Recombinant Glandirana rugosa Gaegurin-6 (GGN6)

What is the molecular structure of Gaegurin-6 and how does it relate to its antimicrobial function?

Gaegurin-6 belongs to the alpha-helix family of antimicrobial peptides isolated from the skin of Glandirana rugosa (formerly Rana rugosa) . Its structure is characterized by an alpha-helical conformation stabilized by a C-terminal disulfide bridge, often referred to as the "Rana box" . This disulfide bridge plays a critical role in maintaining the peptide's antimicrobial activity, as research demonstrates that reduction of this bridge results in complete loss of function, while replacing the cysteine residues with serines can preserve activity .

The alpha-helical structure facilitates the peptide's interaction with microbial membranes, adopting an amphipathic conformation where hydrophobic and hydrophilic residues segregate to opposite sides of the helix. This arrangement enables the peptide to disrupt microbial membranes through various mechanisms, including toroidal pore formation and barrel-stave mechanisms . Circular dichroism spectroscopy studies have confirmed that the alpha-helical forming propensity correlates directly with antimicrobial activity, highlighting the structure-function relationship of this peptide .

How does Gaegurin-6 compare with other antimicrobial peptides from amphibian skin?

Gaegurin-6 shares structural and functional similarities with other ranid antimicrobial peptides but possesses unique characteristics. Like Esculentin-2EM (previously known as gaegurin-4), it contains a conserved C-terminal cyclic region stabilized by a disulfide bond that helps stabilize pore formation and promote antimicrobial action . This structural feature, known as the "Rana box," is common among many antimicrobial peptides isolated from ranid frogs.

Compared to peptides from other amphibian species, Gaegurin-6 demonstrates distinctive pH-dependent activity patterns. Unlike some related peptides such as gad-1 and gad-2 from Gadus morhua that show decreased alpha-helicity at acidic pH, Gaegurin-6 maintains its structural integrity across varying pH conditions while exhibiting modified activity profiles . This pH dependence may represent an evolutionary adaptation synchronized with the acidic microenvironment of frog skin.

The antimicrobial spectrum of Gaegurin-6 includes activity against both Gram-positive and Gram-negative bacteria, which distinguishes it from more selective amphibian peptides like chensinin-1 from Rana chensinensis, which is ineffective against Gram-negative bacteria due to high-affinity binding with lipopolysaccharide .

What experimental evidence demonstrates the importance of the disulfide bridge in Gaegurin-6?

The critical role of the disulfide bridge in Gaegurin-6 has been established through systematic structure-function studies. Research has demonstrated that reduction of the disulfide bridge results in complete loss of antimicrobial activity, providing direct evidence of its functional importance . This finding suggests that the bridge is not merely a structural feature but a critical determinant of the peptide's antimicrobial capability.

Circular dichroism spectroscopy experiments reveal that the disulfide bridge stabilizes the induction of alpha-helical structure when the peptide interacts with lipid membranes . These studies show a clear correlation between the alpha-helical forming propensity and antimicrobial activity, indicating that the disulfide bridge contributes to function by maintaining the optimal secondary structure for membrane interaction.

Interestingly, when the cysteine residues are replaced with serines (maintaining similar spatial occupancy but eliminating the disulfide bond), antimicrobial activity is retained . This suggests that while the conformational constraint provided by the bridge is essential, the specific chemistry of the disulfide bond may be substitutable with appropriate structural mimics that preserve the critical spatial arrangement of the peptide.

What expression systems are most effective for producing recombinant Gaegurin-6?

The selection of an appropriate expression system for recombinant Gaegurin-6 production depends on research objectives, required yield, and downstream applications. Several systems offer distinct advantages:

The choice between these systems should be guided by specific requirements including yield needs, budget constraints, and whether post-translational modifications are essential for the peptide's activity. For most basic research applications, E. coli systems with appropriate fusion partners offer the best balance of yield, cost, and functionality.

What purification strategies effectively isolate recombinant Gaegurin-6 while maintaining its structural integrity?

Purification of recombinant Gaegurin-6 requires a strategic approach that preserves the critical disulfide bridge while achieving high purity. An effective purification protocol typically involves:

• Initial capture:

  • For tagged constructs: Immobilized metal affinity chromatography (IMAC) for His-tagged fusions or glutathione affinity chromatography for GST fusions

  • For tag-free expression: Cation exchange chromatography leveraging the peptide's positive charge

• Tag removal and processing:

  • Enzymatic cleavage with specific proteases (TEV, Factor Xa, thrombin)

  • Second affinity chromatography to separate cleaved peptide from tag

  • Buffer exchange to optimal conditions for disulfide bond formation

• Intermediate purification:

  • Ion exchange chromatography, particularly cation exchange using SP-Sepharose

  • Hydrophobic interaction chromatography to separate based on surface hydrophobicity

• Polishing and final preparation:

  • Reversed-phase HPLC using C18 or C8 columns with acetonitrile gradients

  • Size exclusion chromatography for higher molecular weight aggregates removal

  • Lyophilization in the presence of stabilizers if long-term storage is required

Throughout the purification process, conditions should be carefully controlled to maintain the integrity of the disulfide bridge, with verification through analytical methods such as mass spectrometry and circular dichroism spectroscopy. The purified peptide should undergo activity testing against standard microbial strains to confirm that structural integrity and functional activity have been preserved.

How can the proper formation of the disulfide bridge in recombinant Gaegurin-6 be ensured?

Ensuring correct disulfide bridge formation is critical for recombinant Gaegurin-6 activity, as research has demonstrated that reduction of this bridge results in complete loss of antimicrobial function . Several strategies can be implemented to promote proper disulfide bond formation:

• Expression system selection:

  • Specialized E. coli strains (Origami, SHuffle) with mutations in thioredoxin reductase and glutathione reductase genes create a more oxidizing cytoplasmic environment

  • Periplasmic expression directs the peptide to a cellular compartment more conducive to disulfide bond formation

  • Eukaryotic expression systems (yeast, mammalian cells) with native machinery for disulfide bond formation

• Oxidative folding protocols:

  • Controlled oxidation using buffer systems containing reduced and oxidized glutathione at optimized ratios (typically 1:10 to 1:100 GSH:GSSG)

  • Folding at pH 7.5-8.5 to facilitate thiol-disulfide exchange

  • Gradual removal of denaturants through dialysis or dilution methods

• Verification methods:

  • Mass spectrometry to confirm the presence of the disulfide bond through mass difference

  • Reversed-phase HPLC mobility shifts between reduced and oxidized forms

  • Ellman's reagent for quantification of free sulfhydryl groups

  • Circular dichroism spectroscopy to assess secondary structure formation

  • Antimicrobial activity assays comparing to chemically synthesized standards

The importance of correctly formed disulfide bridges is underscored by research showing that while reduction eliminates activity, cysteine-to-serine substitutions can maintain function, suggesting that the bridge's conformational effects rather than the specific chemistry may be most critical .

What membrane disruption mechanisms does Gaegurin-6 employ against different microbial targets?

Gaegurin-6 employs multiple membrane disruption mechanisms that vary depending on target microorganism membrane composition and environmental conditions. Based on studies of related peptides including Esculentin-2EM (previously gaegurin-4), several models have been proposed :

• Toroidal pore mechanism: In this model, Gaegurin-6 induces the formation of pores where both the peptide and lipid headgroups line the pore interior, creating positive curvature strain that causes lipids to bend continuously from the outer to inner leaflet. This mechanism appears predominant in membranes with anionic lipids typical of bacterial membranes .

• Barrel-stave mechanism: Alternatively, Gaegurin-6 can form barrel-like structures where peptides align perpendicular to the membrane plane, with hydrophobic regions facing the lipid bilayer and hydrophilic regions forming the pore lumen. The C-terminal cyclic region stabilized by the disulfide bond (Rana box) plays a crucial role in stabilizing these pore structures .

• Carpet mechanism: At higher concentrations, the peptide may adsorb parallel to the membrane surface until reaching a threshold concentration, after which the membrane is disrupted in a detergent-like fashion.

The predominant mechanism deployed by Gaegurin-6 depends on multiple factors including:

• Lipid composition of the target membrane

• Peptide concentration

• Environmental pH

• Ionic strength of the medium

These complex interactions explain the differential activity of Gaegurin-6 against various microbial targets and highlight the sophisticated nature of its antimicrobial action.

How does pH influence the antimicrobial activity and structure of Gaegurin-6?

The antimicrobial activity of Gaegurin-6 exhibits significant pH dependence, a characteristic shared with several antimicrobial peptides from amphibian skin. This pH sensitivity is particularly relevant considering the acidic microenvironment of frog skin (typically pH 6.4-6.9) . Several mechanisms contribute to this pH-dependent activity:

• Charge state modulation: At lower pH, protonation of titratable amino acid residues increases the net positive charge of Gaegurin-6, potentially enhancing electrostatic interaction with negatively charged microbial membranes.

• Conformational changes: pH can influence the secondary structure of the peptide. Studies on related peptides from Glandirana emeljanovi (formerly Rana rugosa) show that low pH conditions associated with frog skin can modify the peptide's membrane interaction capabilities .

• Membrane disruption efficiency: Research on related peptides demonstrates that under low pH conditions, membrane lysis capabilities can be altered, with enhanced activity observed at higher pH for some membrane compositions . This suggests that pH affects not only peptide structure but also its mechanism of membrane disruption.

• Selectivity modulation: pH changes may influence Gaegurin-6's selectivity between microbial and host cell membranes, potentially representing an evolutionary adaptation that synchronizes optimal antimicrobial activity with the natural skin environment of the frog.

Understanding this pH dependence is crucial for both fundamental research on antimicrobial mechanisms and potential biotechnological applications of Gaegurin-6 or its derivatives in environments with varying pH conditions.

What is the current understanding of specificity mechanisms that allow Gaegurin-6 to target microbial membranes while sparing host cells?

The selective activity of Gaegurin-6 against microbial membranes while exhibiting minimal toxicity toward host cells involves several sophisticated molecular mechanisms:

• Differential membrane composition targeting: Microbial membranes typically contain a higher proportion of negatively charged phospholipids (phosphatidylglycerol, cardiolipin) compared to mammalian cells, which predominantly display neutral phospholipids (phosphatidylcholine, sphingomyelin) on their outer leaflet. Gaegurin-6, being cationic, preferentially interacts with these negatively charged bacterial surfaces.

• Cholesterol sensitivity: The presence of cholesterol in mammalian membranes, which is absent in bacterial membranes, can inhibit the membrane-disruptive activities of antimicrobial peptides like Gaegurin-6. Cholesterol increases membrane rigidity and may prevent peptide insertion and subsequent pore formation.

• Threshold concentration effects: Antimicrobial peptides frequently exhibit a threshold concentration for activity that differs between microbial and host membranes. The peptide concentration required to disrupt host cell membranes is typically significantly higher than that needed for antimicrobial activity.

• Membrane potential sensitivity: The higher negative-inside membrane potential of bacteria compared to mammalian cells may enhance the driving force for cationic peptide accumulation at bacterial membranes, contributing to selectivity.

• pH-dependent activity modulation: As discussed previously, Gaegurin-6 exhibits pH-dependent activity profiles that may align with the microenvironments encountered during infection, optimizing antimicrobial efficacy while minimizing host cell damage .

Research with related peptides suggests that the C-terminal cyclic region stabilized by the disulfide bond (Rana box) may play a key role in this selectivity, potentially by optimizing membrane interaction specificity or stabilizing pore formation in a membrane composition-dependent manner .

What are the optimal analytical methods for characterizing the structure-function relationship of Gaegurin-6?

Comprehensive characterization of Gaegurin-6's structure-function relationship requires integration of multiple analytical approaches:

• Structural Analysis Techniques:

  • Circular Dichroism (CD) Spectroscopy: Essential for quantifying secondary structure content (α-helix, β-sheet) in solution and membrane-mimetic environments. Particularly valuable for monitoring structural changes induced by membrane interaction or disulfide bridge modification .

  • Nuclear Magnetic Resonance (NMR) Spectroscopy: Provides atomic-level structural information and can determine the three-dimensional structure in solution or membrane-mimetic environments.

  • Fourier Transform Infrared (FTIR) Spectroscopy: Complements CD by providing additional secondary structure information through amide band analysis.

  • Mass Spectrometry: Confirms peptide mass, sequence, and disulfide bridge integrity, particularly useful when combined with proteolytic digestion methods.

• Membrane Interaction Studies:

  • Lipid Vesicle Leakage Assays: Quantify membrane disruption using fluorophore-loaded liposomes of defined composition.

  • Surface Plasmon Resonance: Measures real-time binding kinetics to model membranes.

  • Isothermal Titration Calorimetry: Determines thermodynamic parameters of membrane binding.

  • Fluorescence Spectroscopy: Using intrinsic or introduced fluorophores to monitor conformational changes upon membrane interaction.

• Functional Assessment Methods:

  • Broth Microdilution Assays: Determine minimum inhibitory concentrations against various microbial strains.

  • Time-Kill Kinetics: Assess the rate of antimicrobial action.

  • Membrane Permeabilization Assays: Measure uptake of membrane-impermeable dyes by treated microorganisms.

  • Hemolytic Assays: Evaluate toxicity toward mammalian cells.

  • pH-Dependent Activity Profiling: Characterize activity across physiologically relevant pH ranges .

• Structure-Function Correlation:

  • Site-Directed Mutagenesis: Systematic modification of key residues to establish their contribution to activity.

  • Disulfide Bridge Modification: Compare native, reduced, and substituted variants to determine the role of the disulfide bridge .

  • Truncation Analysis: Identify the minimal peptide fragment required for activity.

Integration of data from these complementary techniques allows construction of a comprehensive model describing how specific structural features contribute to Gaegurin-6's antimicrobial mechanism and selectivity.

What experimental approaches can be used to investigate the synergistic potential of Gaegurin-6 with conventional antibiotics?

Investigating the synergistic potential of Gaegurin-6 with conventional antibiotics requires methodical approaches that address both the fundamental mechanisms and practical applications:

• In Vitro Synergy Screening:

  • Checkerboard Assays: The primary method for quantifying synergy, where Gaegurin-6 and antibiotics are tested in a matrix of concentrations. Results are analyzed using the fractional inhibitory concentration index (FICI), with values ≤0.5 indicating synergy.

  • Time-Kill Kinetics: Measures bacterial killing rates with combination treatments compared to individual agents, providing dynamic information on synergistic interactions.

  • Disk Diffusion Assays: A preliminary screening method where zones of inhibition are measured for combined versus individual treatments.

  • E-test Synergy Methods: Combines E-test strips to evaluate synergistic interactions through zone interpretation.

• Mechanism Investigation:

  • Membrane Permeabilization Assays: Determine if Gaegurin-6 increases antibiotic uptake through enhanced membrane permeability.

  • Biofilm Penetration Studies: Evaluate whether Gaegurin-6 facilitates antibiotic access to bacteria within biofilms.

  • Gene Expression Analysis: RNA-Seq or qPCR to identify changes in resistance gene expression when exposed to combinations.

  • Efflux Pump Inhibition Assays: Assess if Gaegurin-6 inhibits bacterial efflux pumps, increasing intracellular antibiotic concentration.

• Optimization Studies:

  • Sequence Modification: Systematic alteration of Gaegurin-6 to enhance synergistic interactions.

  • Formulation Development: Creation of delivery systems that maintain both peptide and antibiotic activity.

  • Exposure Timing Variations: Testing different sequences of administration (pre-treatment, simultaneous, or sequential).

  • pH and Ionic Strength Modulation: Identifying optimal environmental conditions for synergistic activity.

• Resistance Development Assessment:

  • Serial Passage Experiments: Comparing resistance development rates between single agents and combinations.

  • Mutant Prevention Concentration Determination: Measuring the antibiotic concentration that prevents the selection of resistant mutants when combined with Gaegurin-6.

  • Cross-Resistance Testing: Evaluating whether resistance to the combination confers resistance to other antimicrobial agents.

• Advanced Model Systems:

  • Ex Vivo Tissue Models: Testing combinations on infected tissue explants.

  • Hollow Fiber Infection Models: Simulating pharmacokinetic/pharmacodynamic parameters of combination therapy.

  • Galleria mellonella Infection Model: An invertebrate model for preliminary in vivo efficacy screening.

This multifaceted approach provides comprehensive insights into the potential of Gaegurin-6 to enhance conventional antibiotic efficacy, particularly against resistant pathogens.

How can molecular dynamics simulations contribute to understanding Gaegurin-6 membrane interactions?

Molecular dynamics (MD) simulations provide valuable atomic-level insights into Gaegurin-6 membrane interactions that complement experimental approaches. These computational methods offer unique contributions to understanding antimicrobial peptide function:

• Structural Dynamics Analysis:

  • Conformational Sampling: MD simulations can explore the conformational landscape of Gaegurin-6 in solution and membrane environments, capturing transitions between different structural states.

  • Disulfide Bridge Influence: Comparative simulations of native and modified peptides can elucidate how the disulfide bridge constrains peptide dynamics and influences membrane interactions.

  • pH-Dependent Structural Changes: Simulations with different protonation states can model pH-dependent conformational changes that affect antimicrobial activity .

• Membrane Interaction Characterization:

  • Binding Energy Calculations: Free energy methods like umbrella sampling or steered MD can quantify peptide-membrane binding energetics.

  • Insertion Depth Analysis: Simulations reveal how deeply Gaegurin-6 penetrates into membranes of different compositions.

  • Aggregation Behavior: MD can model peptide oligomerization on membrane surfaces, a critical step in pore formation.

  • Lipid Sorting Effects: Simulations can show how Gaegurin-6 may preferentially interact with specific lipid types, potentially explaining selectivity.

• Pore Formation Mechanisms:

  • Toroidal Pore Visualization: MD can model the formation of toroidal pores, showing how lipids bend to form the pore lining alongside peptide molecules .

  • Barrel-Stave Assembly: Simulations can reveal the structural arrangement of peptides in barrel-stave pores and the role of the disulfide-stabilized C-terminal region.

  • Water Permeation Pathways: MD can track water molecules and ions traversing peptide-induced membrane pores.

  • Pore Stability Assessment: Long-timescale simulations can evaluate the stability of different pore architectures.

• Advanced Simulation Approaches:

  • Coarse-Grained Modeling: Allows simulation of larger systems and longer timescales to capture collective phenomena like large-scale membrane disruption.

  • Constant-pH Molecular Dynamics: Specifically models pH-dependent effects by allowing protonation states to change during simulation.

  • Enhanced Sampling Methods: Techniques like replica exchange or metadynamics accelerate sampling of rare events such as membrane insertion or pore formation.

  • Multi-scale Modeling: Combines atomistic detail in regions of interest with coarse-grained representations elsewhere for computational efficiency.

• Integration with Experimental Data:

  • Validation with NMR Parameters: Simulation-derived order parameters can be compared with experimental NMR measurements.

  • Prediction of Fluorescence Properties: Simulations can model environmental effects on fluorescent probes for comparison with experimental fluorescence data.

  • Hypothesis Generation: MD results can suggest specific residues critical for function that can be tested through experimental mutagenesis.

Through these approaches, MD simulations provide a "computational microscope" that reveals molecular details of Gaegurin-6 action that are difficult or impossible to observe experimentally.

How can recombinant Gaegurin-6 be modified to enhance stability while maintaining antimicrobial activity?

Enhancing the stability of recombinant Gaegurin-6 while preserving its antimicrobial efficacy requires strategic modifications informed by structure-function relationships. Several approaches have demonstrated promise:

• Amino Acid Substitutions:

  • D-amino acid incorporation: Replacing L-amino acids with their D-enantiomers, particularly in non-critical regions, significantly enhances resistance to proteolytic degradation.

  • Strategic non-natural amino acid inclusion: Introduction of amino acids like β-amino acids or peptoid residues at key positions increases stability while maintaining membrane interactions.

  • Terminal modification: N-terminal acetylation or C-terminal amidation protects against exopeptidases and can stabilize secondary structure.

  • Helix-promoting substitutions: Introducing alanine or leucine at positions that enhance helical propensity can strengthen the active conformation.

• Disulfide Engineering:

  • Additional disulfide bridges: Introduction of strategically placed cysteines can create additional stabilizing constraints, particularly valuable in high-protease environments .

  • Diselenide substitution: Replacing the native disulfide with diselenide bonds increases stability while maintaining similar structural constraints.

  • Thioether bridges: Substituting disulfides with more stable thioether linkages provides enhanced resistance to reducing environments.

• Cyclization Strategies:

  • Backbone cyclization: Head-to-tail cyclization via peptide bond formation between N- and C-termini increases resistance to exopeptidases.

  • Stapled peptides: Introduction of hydrocarbon staples that link residues on the same face of the helix reinforces the α-helical structure.

  • Click chemistry cyclization: Using bioorthogonal chemistry to create non-native crosslinks between distant parts of the sequence.

• Formulation Approaches:

  • PEGylation: Conjugation with polyethylene glycol at non-critical residues enhances serum half-life and reduces proteolytic degradation.

  • Liposomal encapsulation: Incorporation into liposomes protects from proteases while potentially enhancing delivery to target membranes.

  • Nanoparticle conjugation: Attachment to nanoparticles can protect the peptide while maintaining surface availability for antimicrobial action.

• Hybrid Peptide Design:

  • Chimeric peptides: Fusion of Gaegurin-6 with complementary antimicrobial peptides to combine stability features.

  • Peptide-small molecule conjugates: Attachment of stabilizing moieties that additionally enhance membrane targeting.

  • Branched peptide architectures: Creation of dendrimeric structures with multiple copies of active regions.

Each modification strategy should be evaluated through systematic testing of:

• Antimicrobial activity spectrum and potency

• Resistance to various proteases (trypsin, chymotrypsin, proteases from target microorganisms)

• Stability under physiologically relevant conditions (temperature, pH, salt concentration)

• Specificity for microbial versus host cell membranes

• Membrane permeabilization efficiency

The optimal approach often involves combinations of these strategies, tailored to the specific application environment and target pathogens.

What potential exists for using Gaegurin-6 structure as a template for designing novel antimicrobial peptides?

The structural features of Gaegurin-6 provide a valuable template for rational design of next-generation antimicrobial peptides with enhanced properties. Several design strategies can leverage this template:

• Structure-Guided Minimization:

  • Identification of pharmacophore: Determining the minimal structural elements required for antimicrobial activity allows design of shorter, more economical peptides.

  • Truncation analysis: Systematic removal of terminal residues while maintaining the core α-helical structure and critical disulfide region.

  • Discontinuous epitope mimetics: Creating scaffolds that position key functional groups in the correct spatial orientation without requiring the full peptide backbone.

  • Cyclic peptide libraries: Generating constrained cyclic variants that maintain the active conformation with fewer residues.

• Selectivity Enhancement:

  • Charge distribution optimization: Fine-tuning the pattern of cationic residues to enhance microbial selectivity.

  • Hydrophobicity gradient engineering: Adjusting the hydrophobic moment to optimize membrane disruption while minimizing hemolytic activity.

  • Target-specific modifications: Incorporating moieties that recognize specific microbial surface components.

  • pH-responsive elements: Designing peptides with enhanced activity in specific microenvironments, such as acidic infection sites .

• Hybrid Peptide Design:

  • Domain fusion: Combining the membrane-active region of Gaegurin-6 with targeting domains from other peptides.

  • Multifunctional peptides: Integrating antimicrobial, anti-biofilm, and immunomodulatory properties.

  • Synergy-optimized variants: Designing peptides specifically engineered to enhance conventional antibiotic efficacy.

  • Membrane-translocating antimicrobials: Creating variants that can deliver additional cargo across microbial membranes.

• Non-Natural Modifications:

  • β-peptide incorporation: Introducing β-amino acids at strategic positions to enhance proteolytic stability.

  • Peptoid segments: Incorporating N-substituted glycine residues to resist proteolytic degradation.

  • Lipidation: Strategic attachment of lipid moieties to enhance membrane affinity and cellular uptake.

  • Fluorination: Introducing fluorinated amino acids to enhance stability and modify membrane interactions.

• Computational Design Approaches:

  • Machine learning optimization: Training algorithms on structure-activity relationships from Gaegurin-6 and related peptides to predict novel sequences with enhanced properties.

  • Molecular dynamics-guided design: Using simulation insights to identify modifications that enhance membrane interaction or pore formation.

  • De novo helix design: Creating novel helical scaffolds inspired by Gaegurin-6's structural features but with optimized stability and activity profiles.

  • Evolutionary algorithms: Applying in silico evolution to optimize peptide sequences for specific targets or conditions.

This template-based design approach can yield antimicrobial peptides with improved pharmaceutical properties, including enhanced stability, reduced production costs, improved safety profiles, and activity against resistant pathogens.

How might research on Gaegurin-6 contribute to understanding the evolution of antimicrobial peptides in amphibians with different sex chromosome systems?

Research on Gaegurin-6 offers a unique opportunity to explore the intersection of antimicrobial peptide evolution and sex chromosome diversity, particularly given the unusual sex determination systems in Glandirana rugosa. This species possesses both XX-XY and ZZ-ZW sex chromosome systems within the same species, making it an exceptional model for evolutionary studies .

• Comparative Genomics Approaches:

  • Sex-linked variation analysis: Investigating whether Gaegurin-6 genes show different patterns of variation in populations with XX-XY versus ZZ-ZW systems.

  • Chromosome location mapping: Determining if antimicrobial peptide genes are located on sex chromosomes or autosomes, and how this varies across populations.

  • Whole genome comparison: Leveraging the sequenced G. rugosa genome (7.08 Gb) to identify evolutionary patterns in antimicrobial peptide gene families across different sex determination systems .

  • Synteny analysis: Examining conservation of gene order and location between sex chromosomes and related species to track evolutionary history.

• Selection Pressure Assessment:

  • Molecular evolution analyses: Calculating selection ratios (dN/dS) for Gaegurin-6 genes across populations with different sex determination systems.

  • Divergence timing estimation: Determining when sequence divergence occurred relative to sex chromosome turnover events.

  • Regulatory element evolution: Examining whether expression control mechanisms differ between populations with different sex determination systems.

  • Copy number variation analysis: Investigating potential differences in gene duplication patterns between XX-XY and ZZ-ZW lineages.

• Expression Pattern Studies:

  • Sex-specific expression analysis: Determining whether Gaegurin-6 expression levels differ between males and females, and how this varies between populations with different sex determination systems.

  • Tissue distribution comparisons: Mapping expression patterns across skin and other tissues in different populations.

  • Developmental regulation: Examining whether sex hormone responsiveness of Gaegurin-6 genes differs between XX-XY and ZZ-ZW systems.

  • Environmental response patterns: Comparing how Gaegurin-6 expression responds to pathogen challenges across populations.

• Functional Diversification Analysis:

  • Activity spectrum comparison: Testing whether Gaegurin-6 variants from different populations show specialized activity against local pathogens.

  • Structural diversity assessment: Comparing the degree of sequence and structural variation in Gaegurin-6 across populations.

  • Biotope correlation: Relating antimicrobial peptide diversity to habitat differences between populations.

  • Microbiome interaction studies: Investigating how different Gaegurin-6 variants shape the skin microbiome in different populations.

• Evolutionary Model Integration:

  • Sex chromosome-antimicrobial peptide coevolution: Developing models to explain potential linkage between sex chromosome evolution and antimicrobial peptide diversification.

  • Sexual selection influences: Investigating whether antimicrobial peptide variation contributes to mate choice or reproductive isolation.

  • Genomic conflict resolution: Examining how antimicrobial peptide genes navigate potential genomic conflicts during sex chromosome turnover.

This research direction not only illuminates the evolution of amphibian immunity but also provides insights into the complex interplay between sex chromosome evolution and adaptive immune functions. Understanding these patterns may reveal broader principles about how genomic architecture influences the evolution of defense systems across vertebrates.