Recombinant Coccidioides immitis Mitochondrial escape protein 2 (YME2)

What are the optimal storage and handling conditions for recombinant YME2 protein?

For optimal stability and activity preservation of recombinant YME2 protein, the following methodological approach is recommended:

• Initial storage: Store lyophilized powder at -20°C to -80°C upon receipt .

• Reconstitution protocol:

  • Briefly centrifuge vial before opening

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

  • Add glycerol to a final concentration of 5-50% (optimally 50%)

• Working aliquots: Store at 4°C for up to one week

• Long-term storage: Prepare aliquots and store at -20°C/-80°C in Tris/PBS-based buffer (pH 8.0) containing 6% trehalose

• Stability considerations: Avoid repeated freeze-thaw cycles as they significantly decrease protein stability and activity

The addition of trehalose is particularly important as this disaccharide acts as a cryoprotectant, preventing protein denaturation during freeze-thaw cycles by stabilizing protein structure through hydrogen bonding with polar residues.

How does YME2 differ between Coccidioides immitis and Coccidioides posadasii?

While the search results don't directly compare YME2 between the two Coccidioides species, we can apply the methodological approaches used to differentiate these closely related fungi:

To specifically characterize differences in YME2 between these species, researchers should consider:

• Comparative sequence analysis of YME2 genes from both species

• Expression profiling under various environmental conditions

• Functional complementation studies to assess interspecies compatibility

What molecular techniques are most effective for detecting YME2 expression in clinical samples?

For detecting YME2 expression in clinical samples, a multi-tiered approach is recommended:

• Real-time quantitative PCR (RT-qPCR):

  • Design primers specific to YME2 gene regions conserved across Coccidioides species

  • Utilize TaqMan chemistry with fluorescent probes for increased specificity

  • Establish detection thresholds (approximately 10 genome copies or 0.1 pg gDNA)

  • Include internal amplification controls to identify PCR inhibition

• Digital droplet PCR (ddPCR):

  • Recommended for low-abundance samples where absolute quantification is necessary

  • Provides enhanced sensitivity for detecting YME2 in complex clinical matrices

• Targeted proteomics:

  • Selected Reaction Monitoring (SRM) mass spectrometry for detection of YME2-specific peptides

  • Requires prior development of specific transitions for YME2 peptides

• Validation approaches:

  • Cross-validate molecular findings with serological test results

  • In a preliminary analysis of CSF and pleural fluid samples, a positive correlation was observed between serology tests and PCR results for only two of 15 samples , underscoring the need for multiple detection methods

What experimental approaches can elucidate the function of YME2 in Coccidioides immitis pathogenesis?

To systematically investigate the role of YME2 in C. immitis pathogenesis, an integrated experimental strategy comprising the following methodological components is recommended:

Genetic Manipulation Approaches:

• CRISPR-Cas9 gene editing:

  • Design sgRNAs targeting multiple sites within the YME2 gene

  • Optimize transformation protocols for Coccidioides (challenging due to its biosafety level)

  • Generate knockout, knockdown, and site-directed mutants

  • Validate modifications by sequencing and expression analysis

• Conditional expression systems:

  • Develop tetracycline-inducible or repressible YME2 constructs

  • Engineer strains with tagged versions (GFP, HA, FLAG) for localization and pull-down studies

Functional Analysis Methods:

• Virulence assessment in animal models:

  • Compare wild-type and YME2-modified strains in murine models

  • Quantify fungal burden, inflammatory markers, and survival rates

  • Implement tissue-specific examination of YME2 expression during infection

• Mitochondrial function assays:

  • Measure oxygen consumption rates, ATP production, and membrane potential

  • Assess mitochondrial morphology in YME2-deficient strains using fluorescence microscopy

  • Evaluate mitochondrial stress responses and their relationship to virulence

• Host-pathogen interaction studies:

  • Co-culture YME2-modified strains with human macrophages and epithelial cells

  • Quantify phagocytosis rates, intracellular survival, and cytokine responses

  • Perform transcriptomic analysis of host cells upon exposure to wild-type vs. modified strains

Structural Biology Approaches:

• Protein-protein interaction mapping:

  • Use yeast two-hybrid or proximity labeling methods (BioID, APEX)

  • Perform co-immunoprecipitation with subsequent mass spectrometry analysis

  • Validate key interactions with bimolecular fluorescence complementation

• High-resolution structural analysis:

  • Express and purify domains of YME2 for crystallization trials

  • Employ cryo-EM for larger complexes involving YME2

  • Use hydrogen-deuterium exchange mass spectrometry to map functional regions

How can researchers optimize PCR-based detection protocols for distinguishing Coccidioides species in complex clinical samples?

For researchers developing optimized PCR protocols to distinguish Coccidioides species in complex clinical samples, the following methodological framework is essential:

Primer and Probe Design Strategy:

• Target selection:

  • Primary target: RRA2 gene (encoding proline-rich antigen 2) for genus-level identification

  • Secondary target: Ci45815 fragment for C. immitis-specific detection

  • Exploit the 86-bp deletion in C. posadasii for definitive species differentiation

• Probe chemistry optimization:

  • Implement dual-labeled probes (Cy5 and FAM) for multiplexed detection

  • Design probes with optimal Tm values (8-10°C higher than primers)

  • Include minor groove binders (MGBs) for increased specificity in AT-rich regions

Assay Development Parameters:

Validation Approach:

• Analytical validation:

  • Determine specificity against panel of fungal and bacterial pathogens (no cross-reactivity observed in published studies)

  • Establish limit of detection (0.1 pg gDNA/reaction)

  • Assess reproducibility across different sample types

• Clinical validation:

  • Test with diverse sample types (CSF, pleural fluid, BAL, tissue)

  • Compare against culture and serological methods

  • Evaluate performance in specimens with mixed microbial populations

• Continuous improvement:

  • Monitor for emerging genetic variants that may affect primer binding

  • Incorporate advances in sample preparation technologies

  • Consider integration with isothermal amplification for point-of-care applications

What are the potential mechanistic relationships between YME2 and the geographic distribution patterns of Coccidioides species?

The distinctive geographic distribution patterns of Coccidioides species (with C. immitis primarily in California and C. posadasii more widely distributed throughout the southwestern US, Mexico, and parts of South America) may be partially explained by mechanisms involving YME2 function:

Hypothesized Mechanisms and Research Approaches:

• Environmental adaptation hypothesis:

  • YME2's role in mitochondrial function may contribute to adaptation to specific environmental stresses

  • Research approach: Compare YME2 expression and activity across isolates from different geographic regions under varying temperature, humidity, and soil composition conditions

  • Methodology: RNA-seq and proteomics of environmental samples with controlled variable manipulation

• Host-specificity mechanisms:

  • YME2 may influence interaction with different host species prevalent in various geographic regions

  • Research approach: Test C. immitis and C. posadasii strains with wild-type and modified YME2 in diverse host cell models

  • Methodology: Infection models using cell lines derived from different host species indigenous to endemic regions

• Genetic drift and selection pressure:

  • YME2 sequence variations may reflect evolutionary divergence under different selection pressures

  • Research approach: Perform population genetics analysis of YME2 sequence from geographically diverse isolates

  • Methodology: Whole genome sequencing with focused analysis of YME2 locus and surrounding genomic regions

Evidence from Distribution Studies:

The retrospective analysis of clinical isolates revealed:

• 168 isolates of C. posadasii compared to 30 isolates of C. immitis from human cases

• Geographic anomalies: Four C. posadasii isolates were identified from California, where C. immitis is expected to predominate

• Animal infections: All eight primary samples from animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii

These findings suggest complex distribution patterns that warrant detailed investigation of molecular determinants, potentially including YME2's role in environmental fitness and host adaptation.

What methodological approaches can be used to study the structural determinants of YME2 function?

To elucidate the structural basis of YME2 function, a comprehensive structural biology workflow is recommended:

Computational Structure Prediction and Analysis:

• Sequence-based predictions:

  • Apply homology modeling using related proteins with known structures

  • Perform ab initio modeling for unique domains without homologs

  • Predict post-translational modifications and their effects on structure

• Molecular dynamics simulations:

  • Simulate protein behavior in membrane environments

  • Model conformational changes upon substrate binding

  • Identify critical residues for function through in silico mutagenesis

Experimental Structure Determination:

Structure-Function Analysis:

• Site-directed mutagenesis:

  • Target conserved residues identified from sequence analysis

  • Focus on predicted active sites and interaction interfaces

  • Generate systematic alanine scanning libraries

• Functional reconstitution:

  • Develop in vitro assays for specific YME2 activities

  • Correlate structural features with biochemical functions

  • Assess the impact of disease-associated mutations

How can researchers address challenges in recombinant YME2 expression and purification?

When encountering difficulties with recombinant YME2 expression and purification, researchers should systematically implement the following troubleshooting methodology:

Expression Optimization Strategy:

Purification Strategy Optimization:

• Initial capture conditions:

  • Optimize imidazole concentration in binding and wash buffers

  • Test range of pH conditions (7.0-8.5) for optimal His-tag binding

  • Evaluate different metal ions (Ni²⁺, Co²⁺, Cu²⁺) for affinity chromatography

• Buffer optimization:

  • Screen additives (glycerol, detergents, salt concentrations)

  • Test protein stabilizers (arginine, sucrose, Tris/PBS-based buffer with 6% trehalose)

  • Evaluate reducing agents (DTT, TCEP, β-mercaptoethanol)

• Advanced purification steps:

  • Implement ion exchange chromatography for higher purity

  • Apply size exclusion chromatography to remove aggregates

  • Consider affinity tag removal and subsequent polishing steps

• Quality control checkpoints:

  • Verify identity by mass spectrometry

  • Assess purity by SDS-PAGE (target >90%)

  • Confirm activity through functional assays

What are the key considerations for designing experiments to investigate YME2's role in Coccidioides pathogenesis?

When designing experiments to investigate YME2's role in Coccidioides pathogenesis, researchers should consider this comprehensive experimental design framework:

Experimental Controls and Variables:

• Essential controls:

  • Wild-type strain (positive control)

  • YME2 knockout/knockdown strain

  • Complemented strain (knockout with reintroduced YME2)

  • Heterologous expression (YME2 in non-pathogenic model)

• Critical variables to monitor:

  • Growth conditions (temperature, pH, oxygen levels)

  • Host cell types and activation states

  • Infection time course points

  • Animal model genetic background

Biosafety and Containment Considerations:

• Biosafety level requirements:

  • Work with Coccidioides requires BSL-3 containment

  • Develop surrogate models in related but less pathogenic fungi

  • Establish clear protocols for sample inactivation and transfer

• Alternative approaches for lower containment levels:

  • Use of heterologous expression in Saccharomyces cerevisiae

  • Cell-free protein synthesis for biochemical studies

  • Computational modeling and simulation

Specific Experimental Design Elements:

Analytical Framework:

• Data integration approach:

  • Combine transcriptomic, proteomic, and metabolomic data

  • Develop computational models of YME2's role in cellular networks

  • Correlate molecular findings with phenotypic observations

• Statistical considerations:

  • Conduct power analyses to determine appropriate sample sizes

  • Apply correction for multiple comparisons in large-scale data

  • Use appropriate statistical tests for data distribution types

How might YME2 be exploited for developing novel diagnostic approaches for coccidioidomycosis?

The potential exploitation of YME2 for novel diagnostic approaches for coccidioidomycosis can be systematically explored through the following research strategy:

Diagnostic Target Assessment:

• YME2 as a direct detection target:

  • Evaluate YME2 gene conservation across clinical isolates

  • Determine expression levels during different infection stages

  • Assess specificity compared to other fungal pathogens

• YME2 as an antigenic target:

  • Screen for immunodominant epitopes within YME2 sequence

  • Evaluate serological response to YME2 in patient cohorts

  • Compare with current diagnostic antigens (e.g., proline-rich antigen)

Technological Platforms for Development:

Validation Strategy:

• Analytical validation:

  • Determine limit of detection in various clinical matrices

  • Assess cross-reactivity with other fungal and bacterial pathogens

  • Evaluate reproducibility across different laboratory settings

• Clinical validation:

  • Test retrospective samples with confirmed diagnosis

  • Perform prospective studies in endemic regions

  • Compare performance against current gold standard methods

The duplex real-time PCR approach that successfully distinguishes C. immitis and C. posadasii provides a methodological framework that could be adapted for YME2-based diagnostics, with demonstrated sensitivity reaching approximately ten genome copies .

What comparative genomics approaches could reveal evolutionary insights about YME2 across fungal pathogens?

To elucidate the evolutionary history and functional divergence of YME2 across fungal pathogens, the following comparative genomics methodology is recommended:

Sequence-Based Evolutionary Analysis:

• Homology identification:

  • Perform reciprocal BLAST searches across fungal genomes

  • Apply hidden Markov model (HMM) profiles for sensitive detection

  • Identify orthologs and paralogs of YME2 across fungal kingdom

• Phylogenetic reconstruction:

  • Align sequences using structure-aware methods

  • Employ maximum likelihood and Bayesian inference approaches

  • Test alternative evolutionary models and tree topologies

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify signatures of selection

  • Perform branch-site tests for lineage-specific selection

  • Map selection signatures onto protein structural models

Functional Divergence Assessment:

• Domain architecture comparison:

  • Identify conserved and lineage-specific domains

  • Map sequence variations onto structural models

  • Correlate domain changes with ecological niches

• Regulatory element analysis:

  • Compare promoter regions across species

  • Identify conserved transcription factor binding sites

  • Correlate regulatory differences with expression patterns

Integration with Biological Context:

• Correlation with pathogenicity:

  • Compare YME2 sequence features between pathogenic and non-pathogenic fungi

  • Identify YME2 variations associated with host range differences

  • Correlate genomic findings with experimental virulence data

• Environmental adaptation signals:

  • Compare YME2 sequences from fungi in different ecological niches

  • Identify adaptations associated with specific environmental stresses

  • Correlate with the broader geographic distribution patterns observed in Coccidioides species

This evolutionary analysis could provide critical insights into why C. posadasii appears to have a larger population size and more diverse distribution compared to C. immitis , potentially relating these patterns to functional differences in key proteins like YME2.