Recombinant Coccidioides posadasii Putative dipeptidase CPC735_014430 (CPC735_014430)

What is Coccidioides posadasii Putative dipeptidase CPC735_014430?

CPC735_014430 is annotated as a putative dipeptidase from the fungal pathogen Coccidioides posadasii. It belongs to the metallopeptidase family and is hypothesized to hydrolyze dipeptides into amino acids. The native protein consists of 461 amino acid residues, with computational analyses suggesting conserved domains typical of metallopeptidases. The recombinant form is produced with an N-terminal histidine tag, facilitating purification and detection in experimental settings .

The protein's sequence begins with MSARDNEKGSARSQPSHAAASEIENVPRPSRQQSWTGTMIKVFIICACAGIVSKYIIPL and extends to a full length of 464 amino acids in the recombinant form. While its enzymatic function has been computationally predicted, experimental validation of its specific activity and substrate specificity remains pending .

What is the proposed biological function of CPC735_014430 in Coccidioides posadasii?

Based on sequence homology and computational predictions, CPC735_014430 likely functions as a dipeptidase involved in protein metabolism pathways within C. posadasii. In the context of fungal physiology, this enzyme may contribute to:

• Nutrient acquisition through the breakdown of environmental peptides

• Cell wall remodeling during morphological transitions

• Stress adaptation, particularly in high-salt environments, which is a characteristic ecological niche for C. posadasii

The strain C735, from which this protein originates, is a clinical isolate from Texas that serves as a reference for genomic studies. Comparative analysis between C. posadasii C735 and related species such as C. immitis RMSCC 2006 reveals distinctive ecological adaptations:

While the precise role of CPC735_014430 in these adaptations remains to be fully characterized, its conservation in the C. posadasii genome suggests functional importance.

How can researchers experimentally validate the dipeptidase activity of CPC735_014430?

To validate the putative dipeptidase activity of CPC735_014430, researchers should employ a systematic experimental approach:

• Substrate specificity assays: Test the recombinant protein against a panel of synthetic dipeptides with varying amino acid compositions. Monitor hydrolysis using chromatographic methods (HPLC) or colorimetric assays that detect free amino acid release.

• Enzyme kinetics characterization: Determine Km, Vmax, and kcat values using varying substrate concentrations under optimized reaction conditions. Plot Michaelis-Menten curves to establish the catalytic efficiency (kcat/Km) of the enzyme.

• Metal ion dependency tests: As a putative metallopeptidase, activity should be assessed in the presence of various divalent cations (Zn2+, Mg2+, Mn2+, Ca2+) and metal chelators (EDTA, 1,10-phenanthroline) to identify cofactor requirements.

• pH and temperature optima determination: Establish optimal reaction conditions by measuring enzyme activity across pH ranges (5.0-9.0) and temperatures (25-45°C), which may provide insights into its physiological function in C. posadasii.

• Inhibitor profiling: Test class-specific protease inhibitors to confirm the metallopeptidase classification and identify compounds that could serve as experimental tools for functional studies.

These methodological approaches will provide definitive evidence of CPC735_014430's enzymatic function beyond computational predictions.

What role might CPC735_014430 play in Coccidioides pathogenesis and host-pathogen interactions?

The potential role of CPC735_014430 in C. posadasii pathogenesis can be investigated through several experimental approaches:

• Gene knockout/knockdown studies: Generate CPC735_014430-deficient strains using CRISPR-Cas9 or RNA interference techniques. Compare virulence, morphological transitions, and growth characteristics between mutant and wild-type strains in both in vitro and murine infection models.

• Transcriptional profiling: Analyze CPC735_014430 expression during different stages of infection (conidia, spherule formation, endosporulation) using qRT-PCR or RNA-seq to identify infection-specific regulation patterns.

• Immunological studies: Assess whether CPC735_014430 elicits immune responses in infected hosts by testing patient sera for antibodies against the recombinant protein. This could indicate exposure of the protein to the host immune system during infection.

• Protein localization: Determine the cellular localization of CPC735_014430 using immunofluorescence microscopy or cell fractionation techniques. Surface-exposed proteins in C. posadasii spherules, including certain proteases, are prioritized as vaccine candidates.

• Host substrate identification: Investigate whether CPC735_014430 can cleave host-derived peptides that might contribute to immune evasion or tissue invasion.

Current evidence suggests that surface-exposed proteins in C. posadasii spherules, such as aspartyl proteases and superoxide dismutases, are immunogenic. While CPC735_014430 has not been directly tested, its homology to immunogenic proteins suggests potential utility in vaccine formulations, particularly when combined with adjuvants like glucan-chitin particles (GCP).

What are the optimal conditions for handling and storing recombinant CPC735_014430 to maintain enzymatic activity?

Maintaining the stability and activity of recombinant CPC735_014430 requires careful attention to storage and handling conditions:

• Storage recommendations:

  • Store lyophilized protein at -20°C to -80°C upon receipt

  • After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

  • For short-term use, store working aliquots at 4°C for up to one week

• Reconstitution protocol:

  • Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

  • Add glycerol to a final concentration of 5-50% (with 50% being optimal) for long-term storage at -20°C/-80°C

• Activity preservation:

  • Monitor protein stability using analytical techniques such as size-exclusion chromatography or dynamic light scattering

  • Assess enzymatic activity periodically using standardized dipeptide substrates

  • Consider addition of metal cofactors (e.g., Zn2+) if activity diminishes during storage

  • Avoid buffer components that might chelate metal ions, which could impact metallopeptidase activity

How can researchers design expression systems to optimize yield and activity of recombinant CPC735_014430?

Optimizing the expression and purification of functionally active recombinant CPC735_014430 requires careful consideration of several factors:

• Expression system selection:

  • While E. coli is commonly used and successful for this protein , alternative systems like Pichia pastoris might provide better folding for fungal proteins

  • Compare codon-optimized constructs for different expression hosts to maximize translation efficiency

  • Test various fusion tags (His6, GST, MBP) for their effects on solubility and activity

• Induction conditions optimization:

  • Perform factorial experiments varying temperature (16-37°C), inducer concentration, and duration of induction

  • Lower temperatures (16-25°C) often favor proper folding of complex proteins

  • Consider auto-induction media to achieve higher cell densities before protein expression initiates

• Purification strategy development:

  • Implement two-step purification combining immobilized metal affinity chromatography (IMAC) with size exclusion or ion exchange chromatography

  • Include low concentrations of zinc or other divalent cations in purification buffers to maintain metallopeptidase structure

  • Evaluate activity retention after each purification step to identify potential activity-compromising conditions

• Structural integrity assessment:

  • Use circular dichroism spectroscopy to evaluate secondary structure content

  • Employ thermal shift assays to identify stabilizing buffer components

  • Consider limited proteolysis coupled with mass spectrometry to identify flexible regions that might affect crystallization

These methodological considerations should guide experimental design for obtaining functionally active CPC735_014430 suitable for downstream enzymatic and structural studies.

What are the best approaches for analyzing potential substrates and inhibitors of CPC735_014430?

Comprehensive substrate and inhibitor profiling for CPC735_014430 requires systematic methodological approaches:

• Substrate specificity mapping:

  • Screen positional scanning synthetic combinatorial libraries of dipeptides to determine preferred amino acid residues at P1 and P2 positions

  • Employ fluorescent resonance energy transfer (FRET) peptides containing various dipeptide sequences for high-throughput activity screening

  • Validate preferences using individual synthetic substrates and steady-state kinetic analysis

• Physiologically relevant substrate identification:

  • Perform in silico analysis of the C. posadasii proteome to identify potential native substrates based on established cleavage preferences

  • Conduct peptidomic analysis comparing wild-type and CPC735_014430-deficient strains to identify differentially processed peptides

  • Use MALDI-TOF mass spectrometry to map cleavage sites in candidate physiological substrates

• Inhibitor discovery and characterization:

  • Screen metallopeptidase inhibitor libraries to identify compounds with activity against CPC735_014430

  • Determine inhibition mechanisms (competitive, non-competitive, uncompetitive) through enzyme kinetics studies

  • Measure IC50 and Ki values for promising inhibitors under standardized conditions

  • Assess selectivity by testing active inhibitors against related human metallopeptidases

• Structure-function correlation:

  • Generate homology models based on structurally characterized metallopeptidases to predict substrate binding sites

  • Design site-directed mutagenesis experiments targeting predicted catalytic and substrate-binding residues

  • Use computational docking to visualize substrate and inhibitor interactions with the enzyme active site

These approaches provide a systematic framework for unraveling the molecular function of CPC735_014430 and its potential as a therapeutic target.

How can researchers investigate the immunological properties of CPC735_014430 for vaccine development?

Investigating CPC735_014430 as a potential vaccine candidate requires systematic evaluation of its immunological properties:

• Antigenicity assessment:

  • Screen sera from confirmed coccidioidomycosis patients for antibodies recognizing recombinant CPC735_014430

  • Perform epitope mapping using overlapping peptide arrays to identify immunodominant regions

  • Compare antibody responses between different clinical manifestations (asymptomatic, pulmonary, disseminated)

• Immunization studies in animal models:

  • Evaluate various adjuvant formulations, with special consideration for glucan-chitin particles (GCP) which have shown promise with other C. posadasii antigens

  • Compare humoral and cell-mediated immune responses following immunization

  • Establish correlates of protection by challenging immunized animals with infectious C. posadasii spores

• T-cell response characterization:

  • Identify MHC class I and II epitopes using prediction algorithms and experimental validation

  • Analyze T-cell proliferation, cytokine profiles, and memory cell generation following stimulation with recombinant CPC735_014430

  • Compare the relative contribution of CD4+ and CD8+ T-cell responses to protection

• Combination vaccine strategies:

  • Test CPC735_014430 in combination with other C. posadasii immunogens (e.g., aspartyl proteases, superoxide dismutases) that have shown promise as vaccine candidates

  • Evaluate potential synergistic effects on protective immunity

  • Assess cross-protection against both C. posadasii and C. immitis challenge

• Safety assessment:

  • Evaluate potential cross-reactivity with human proteins using bioinformatic approaches and in vitro assays

  • Monitor for adverse reactions in immunized animals across different dosing regimens

These methodological approaches provide a framework for evaluating the utility of CPC735_014430 in vaccine development against coccidioidomycosis, a significant fungal disease in endemic regions.

How might comparative genomic approaches enhance our understanding of CPC735_014430 function?

Leveraging comparative genomics can provide valuable insights into the evolutionary context and functional significance of CPC735_014430:

• Ortholog identification and analysis:

  • Identify CPC735_014430 orthologs across diverse fungal species, particularly pathogenic and non-pathogenic relatives

  • Calculate selection pressures (dN/dS ratios) to identify regions under positive or purifying selection

  • Correlate conservation patterns with predicted functional domains and catalytic sites

• Genomic context analysis:

  • Examine the genomic neighborhood of CPC735_014430 across different fungal species to identify functionally related genes through synteny

  • Investigate co-expression patterns with other genes involved in pathogenesis or stress responses

  • Identify potential transcriptional regulatory elements through promoter region analysis

• Strain variation studies:

  • Compare CPC735_014430 sequences across clinical isolates of C. posadasii with varying virulence profiles

  • Correlate sequence polymorphisms with geographic distribution and ecological adaptations

  • Analyze expression levels across strains under standardized growth conditions and during host infection

• Interspecies comparison with C. immitis:

  • Build upon established genomic divergence (4-5%) between C. posadasii and C. immitis

  • Identify species-specific adaptations in CPC735_014430 that might correlate with differential growth rates in high-salt environments

  • Investigate functional differences between orthologous proteins through heterologous expression and biochemical characterization

These comparative approaches can provide evolutionary context for CPC735_014430 function and may identify selective pressures shaping its role in C. posadasii biology and pathogenesis.

What high-throughput approaches could accelerate functional characterization of CPC735_014430?

Modern high-throughput methodologies can expedite the functional characterization of CPC735_014430:

• Proteomics approaches:

  • Activity-based protein profiling using metallopeptidase-targeted probes to assess CPC735_014430 activity in complex biological samples

  • Protein interactome mapping through proximity labeling methods (BioID, APEX) to identify interaction partners

  • Quantitative proteomics comparing wild-type and CPC735_014430-deficient strains to identify downstream effectors

• Transcriptomics applications:

  • RNA-seq analysis of gene expression changes in response to CPC735_014430 overexpression or deletion

  • Single-cell RNA-seq of C. posadasii populations during host interaction to identify cell-to-cell variability in CPC735_014430 expression

  • Dual RNA-seq of host-pathogen interactions to correlate CPC735_014430 expression with host response patterns

• Chemical biology screening:

  • High-throughput screening of chemical libraries to identify selective inhibitors of CPC735_014430

  • CRISPR-Cas9 screens to identify genetic interactors that modify CPC735_014430 function

  • Phenotypic screening of CPC735_014430 variants with site-directed mutations to map structure-function relationships

• Structural biology approaches:

  • Cryo-electron microscopy to visualize CPC735_014430 structure at high resolution

  • X-ray crystallography of CPC735_014430 in complex with substrates or inhibitors

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions and conformational changes upon substrate binding

These high-throughput approaches would significantly accelerate our understanding of CPC735_014430 function in C. posadasii biology and pathogenesis, potentially identifying new therapeutic strategies for coccidioidomycosis.