Recombinant Rana temporaria Temporin-B

What is Temporin-B and where is it naturally found?

Temporin-B (TB) is an antimicrobial peptide belonging to the temporin family, naturally secreted from the skin of the common European frog Rana temporaria, particularly under stressful conditions. Temporins are important components of the amphibian innate immune system, protecting against microbial infections. R. temporaria is a semi-aquatic amphibian of the Ranidae family, which has the highest distribution among all frog families throughout European regions .

Temporins are synthesized as precursors with a highly conserved N-terminal domain and acidic propeptide region containing a Lys-Arg processing site. The mature temporin peptide is generated through post-translational processing, including cleavage at this site. Typically, the Glycine residue present at the C-terminus serves as a substrate for peptidyl-glycine α-amidating monooxygenase to produce C-terminally amidated temporins, which contributes to their antimicrobial activity .

What are the structural characteristics of Temporin-B?

Temporin-B is a short antimicrobial peptide with several distinct structural features:

• Typically 10-14 amino acids in length

• Predominantly adopts an amphipathic α-helical structure when interacting with bacterial membranes

• Post-translationally amidated at the C-terminus, enhancing stability and activity

• Possesses a low net positive charge due to the presence of only one or two basic residues

• Relatively high hydrophobicity compared to other antimicrobial peptide families

The amphipathic nature of Temporin-B is crucial for its antimicrobial mechanism, as it allows the hydrophobic residues to interact with membrane lipids while the positively charged residues interact with negatively charged components of bacterial membranes . The peptide's relatively simple sequence makes it an excellent candidate for structure-function studies and targeted modifications to enhance specific properties.

What is the antimicrobial spectrum of native Temporin-B?

Native Temporin-B exhibits a relatively narrow antimicrobial spectrum compared to some other antimicrobial peptides:

• Generally more active against Gram-positive bacteria, including Staphylococcus and Streptococcus species

• Typical minimum inhibitory concentrations (MICs) range from 2.5-20 μM for Gram-positive bacteria

• Limited activity against Gram-negative bacteria due to their complex outer membrane structure

• Modest efficacy against methicillin-resistant Staphylococcus aureus (MRSA) when used alone

• Limited antifungal activity

This restricted spectrum has motivated extensive research into modified Temporin-B variants with enhanced activity profiles, particularly against Gram-negative bacteria which are increasingly associated with antibiotic resistance . The relatively narrow spectrum of native Temporin-B makes it an ideal template for structural modifications aimed at broadening antimicrobial activity while maintaining its favorable properties.

What expression systems are effective for recombinant production of Temporin-B?

Several expression systems have been developed for the recombinant production of Temporin-B, each with unique advantages:

• E. coli-based expression systems:

  • IMPACT-TWIN plasmid expression vector system that utilizes intein-mediated purification via affinity chitin-binding tags

  • Fusion protein approaches using partners such as thioredoxin, SUMO, or glutathione S-transferase

  • Intein self-cleavage systems that enable obtaining the native peptide without additional amino acids

• Plant-based expression systems:

  • Transient expression in tobacco leaves using specific fusion constructs

  • Stable transgenic barley lines with grain-specific promoters

The choice of expression system depends on research objectives, required yield, and downstream applications. The E. coli system remains most widely used due to its simplicity, cost-effectiveness, and potential for high yields, while plant-based systems offer advantages for scale-up and certain post-translational modifications . Each system requires optimization of expression conditions, purification strategies, and verification methods to ensure production of functional Temporin-B.

What challenges exist in recombinant expression of Temporin-B?

Recombinant expression of Temporin-B faces several significant challenges:

• Toxicity to host cells: The antimicrobial activity of Temporin-B can be toxic to expression hosts, particularly E. coli, limiting viable expression and yield.

• Proteolytic degradation: Small peptides like Temporin-B are highly susceptible to degradation by host proteases, reducing recovery of intact peptide.

• Improper folding: Ensuring correct secondary structure formation can be challenging in recombinant systems, affecting bioactivity.

• Formation of inclusion bodies: High-level expression often leads to aggregation and inclusion body formation, necessitating refolding steps.

• Post-translational modifications: Native Temporin-B is C-terminally amidated, which is difficult to achieve in bacterial expression systems .

• Obtaining the native sequence: Expression often results in fusion proteins requiring additional processing steps to release the native peptide without extra amino acids .

Most of these challenges are addressed using fusion protein strategies, where Temporin-B is expressed as part of a larger protein to improve stability and reduce toxicity, followed by specific cleavage methods to release the native peptide. These approaches require careful optimization of expression conditions, cleavage efficiency, and recovery protocols.

How do sequence modifications affect the antimicrobial activity of Temporin-B?

Sequence modifications of Temporin-B can dramatically alter its antimicrobial profile:

A notable example is TB_KKG6A, which displays anti-Pseudomonas aeruginosa activity at 5 μM concentration while the native peptide shows no activity against this pathogen . This demonstrates how strategic sequence modifications can create temporin derivatives with broader antimicrobial spectra and enhanced potency against specific pathogens.

What structural features correlate with enhanced antimicrobial activity in Temporin-B analogues?

Several structural features have been identified that correlate with enhanced antimicrobial activity in Temporin-B analogues:

These structural features allow for rational design of Temporin-B analogues with specific activity profiles against different bacterial pathogens, including multi-drug resistant strains that are increasingly problematic in clinical settings.

How does Temporin-B interact with bacterial membranes at the molecular level?

Temporin-B's interaction with bacterial membranes is complex and has been studied using various techniques including molecular dynamics simulations, circular dichroism, solid-state NMR, and patch clamp experiments:

• Initial attraction and binding:

  • Electrostatic attraction between the positively charged peptide and negatively charged bacterial membrane components

  • Initial binding is followed by conformational changes, with the peptide adopting an α-helical structure

• Membrane insertion mechanism:

  • Insertion of hydrophobic residues into the lipid bilayer

  • The depth and angle of insertion depend on the peptide's amphipathicity and the membrane composition

• Pore formation and membrane disruption:

  • At sufficient concentrations, Temporin-B can form transient or stable pores

  • Channel conductance studies show that native Temporin-B can trigger ion channel-like activity

  • Modified versions may have altered pore-forming capabilities; for example, enhancing the cationicity of the N-terminus can abrogate channel conductance

• Differential interaction with bacterial types:

  • Interaction with Gram-positive bacteria typically involves direct binding to the cytoplasmic membrane

  • Interaction with Gram-negative bacteria requires penetration or disruption of the outer membrane before accessing the inner membrane

These molecular interactions explain why relatively modest modifications to Temporin-B's primary sequence can induce substantial changes in potency and spectrum of activity by altering the nature of membrane interaction .

What synergistic effects are observed between Temporin-B and other antimicrobial peptides?

Temporin-B exhibits significant synergistic effects when combined with other antimicrobial peptides:

• Synergy with Temporin L:

  • Temporin B forms hetero-oligomers with Temporin L

  • This combination enhances cooperativity of antibacterial activity against EMRSA-15 (epidemic methicillin-resistant Staphylococcus aureus)

  • Temporin B modifies the pharmacodynamic profile of Temporin L, improving its killing efficacy

• Synergy with modified temporins:

  • TB-KK (Temporin B with additional lysines at the N-terminus) acts synergistically with Temporin A against both Gram-positive and Gram-negative bacteria

  • This synergy has been demonstrated both in vitro and in vivo models

• Synergy with other antimicrobial peptides:

  • TB-KK in combination with an analog of royal jellein I (RJI-C), an antimicrobial peptide from bee jelly, shows strong activity against S. epidermidis while sparing probiotic bacteria

The mechanisms underlying these synergistic interactions may include complementary membrane targeting, enhanced pore formation through hetero-oligomerization, improved access to target membranes, and sequential attack on different bacterial targets. These findings suggest combination therapy approaches using Temporin-B with other antimicrobial peptides could be more effective than monotherapy, potentially reducing required dosages and minimizing side effects .

What methods are used to assess the antimicrobial activity of recombinant Temporin-B?

Several standardized and specialized methods are used to assess the antimicrobial activity of recombinant Temporin-B:

• Minimum Inhibitory Concentration (MIC) determination:

  • Broth microdilution method: Bacteria are exposed to serial dilutions of the peptide in growth medium

  • The MIC is defined as the lowest concentration that prevents visible bacterial growth after incubation

  • This is typically performed against standard strains as well as clinical isolates

• Time-kill kinetics:

  • Bacteria are exposed to the peptide at different concentrations over time

  • Samples are taken at various time points, diluted, and plated to count viable bacteria

  • This reveals the rate of bacterial killing and whether the peptide is bacteriostatic or bactericidal

• Anti-biofilm activity assessment:

  • Crystal violet staining to quantify biofilm formation in the presence of the peptide

  • Confocal microscopy to visualize biofilm structure and bacterial viability within biofilms

  • Quantification of biofilm inhibition and biofilm eradication activities

• Membrane permeabilization assays:

  • Fluorescent dye uptake (e.g., SYTOX Green, propidium iodide) to measure membrane integrity

  • Membrane potential-sensitive dyes to assess depolarization effects

  • Liposome leakage assays using fluorescent dye-loaded vesicles

• In vivo efficacy studies:

  • Insect larvae models (e.g., Galleria mellonella) for preliminary in vivo efficacy

  • Assessment of bacterial burden in infected tissues after peptide treatment

These methods provide comprehensive data on the antimicrobial properties, mechanism of action, and potential therapeutic applications of recombinant Temporin-B and its analogues.

How can molecular dynamics simulations contribute to Temporin-B research?

Molecular dynamics (MD) simulations provide powerful insights into Temporin-B interactions with membranes at the atomic and molecular levels:

• Setting up the simulation system:

  • Modeling of Temporin-B peptide structure, typically as an α-helix

  • Creation of model membranes representing bacterial or mammalian cell membranes

  • Inclusion of water molecules and ions to mimic physiological conditions

  • Positioning of the peptide in relation to the membrane

• Key observations and analyses:

  • Peptide-membrane binding energy calculations

  • Tracking peptide insertion depth and orientation in the membrane

  • Monitoring conformational changes in both peptide and membrane

  • Quantifying water penetration and membrane thinning or disruption

  • Analyzing potential pore formation and ion/water permeation

• Comparing different Temporin-B variants:

  • Simulating native Temporin-B alongside modified analogues

  • Correlating simulation results with experimental antimicrobial and hemolytic activities

  • Identifying molecular determinants of enhanced or altered activity

MD simulations have revealed that even closely related Temporin-B analogues may interact with membranes through fundamentally different mechanisms, explaining their distinct antimicrobial profiles against different bacterial types . This approach has proven invaluable for rational design of improved analogues based on molecular interaction patterns, reducing the need for extensive trial-and-error in peptide design.

What anti-inflammatory properties have been observed for Temporin-B derivatives?

Several Temporin-B derivatives have demonstrated significant anti-inflammatory properties in addition to their antimicrobial activities:

• TB_KKG6A:

  • Inhibits P. aeruginosa-induced upregulation of pro-inflammatory cytokine genes in cystic fibrosis bronchial cells (IB3-1 cell line)

  • Significantly reduces expression of IL-8, a key neutrophil chemoattractant

  • Inhibits expression of other pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α

  • Active at 5 μM concentration, showing both anti-P. aeruginosa activity and anti-inflammatory effects

• TB-KK combined with RJI-C (royal jellein I analog):

  • Down-regulates the levels of pro-inflammatory cytokines TNF-α and IFN-γ in cells stimulated with lipopolysaccharide (LPS)

  • Enhances expression of the anti-inflammatory cytokine IL-10

  • Effects comparable to those of gentamicin, a conventional antibiotic used in cystic fibrosis patients

The mechanisms of anti-inflammatory action may include:

• Direct binding to bacterial LPS, neutralizing its pro-inflammatory effects

• Modulation of intracellular signaling pathways leading to reduced pro-inflammatory gene expression

• Dual antimicrobial and anti-inflammatory activities through the peptide's ability to bind bacterial components like LPS

These anti-inflammatory properties add significant value to Temporin-B derivatives as potential therapeutic agents, offering a dual benefit that conventional antibiotics typically lack, particularly for conditions like cystic fibrosis where both infection and inflammation contribute to pathology .

How do Temporin-B analogues compare in terms of therapeutic index?

The therapeutic index (TI), representing the ratio between the concentration causing mammalian cell toxicity and the effective antimicrobial concentration, is a critical parameter for evaluating the potential clinical utility of Temporin-B analogues:

• Native Temporin-B:

  • Generally shows low hemolytic activity

  • Limited activity against Gram-negative bacteria

  • Moderate activity against Gram-positive bacteria

  • Favorable therapeutic index for Gram-positive infections

• Modified analogues with increased cationicity (e.g., TB_KK, TB_KKG6A):

  • Enhanced activity against both Gram-positive and Gram-negative bacteria

  • Typically show slightly increased hemolytic activity compared to native Temporin-B

  • Still maintain favorable therapeutic indices, particularly against Gram-negative bacteria

  • TB_KKG6A shows anti-P. aeruginosa activity at 5 μM while maintaining acceptable cytotoxicity profiles

• Lysine-rich analogues (e.g., 6K-WY2, 6K-1426):

  • 8-64 fold more potent against Gram-negative bacteria than parent peptides

  • Show more rapid killing kinetics

  • Despite slightly increased hemolytic activity and cytotoxicity, they demonstrate improved therapeutic indices

  • Enhanced antiproliferation activities against cancer cell lines (>100-fold more potent than parent peptides)

The goal in designing Temporin-B analogues is to maximize the therapeutic index by enhancing antimicrobial activity while minimizing cytotoxicity. This often involves strategic modifications that enhance selective interaction with bacterial membranes over mammalian cell membranes, as demonstrated by the successful engineering of analogues like QUB-1426 and 6K-WY2 .

What are the major obstacles in translating Temporin-B research to clinical applications?

Several significant challenges must be overcome to translate Temporin-B research into clinical applications:

• Production challenges:

  • Scaling up recombinant production for clinical applications

  • Achieving consistent post-translational modifications, particularly C-terminal amidation

  • Reducing production costs to make therapeutic applications economically viable

• Stability and delivery issues:

  • Susceptibility to proteolytic degradation in vivo

  • Limited systemic half-life

  • Challenges in delivering peptides to infection sites, particularly for systemic infections

  • Need for formulations that enhance stability without compromising activity

• Resistance concerns:

  • Potential for resistance development with widespread use

  • Limited understanding of resistance mechanisms specific to Temporin-B

  • Need for studies on resistance development to guide rational design of resistance-proof analogues

• Regulatory hurdles:

  • Complex regulatory pathway for peptide-based antimicrobials

  • Need for comprehensive safety and efficacy data

  • Limited precedent for antimicrobial peptide therapeutics in clinical practice

Addressing these challenges requires multidisciplinary approaches combining expertise in peptide chemistry, molecular biology, pharmaceutical formulation, and clinical medicine. Despite these obstacles, the pressing need for new antimicrobial agents in the face of increasing resistance to conventional antibiotics continues to drive research into Temporin-B and other antimicrobial peptides as potential therapeutic solutions .

What future research directions are most promising for Temporin-B?

Several promising research directions could advance the development of Temporin-B as a therapeutic agent:

• Optimized expression systems:

  • Development of more efficient bacterial or eukaryotic expression systems

  • Novel fusion partners that enhance expression while facilitating purification

  • Scalable production methods suitable for clinical development

• Rational design of multi-functional analogues:

  • Peptides combining enhanced antimicrobial activity with anti-inflammatory properties

  • Temporin-B derivatives with targeted activity against specific pathogens

  • Analogues with enhanced stability and reduced cytotoxicity

• Delivery systems and formulations:

  • Nanoparticle-based delivery systems for targeted release

  • Topical formulations for skin and wound infections

  • Inhalation formulations for respiratory infections, particularly for cystic fibrosis

• Combination approaches:

  • Further exploration of synergistic combinations with other antimicrobial peptides

  • Combinations with conventional antibiotics to enhance efficacy and reduce resistance

  • Development of optimized peptide cocktails for specific clinical applications

• Anti-biofilm applications:

  • Enhanced anti-biofilm formulations for medical device coatings

  • Temporin-B derivatives specifically engineered for biofilm penetration

  • Combination strategies for biofilm eradication

The most promising approach likely involves developing Temporin-B not as a standalone therapeutic but as a component of combination therapies or specialized formulations targeting specific clinical needs where conventional antibiotics are failing due to resistance or biofilm formation .

What makes Temporin-B a valuable research subject in antimicrobial peptide development?

Temporin-B represents a valuable research subject in antimicrobial peptide development for several compelling reasons:

• Structural simplicity with functional complexity:

  • The relatively short sequence of Temporin-B makes it amenable to chemical synthesis and recombinant production

  • Despite its simplicity, it displays complex membrane interactions and antimicrobial mechanisms

  • The straightforward structure facilitates structure-activity relationship studies

• Modifiable template:

  • Temporin-B serves as an excellent template for rational design of improved antimicrobial peptides

  • Small modifications can dramatically alter activity spectrum and potency

  • Strategic alterations can create multi-functional peptides with both antimicrobial and immunomodulatory properties

• Dual antimicrobial and anti-inflammatory activity:

  • Modified versions like TB_KKG6A exhibit both direct antimicrobial effects and anti-inflammatory properties

  • This dual functionality offers advantages over conventional antibiotics, particularly for conditions with inflammatory components

• Synergistic potential:

  • Demonstrated synergy with other antimicrobial peptides opens possibilities for combination therapies

  • The ability to form hetero-oligomers with other temporins creates unique opportunities for peptide engineering

• Activity against resistant pathogens:

  • Modified temporins can target multi-drug resistant bacteria through mechanisms distinct from conventional antibiotics

  • The membrane-disrupting action provides a high barrier to resistance development

These characteristics position Temporin-B as a valuable model for developing novel antimicrobial strategies to address the growing crisis of antimicrobial resistance, particularly for difficult-to-treat infections caused by biofilm-forming or multi-drug resistant pathogens .