Recombinant Trichophyton verrucosum Formation of crista junctions protein 1 (FCJ1)

What is Trichophyton verrucosum and its significance in research?

Trichophyton verrucosum is a zoophilic dermatophyte that primarily infects cattle, causing approximately 98% of their dermatophytic infections . This fungal pathogen has notable research significance due to its ability to transmit to humans, particularly individuals working with farm animals or in animal-adjacent environments . While historically rare in humans, infections have increased potentially due to decreased immunization of cattle .

T. verrucosum belongs to the broader group of dermatophytes that cause superficial infections in keratinized tissues. It is classified specifically as a zoophilic dermatophyte, indicating its primary adaptation to non-human animal hosts . The organism has a worldwide distribution and primarily causes ringworm infections, with human infections typically resulting in more significant inflammation compared to anthropophilic dermatophyte infections, likely due to less host-pathogen adaptation .

From a genomic perspective, T. verrucosum has been subject to comparative genome analysis alongside other dermatophytes such as T. rubrum, T. tonsurans, and T. equinum . These genomic studies reveal important insights about evolutionary relationships, gene content, and transposable elements that contribute to phenotypic differences among dermatophytes.

What is the Formation of Crista Junctions Protein 1 (FCJ1) and its functional role?

The Formation of Crista Junctions Protein 1 (FCJ1) is a mitochondrial protein involved in maintaining the architecture of mitochondrial inner membrane structures known as crista junctions. In T. verrucosum, the mature FCJ1 protein spans amino acids 12-683 of the full protein sequence . The protein plays a crucial role in mitochondrial function by helping to form and maintain the characteristic folded structure of the inner mitochondrial membrane.

From a research perspective, understanding FCJ1 function provides insights into basic mitochondrial biology, fungal physiology, and potentially novel antifungal treatment approaches that could target mitochondrial function. The protein represents an important component of cellular energetics that may influence pathogenicity and stress responses in T. verrucosum.

How should recombinant T. verrucosum FCJ1 protein be expressed and purified?

The recommended expression system for recombinant T. verrucosum FCJ1 protein is E. coli . When designing expression constructs, researchers should include an N-terminal His-tag to facilitate purification via affinity chromatography. The optimal expression conditions typically involve induction at reduced temperatures (16-20°C) to minimize inclusion body formation and maximize soluble protein yield.

For purification, a multi-step protocol is recommended:

• Initial capture via immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins

• Secondary purification using size exclusion chromatography to remove aggregates

• Final polishing step using ion exchange chromatography if higher purity is required

During purification, buffers should be optimized to maintain protein stability, typically containing:

• 50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0)

• 150-300 mM NaCl to maintain solubility

• 5-10% glycerol as a stabilizing agent

• 1-5 mM reducing agent (DTT or β-mercaptoethanol)

• Protease inhibitors during initial extraction steps

The purified protein is typically obtained as a lyophilized powder that requires proper reconstitution in appropriate buffers before use in experiments .

What are the optimal storage and handling conditions for FCJ1 protein preparations?

For optimal stability, recombinant FCJ1 protein should be stored as aliquots at -20°C to -80°C, with -80°C preferred for long-term storage . Before freezing, the addition of 5-50% glycerol (with 50% recommended as the standard final concentration) helps maintain protein integrity during freeze-thaw cycles .

When handling the lyophilized protein:

• Briefly centrifuge the vial before 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 the recommended final concentration

• Divide into small working aliquots to avoid repeated freeze-thaw cycles

How does FCJ1 protein relate to T. verrucosum pathogenicity and infection mechanisms?

The relationship between FCJ1 function and T. verrucosum pathogenicity remains an area requiring further research, but several connections can be hypothesized based on known dermatophyte biology. T. verrucosum causes specific infection patterns in humans, presenting as disseminated, sharply defined, bluish-red, partly edematous nodules and plaques, particularly on the thighs, trunk, and arms . These infections can mimic perniosis, especially in individuals frequently exposed to cold or undergoing long-term corticosteroid therapy .

Mitochondrial proteins like FCJ1 likely contribute to pathogenicity through:

• Energy provision for invasive growth into keratinized tissues

• Adaptation to host microenvironments, including temperature shifts

• Response to oxidative stress during host immune responses

• Production of virulence factors requiring mitochondrial metabolism

Research approaches to investigate these connections should include comparative gene expression studies between infective and non-infective conditions, mitochondrial morphology analysis during infection, and targeted gene modification studies to assess the impact of FCJ1 alterations on virulence.

What experimental approaches are most effective for studying FCJ1 function in dermatophytes?

Investigating FCJ1 function in T. verrucosum and other dermatophytes requires a multi-faceted experimental approach:

• Gene expression analysis: Quantitative PCR and RNA-sequencing to determine FCJ1 expression levels under various conditions (temperature stress, antifungal exposure, different growth phases)

• Localization studies: Fluorescent protein tagging of FCJ1 combined with mitochondrial markers to confirm subcellular localization and dynamic distribution patterns

• Protein interaction analysis: Co-immunoprecipitation followed by mass spectrometry to identify interaction partners, or yeast two-hybrid screening with components of mitochondrial protein complexes

• Functional assays:

  • Oxygen consumption rate measurements

  • Membrane potential assays using fluorescent dyes

  • ATP production quantification

  • Mitochondrial morphology assessment via electron microscopy

• Genetic manipulation: CRISPR-Cas9 or RNAi-based approaches to modulate FCJ1 expression levels and assess phenotypic consequences

These approaches can be complemented by comparative studies with FCJ1 homologs from other dermatophytes, including anthropophilic species like T. rubrum, which might reveal host-specific adaptations of mitochondrial function.

How does T. verrucosum FCJ1 compare to homologous proteins in other dermatophytes?

Comparative genomic analysis reveals several interesting features about T. verrucosum in relation to other dermatophytes. While specific FCJ1 comparisons are not directly provided in the search results, the broader genomic context offers valuable insights.

T. verrucosum has distinct transposable element profiles compared to other dermatophytes. Notably, helitron family elements are most frequently found in T. verrucosum compared to other dermatophyte species . This genomic feature may influence gene regulation and protein evolution, potentially including genes like FCJ1.

When comparing dermatophyte genomes, significant differences in structural arrangements are observed. For instance, M. canis, which is distantly related to T. rubrum, displays 10 instances of inversions, whereas T. verrucosum likely has a distinct pattern of genomic rearrangements that may affect gene context and expression .

The table below summarizes key comparative features of T. verrucosum alongside other dermatophytes:

These differences in host adaptation likely influence the evolution and function of proteins involved in cellular metabolism, including FCJ1.

What evolutionary insights can be gained from studying FCJ1 across different fungal species?

Evolutionary analysis of FCJ1 across fungal species provides valuable insights into mitochondrial adaptation and host specialization. T. verrucosum, being a zoophilic dermatophyte, represents an evolutionary position where it maintains adaptations for specific animal hosts while retaining capacity for human infection .

Unlike some anthropophilic dermatophytes that rely exclusively on asexual reproduction, zoophilic species like T. verrucosum maintain sexual reproduction capabilities, with both mating types potentially present in nature . This reproductive flexibility may influence genetic diversity and protein evolution rates, including for mitochondrial proteins like FCJ1.

Researchers investigating FCJ1 evolution should consider:

• Constructing phylogenetic trees using FCJ1 sequences from diverse fungi

• Calculating selection pressures (dN/dS ratios) across functional domains

• Identifying conserved motifs that suggest critical functional regions

• Correlating FCJ1 sequence divergence with host adaptation patterns

Such evolutionary analyses could reveal whether FCJ1 undergoes adaptive evolution during host switching or specialization, potentially contributing to the understanding of how dermatophytes adapt to different mammalian hosts.

How should researchers address solubility issues with recombinant FCJ1 protein?

Recombinant FCJ1 protein can present solubility challenges due to its membrane-associated nature. To address these issues, researchers should consider the following methodological approaches:

• Optimization of expression conditions:

  • Reduce induction temperature to 16-18°C

  • Use lower inducer concentrations (e.g., 0.1-0.5 mM IPTG instead of 1 mM)

  • Extend expression time to 16-24 hours at reduced temperature

  • Test different E. coli strains specialized for membrane protein expression (C41, C43, or Rosetta)

• Buffer optimization strategies:

  • Include mild detergents (0.05-0.1% n-Dodecyl β-D-maltoside or CHAPS)

  • Test different salt concentrations (150-500 mM NaCl)

  • Include stabilizing agents (5-10% glycerol, 1 mM EDTA)

  • Adjust pH to optimize solubility (typically pH 7.5-8.0 works well)

• Protein engineering approaches:

  • Express truncated versions lacking the most hydrophobic regions

  • Use solubility-enhancing fusion partners (MBP, SUMO, or thioredoxin)

  • Introduce surface mutations to increase solubility without affecting function

When reconstituting lyophilized FCJ1 protein, researchers should follow the recommended protocol of reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL, followed by the addition of glycerol to a final concentration of 50% .

What controls should be included when studying FCJ1 function in experimental systems?

When designing experiments to study FCJ1 function, proper controls are essential for reliable interpretation of results:

• Positive controls:

  • Well-characterized mitochondrial proteins with known functions

  • FCJ1 homologs from model organisms with established functions

  • Commercial mitochondrial function markers

• Negative controls:

  • Inactive FCJ1 mutants (create by site-directed mutagenesis of key residues)

  • Non-related proteins with similar size/properties but different functions

  • Buffer-only treatments for baseline measurements

• Specificity controls:

  • Competitive binding assays to confirm specific interactions

  • Dose-response relationships to establish specificity

  • Antibody validation using knockout/knockdown systems

• Experimental validation approaches:

  • Use multiple independent methods to confirm findings

  • Complementation studies in FCJ1-deficient systems

  • Reproducibility across different dermatophyte species

These control strategies ensure that experimental observations are specifically attributable to FCJ1 function rather than artifacts or non-specific effects.