• Mouse infection models using either aerosol exposure or intranasal instillation

• Careful timing of sample collection to avoid healthy survivor bias

• RNA extraction from infected tissues

• Real-time RT-PCR with specific primers for genes of interest

• Normalization to housekeeping genes for accurate quantification

The study revealed distinct temporal expression patterns for different genes during infection, with some genes showing similar patterns in vitro and in vivo, while others displayed infection-specific regulation . These methods would be applicable to investigating cpr2 expression during different phases of infection.

How can proteomics approaches advance our understanding of surface proteins like cpr2?

Proteomics techniques have been instrumental in identifying surface-expressed proteins (the "surfome") of A. fumigatus. A recent phylogenetic study compared proteins present on the A. fumigatus conidial surface with those of closely related non-pathogenic species and a far-related pathogenic species .

The methodological approach included:

• Isolation of conidial surface proteins from multiple Aspergillus species

• Mass spectrometry analysis using LTQ Orbitrap instruments

• Comparative genomics to identify species-specific proteins

• Functional characterization through gene deletion studies

• Assessment of mutant phenotypes in infection models

This approach identified 62 proteins specifically expressed on the A. fumigatus conidial surface. Subsequent deletion of 42 encoding genes revealed their roles in evading macrophage killing, epithelial cell penetration and damage, and cytokine production modulation . Similar approaches could elucidate cpr2's potential role in pathogenesis if it is indeed expressed on the conidial surface.

How do researchers evaluate the contribution of specific A. fumigatus proteins to virulence?

Researchers employ several methodological approaches to assess the contribution of specific proteins to A. fumigatus virulence:

• Gene knockout studies followed by virulence assessment in animal models

• Comparative analysis between pathogenic and non-pathogenic species

• Protein localization studies during infection

• Host-pathogen interaction assays

• Immune response measurement following exposure to specific proteins

A study investigating ergot alkaloids exemplifies this approach, using a Galleria mellonella larval model to assess virulence. Researchers injected larvae with conidia from wild-type A. fumigatus strains and various mutants, then monitored mortality rates. This revealed that elimination of ergot alkaloids significantly reduced virulence (P < 0.0001), and mutants accumulating intermediates but not the end product fumigaclavine C were also less virulent than wild type (P < 0.0003) .

What insights can be gained from comparing A. fumigatus proteins with those of related species?

Comparative analysis between pathogenic and non-pathogenic Aspergillus species can reveal proteins that contribute specifically to virulence. A recent proteomics study employed this approach to identify A. fumigatus-specific conidial surface proteins .

The methodology involved:

• Comparing protein expression between:

  • Pathogenic A. fumigatus

  • Closely related non-pathogenic species (A. fischeri and A. oerlinghausenensis)

  • Far-related pathogenic A. lentulus

• Functional characterization of species-specific proteins through:

  • Gene deletion studies

  • Assessment of:

    • Susceptibility to macrophage killing

    • Penetration and damage to epithelial cells

    • Cytokine production

This approach demonstrated that one A. fumigatus-specific gene encoding a glycosylasparaginase modulates IL-1β levels and is important for infection in an immunocompetent murine model . Similar comparative studies could reveal whether cpr2 has unique features in A. fumigatus compared to homologs in non-pathogenic species.

How can researchers assess the immunomodulatory effects of fungal proteins like cpr2?

Assessment of immunomodulatory effects typically involves:

• In vitro assays with immune cells:

  • Cytokine production measurement following exposure to purified proteins

  • Cell surface marker analysis to assess activation states

  • Phagocytosis and killing assays with macrophages/neutrophils

• Ex vivo tissue models to evaluate:

  • Epithelial cell damage and penetration

  • Barrier function disruption

  • Local immune response

• In vivo models to assess:

  • Systemic and local cytokine profiles

  • Immune cell recruitment and activation

  • Disease progression and outcome

The surfome study mentioned earlier identified proteins that modulate cytokine production, including a glycosylasparaginase that specifically affects IL-1β levels . Such methodologies could be applied to investigate potential immunomodulatory effects of cpr2.

What approaches can be used to evaluate the potential of cpr2 as a therapeutic target?

Evaluating cpr2 as a potential therapeutic target would involve several stages:

• Target validation:

  • Confirming essentiality through conditional knockdown/knockout studies

  • Assessing virulence attenuation in cpr2-deficient mutants

  • Determining whether cpr2 is accessible to drugs during infection

• Inhibitor development:

  • Structure-based drug design if crystal structure is available

  • High-throughput screening for small molecule inhibitors

  • Peptide-based inhibitor design based on substrate interactions

• Efficacy testing:

  • In vitro enzymatic assays to confirm target engagement

  • Cell-based assays to assess antifungal activity

  • Animal models to evaluate in vivo efficacy

While the search results don't specifically address cpr2 as a therapeutic target, studies on other A. fumigatus proteins provide methodological frameworks that could be applied.

How do researchers investigate resistance development against antifungal compounds?

A recent study investigating resistance development against an antifungal protein from Neosartorya fischeri (NFAP2) demonstrates methodological approaches applicable to studying resistance:

• Microevolution experiments:

  • Exposing fungi to increasing concentrations of antifungal agents

  • Comparing adaptation rates between different compounds

  • Genome analysis to identify mutations in resistant strains

• Cross-resistance assessment:

  • Testing susceptibility of resistant strains to other antifungal agents

  • Evaluating mechanisms of resistance through binding and uptake studies

• Fitness cost analysis:

  • Assessing growth rates and stress tolerance of resistant strains

  • Evaluating virulence potential in animal models

The study found that Candida albicans adapted to only 1× minimum inhibitory concentration of NFAP2 compared with 32× MIC of fluconazole, suggesting a lower potential for resistance development . Similar approaches could evaluate whether targeting cpr2 might lead to resistance development.

What are the key considerations when designing experiments using recombinant A. fumigatus proteins?

When designing experiments with recombinant A. fumigatus proteins like cpr2, researchers should consider:

• Protein quality and characteristics:

  • Verification of protein identity and purity

  • Assessment of proper folding and activity

  • Endotoxin testing for immunological studies

• Experimental controls:

  • Heat-inactivated protein controls

  • Related proteins from non-pathogenic species

  • Blocking experiments to confirm specificity

• Physiological relevance:

  • Using physiologically relevant concentrations

  • Considering the protein's natural context (e.g., surface-exposed vs. intracellular)

  • Accounting for potential post-translational modifications

The search results indicate that recombinant cpr2 is commercially available as an E. coli-expressed product , which may lack fungal-specific post-translational modifications that could affect function or immunogenicity.

What in vivo models are most appropriate for studying A. fumigatus infections?

Based on the search results, several in vivo models are used to study A. fumigatus infections:

• Murine models:

  • Aerosol exposure: Allows precise control of inoculum and mimics natural infection route

  • Intranasal instillation: Useful when aerosol exposure is impractical, such as with mutants producing insufficient conidia

  • Immunocompetent vs. immunocompromised models: Different models can address different aspects of pathogenesis

• Insect models:

  • Galleria mellonella (wax moth) larvae: Useful for initial virulence screening

  • Allows high-throughput assessment of mortality rates following infection with different strains

• Key considerations for model selection:

  • Research question (e.g., initial infection vs. dissemination)

  • Immunological aspects under investigation

  • Need for statistical power vs. ethical considerations

  • Availability of specific knockout or transgenic models

The appropriate model depends on the specific aspect of A. fumigatus biology being investigated, with each model offering distinct advantages and limitations.

Comparative Analysis of rAsp Antigen Positivity in Asthma and ABPA

The data demonstrates that recombinant A. fumigatus antigens can more specifically identify true Aspergillus sensitization compared to crude antigen preparation, with 19 ASA subjects showing potential misclassification due to cross-reactivity with other fungi .

Key Proteins Identified in A. fumigatus Conidial Surface

Recent proteomic analysis identified 62 proteins specifically expressed on the A. fumigatus conidial surface. Key findings from this study include:

• Functional impact of gene deletions:

  • Altered susceptibility to macrophage killing

  • Changed ability to penetrate and damage epithelial cells

  • Modified cytokine production profiles

• Specific finding: A glycosylasparaginase was found to modulate IL-1β levels and demonstrated importance for infection in an immunocompetent murine model .

This proteomic approach provides a framework for investigating whether cpr2 is present on the conidial surface and what role it might play in initial host-pathogen interactions.

Comparative Resistance Development to Antifungal Agents

This comparative data suggests that proteins like NFAP2 from Neosartorya species have lower potential to trigger resistance development compared to conventional antifungals like fluconazole , which could be relevant when considering cpr2 as a potential therapeutic target or diagnostic marker.

What are the most promising future research directions for understanding cpr2 function?

Based on the available information, several research directions appear promising:

• Structural and functional characterization:

  • Determining the three-dimensional structure of cpr2

  • Identifying natural substrates and interaction partners

  • Elucidating its enzymatic mechanism and regulation

• Role in pathogenesis:

  • Investigating expression patterns during different infection stages

  • Creating and characterizing cpr2 knockout mutants

  • Assessing virulence in appropriate animal models

• Immunological aspects:

  • Evaluating cpr2's potential as a diagnostic marker for Aspergillus sensitization

  • Investigating its immunomodulatory properties

  • Assessing its potential as a vaccine component

• Therapeutic applications:

  • Screening for specific inhibitors

  • Evaluating resistance development potential

  • Assessing efficacy in combination with existing antifungals

These research directions would contribute to a more comprehensive understanding of cpr2's biological role and potential applications in diagnosis and treatment of Aspergillus-related diseases.