Recombinant Rana sevosa Ranatuerin-2SEa

What is the primary structure and biochemical profile of Ranatuerin-2SEa?

Ranatuerin-2SEa has the amino acid sequence GFISTVKNLA TNVAGTVIDT IKCKVTGGC, with a molecular structure featuring a C-terminal cyclic domain formed by a disulfide bridge . Like other members of the ranatuerin-2 family, it contains a characteristic hexapeptide structure formed by 6 amino acid residues . Structurally, Ranatuerin-2SEa is predicted to form an amphipathic, alpha-helical conformation that facilitates membrane interaction, similar to other amphibian antimicrobial peptides that insert into lipid bilayers . The peptide was initially isolated alongside six other novel peptides from R. sevosa skin secretions based on antimicrobial activity and histamine release from rat peritoneal mast cells .

How does Ranatuerin-2SEa compare structurally to other antimicrobial peptides from amphibians?

Ranatuerin-2SEa belongs to one of four established families of ranid frog antimicrobial peptides identified in R. sevosa: esculentin-1, esculentin-2, brevinin-1, and ranatuerin-2 . A notable comparison can be made with ranatuerin-2TRa from the Tarahumara frog (Rana tarahumarae), which has the sequence GIMDSIKGAAKEIAGHLLDNLKCKITGC and shows activity against the chytrid fungus Batrachochytrium dendrobatidis with an MIC of 50 μM against zoospores . Of particular evolutionary interest is the structural similarity observed between brevinin-1SE (another R. sevosa peptide) and ponericin W5, an antibacterial venom peptide from the ant Pachyconyla goeldii, suggesting convergent evolution between amphibian defensive peptides and insect antimicrobial compounds .

What expression systems are most effective for producing recombinant Ranatuerin-2SEa?

Recombinant Ranatuerin-2SEa is effectively produced using yeast expression systems, which allow for proper folding and post-translational modifications necessary for biological activity . The typical production workflow involves:

• Gene synthesis or cloning of the Ranatuerin-2SEa coding sequence

• Insertion into an appropriate yeast expression vector

• Transformation of the expression construct into the yeast host

• Induction of protein expression under optimized conditions

• Cell lysis and initial purification steps

• Chromatographic purification to achieve >85% purity as verified by SDS-PAGE

The expression region typically encompasses amino acids 1-29 of the peptide sequence, and the final product may include tag sequences depending on the specific manufacturing process .

What are the optimal storage and handling conditions for maintaining Ranatuerin-2SEa stability?

For optimal stability and activity retention, Recombinant Ranatuerin-2SEa requires specific storage and handling conditions:

Prior to reconstitution, it is recommended to briefly centrifuge the vial to ensure all material is at the bottom .

What methodologies are most appropriate for evaluating the antimicrobial efficacy of Ranatuerin-2SEa?

Several established methodologies can effectively assess the antimicrobial activity of Ranatuerin-2SEa:

• Minimum Inhibitory Concentration (MIC) determination: Broth microdilution assays using 2-fold serial dilutions of the peptide against target microorganisms. Similar peptides like ranatuerin-2TRa have shown MIC values of 50 μM against B. dendrobatidis zoospores .

• Disk diffusion assays: Particularly useful for screening activity against multiple organisms simultaneously. This method has been effectively applied to assess skin extract activity from stress-challenged amphibians against various microbes including E. coli, B. subtilis, S. cerevisiae, and fungal pathogens .

• Time-kill kinetics: For determining the rate and extent of microbial killing over time, distinguishing between bacteriostatic and bactericidal effects.

• Membrane permeabilization assays: Using fluorescent dyes (e.g., propidium iodide, SYTOX Green) to assess the peptide's ability to compromise microbial membranes.

• Growth curve analysis: Monitoring microbial growth in the presence of varying peptide concentrations over time using spectrophotometric methods.

When comparing results across different antimicrobial peptide studies, it is critical to standardize experimental conditions, including medium composition, inoculum size, and incubation conditions, as these factors can significantly influence observed activity .

How does environmental stress influence the expression and activity of antimicrobial peptides like Ranatuerin-2SEa?

Environmental stressors significantly modulate amphibian antimicrobial peptide expression and activity, as demonstrated in studies with other frog species:

• Dehydration stress: In Rana sylvatica, 40% dehydration resulted in significantly increased brevinin-1SY mRNA levels in both dorsal and ventral skin compared to control conditions .

• Anoxic conditions: 24-hour anoxia exposure led to increased brevinin-1SY mRNA levels specifically in ventral skin of R. sylvatica, with corresponding increases in antimicrobial activity .

• Freezing stress: While 24-hour freezing did not significantly alter brevinin-1SY mRNA levels in R. sylvatica, peptide extracts from frozen animals demonstrated enhanced antimicrobial activity against E. coli and P. sulcatum, suggesting post-transcriptional or post-translational regulation mechanisms .

• Developmental regulation: Antimicrobial peptide expression follows distinct developmental patterns, with brevinin-1SY transcripts significantly increasing during later developmental stages in tadpoles .

These findings suggest that Ranatuerin-2SEa expression and activity may similarly be regulated by environmental stressors, potentially providing enhanced protection against pathogens during physiologically challenging conditions. This environmental responsiveness may have important implications for designing optimal experimental conditions when studying these peptides .

What is the molecular mechanism underlying Ranatuerin-2SEa's antimicrobial activity?

Ranatuerin-2SEa likely employs multiple mechanisms to exert its antimicrobial effects:

• Membrane disruption: As an amphipathic α-helical peptide, Ranatuerin-2SEa primarily targets microbial membranes through:

  • Initial electrostatic interaction between the peptide's cationic regions and negatively charged microbial membrane components

  • Hydrophobic interactions leading to peptide insertion into the lipid bilayer

  • Subsequent membrane permeabilization via pore formation (toroidal or barrel-stave) or carpet-like disruption

• Dual functional activity: Ranatuerin-2SEa exhibits both antimicrobial activity and the ability to induce histamine release from mast cells, suggesting a multifunctional role in host defense that may link innate immunity with inflammatory responses .

• Selective toxicity: The peptide likely exhibits preferential activity against microbial cells over host cells due to differences in membrane composition, particularly the presence of negatively charged phospholipids and absence of cholesterol in microbial membranes.

• Potential intracellular targets: Beyond membrane disruption, some antimicrobial peptides can enter microbial cells and interfere with essential processes such as DNA, RNA, or protein synthesis, though this has not been specifically established for Ranatuerin-2SEa.

This multifaceted mechanism of action may help explain why antimicrobial peptides often remain effective against pathogens that have developed resistance to conventional antibiotics .

How do structural modifications affect the stability and antimicrobial efficacy of Ranatuerin-2SEa?

Structural modifications can significantly impact both stability and antimicrobial activity of peptides like Ranatuerin-2SEa:

When designing Ranatuerin-2SEa derivatives, researchers must carefully balance modifications that improve pharmacokinetic properties with those that maintain or enhance antimicrobial efficacy .

What pathogens are most susceptible to Ranatuerin-2SEa and related amphibian antimicrobial peptides?

Based on studies of related peptides from ranid frogs, Ranatuerin-2SEa likely exhibits activity against diverse microorganisms:

• Bacterial targets: Related peptides show activity against both:

  • Gram-positive bacteria (including Staphylococcus aureus)

  • Gram-negative bacteria (including Escherichia coli)

• Fungal susceptibility: Ranatuerin-2TRa from R. tarahumarae demonstrated activity against Batrachochytrium dendrobatidis (the chytrid fungus implicated in amphibian population declines) with an MIC of 50 μM against zoospores . Similar activity might be expected for Ranatuerin-2SEa.

• Pathogen-specific efficacy: Studies with R. sylvatica skin extracts revealed differential effectiveness against various microorganisms:

  • Significant inhibition of E. coli and Pythium sulcatum from stress-challenged animals

  • Activity against Bacillus subtilis and Botrytis cinerea from anoxic animals

  • Variable effectiveness against Saccharomyces cerevisiae and Rhizopus stolonifer

• Multi-drug resistant organisms: Amphibian antimicrobial peptides often maintain effectiveness against pathogens resistant to conventional antibiotics, making them valuable subjects for research into novel anti-infective agents .

The spectrum of activity suggests Ranatuerin-2SEa could have broad applications in both amphibian conservation efforts and human therapeutic development .

What are the key challenges in translating Ranatuerin-2SEa from basic research to therapeutic applications?

Several significant challenges must be addressed to develop Ranatuerin-2SEa as a therapeutic agent:

• Pharmacokinetic limitations:

  • Susceptibility to proteolytic degradation in vivo

  • Poor tissue penetration and systemic bioavailability

  • Rapid renal clearance due to small molecular size

• Stability constraints: While cyclization can improve stability, studies have shown this may reduce antimicrobial activity . Finding the optimal balance between stability enhancement and activity preservation remains challenging.

• Formulation complexities: Developing suitable formulations for different administration routes requires addressing:

  • Aggregation tendencies in physiological environments

  • Interactions with delivery vehicle components

  • Maintaining activity during storage and administration

• In vivo efficacy translation: Similar peptides have shown disconnects between in vitro and in vivo activity. While some anuran peptides showed limited in vitro activity against S. aureus, they demonstrated strong antibacterial activities in a murine wound infection model . This highlights the importance of comprehensive in vivo testing.

• Immunogenicity concerns: Potential immunogenic responses to peptide therapeutics require careful immunological profiling during development.

• Effective concentration delivery: The immunomodulatory peptide pYR from R. sevosa achieved complete bacterial clearance at 3 mg·kg⁻¹ in a murine model . Determining similar effective concentrations for Ranatuerin-2SEa while minimizing toxicity will be crucial for therapeutic development.

Addressing these challenges through strategic peptide engineering and advanced delivery systems represents a promising direction for future research .

What novel experimental approaches could enhance our understanding of Ranatuerin-2SEa functions?

Several innovative research approaches could advance our understanding of Ranatuerin-2SEa:

• Advanced structural analysis techniques:

  • High-resolution NMR studies in membrane-mimicking environments

  • Cryo-electron microscopy to visualize peptide-membrane interactions

  • Molecular dynamics simulations to examine conformation transitions during membrane insertion

• Systems biology approaches:

  • Transcriptomic analysis of microbial responses to Ranatuerin-2SEa exposure

  • Metabolomic profiling to identify metabolic pathways disrupted by the peptide

  • Proteomic studies to detect potential intracellular targets beyond membrane disruption

• Comparative immunological studies:

  • Investigation of synergistic effects between Ranatuerin-2SEa and other antimicrobial peptides from R. sevosa

  • Analysis of interactions between the peptide and components of the amphibian immune system

  • Examination of the dual antimicrobial and histamine-releasing activities in immunological contexts

• Host-pathogen interaction models:

  • Development of ex vivo skin models to study peptide activity in a tissue context

  • Examination of peptide efficacy against intracellular pathogens

  • Investigation of peptide activity against polymicrobial communities and biofilms

• Evolutionary perspectives:

  • Comparative analysis with structurally similar insect peptides like ponericin W5 to understand evolutionary convergence

  • Examination of selective pressures driving antimicrobial peptide diversification in amphibians

These approaches would provide valuable insights into both fundamental biological questions and applied therapeutic development opportunities .

What are the most promising strategies for enhancing Ranatuerin-2SEa stability while preserving antimicrobial activity?

Several promising strategies could optimize Ranatuerin-2SEa's pharmaceutical properties:

• Rational peptide engineering:

  • Site-directed mutagenesis to replace proteolytically vulnerable residues

  • Enhancement of helical propensity through strategic amino acid substitutions

  • Charge distribution optimization to maintain membrane selectivity while reducing toxicity

• Advanced cyclization approaches:

  • Selective backbone cyclization that preserves the critical amphipathic structure

  • Incorporation of non-natural amino acids at cyclization points

  • Development of stapled peptide variants with enhanced stability

• Hybrid peptide design:

  • Creation of chimeric peptides combining regions of Ranatuerin-2SEa with stable elements from other antimicrobial peptides

  • Development of peptide-small molecule conjugates with improved pharmacokinetics

  • Exploration of lipopeptide derivatives with enhanced membrane association

• Formulation innovations:

  • Encapsulation in nanoparticle delivery systems for sustained release

  • Development of site-specific targeting strategies for improved bioavailability

  • Creation of prodrug forms that activate in specific microenvironments

• Synergistic combinations:

  • Investigation of combinations with conventional antibiotics for enhanced efficacy

  • Exploration of peptide mixtures that mimic the natural amphibian skin secretion composition

  • Development of multi-peptide formulations targeting different aspects of microbial physiology

While cyclization has been shown to improve stability in similar peptides, the challenge remains to implement such modifications without significantly compromising antimicrobial activity . Integrating multiple approaches and utilizing structure-guided design will likely yield the most promising candidates for further development.