Recombinant Lactobacillus paracasei Bacteriocin lactocin-705

What is the molecular structure of Lactobacillus paracasei Bacteriocin lactocin-705?

Lactocin-705 is a two-component bacteriocin whose activity depends on the complementary action of two peptides: Lac705α and Lac705β. Each peptide consists of 33 amino acid residues . The Lac705α peptide has a molecular formula of C152H242N44O40S, with a molecular weight of 3357.8 Da, and its sequence is GMSGYIQGIPDFLKGYLHGISAANKHKKGRL . The Lac705β peptide has the sequence GFWGGLGYIAGRVGAAYGHAQASANNHHSPING . These peptides have isoelectric points of pI = 9.87 for Lac705α and pI = 8.61 for Lac705β . Both peptides are required to work in concert, as neither displays bacteriocin activity independently when tested against sensitive bacterial strains .

How do the two peptide components of lactocin-705 work together?

The two peptides of lactocin-705 function synergistically, with an optimal Lac705α/Lac705β peptide ratio of 1:4 for maximum inhibitory effect . FTIR spectroscopy studies reveal that these peptides interact differently with bacterial membranes. Lac705α slightly increases the melting temperature of DPPC bilayers by approximately 2°C, while Lac705β significantly modifies the melting temperature from 42°C to 33°C . Additionally, Lac705β disturbs the gel phase organization of the membrane, decreasing the conformational order of lipid acyl chains .

When both peptides are present together, they form a transmembrane oligomer that creates pores in the cytoplasmic membrane of target bacteria . This pore formation leads to the dissipation of both membrane potential (ΔΨ) and pH gradient (ΔpH), resulting in the efflux of intracellular ions such as K+ and inorganic phosphate . This combined action effectively kills target bacteria by disrupting their membrane integrity and cellular homeostasis.

What is the mechanism of action of lactocin-705 against sensitive bacteria?

Lactocin-705 exerts its antimicrobial effect through a membrane-disrupting mechanism. When added to sensitive cells like Lactobacillus plantarum CRL691 at a concentration of 90 nmol/l, it dissipates both the membrane potential (ΔΨ) and the pH gradient (ΔpH) . This action is particularly effective against energized membranes, such as those obtained after the addition of glucose, suggesting that the presence of a proton motive force (PMF) enhances the bacteriocin's interaction with the cytoplasmic membrane .

The disruption of membrane integrity leads to the immediate release of intracellular K+ and inorganic phosphate through the pores formed by the bacteriocin . Interestingly, when various ions were tested for their effect on this process, only Ca2+ ions exhibited a protective effect against lactocin-705 activity . This suggests that calcium may interfere with the bacteriocin's ability to bind to or disrupt the membrane.

FTIR spectroscopy provides evidence that Lac705α and Lac705β peptides form a transmembrane oligomer, creating channels that allow ion efflux and disrupt cellular homeostasis . The ability of these peptides to reorganize and insert into membranes is a critical aspect of their killing mechanism.

How does the membrane interaction of Lac705α differ from Lac705β based on biophysical studies?

FTIR spectroscopy reveals significant differences in how the two peptide components interact with membrane models:

In contrast, Lac705β demonstrates much more pronounced membrane interactions. It significantly decreases the melting temperature of DPPC from 42°C to 33°C and disturbs the gel phase organization . The wave numbers of methylene stretching bands are higher in the presence of Lac705β compared to pure DPPC bilayers, indicating a decrease in the conformational order of lipid acyl chains . Furthermore, Lac705β undergoes substantial conformational reorganization upon interaction with DPPC vesicles, with its FTIR band shifting from 1,620 cm-1 to 1,628 cm-1, suggesting different hydrogen bond patterns in hydrophobic environments .

These differences suggest a model where Lac705β inserts more deeply into the membrane core, while Lac705α interacts more with the polar headgroup region. Together, they form a functional transmembrane pore that disrupts membrane integrity.

What analytical methods are most effective for studying lactocin-705's interaction with bacterial membranes?

FTIR spectroscopy has proven particularly valuable for studying lactocin-705's interaction with membranes. This technique allows researchers to monitor several key parameters:

• CH2 stretching vibrations (2,800-3,000 cm-1): These bands, dominated by peaks at approximately 2,920 cm-1 and 2,850 cm-1, provide information about the antisymmetric and symmetric methylene stretching modes respectively . By tracking the wave number shifts of these modes as a function of temperature, researchers can monitor lipid phase transitions and detect changes in membrane fluidity caused by the bacteriocin peptides .

• Ester carbonyl stretching (C=O): Analysis of this band, composed of two overlapped components near 1,745 and 1,730 cm-1, provides insights into the hydration level of the polar-apolar interface of lipid bilayers . Changes in the relative intensity of these components can indicate alterations in water accessibility to the membrane interface.

• Amide I' region: This spectral region (1,600-1,700 cm-1) offers valuable information about peptide secondary structure and conformational changes upon membrane interaction . The shift of Lac705β's band from 1,620 cm-1 to 1,628 cm-1 upon interaction with DPPC vesicles reveals important conformational reorganization .

Other complementary techniques include differential scanning calorimetry to correlate with phase transition data, membrane potential measurements using fluorescent probes to monitor changes in membrane potential (ΔΨ) and pH gradient (ΔpH), and ion flux measurements to track the efflux of intracellular K+ and other ions.

How can recombinant expression systems be optimized for lactocin-705 production?

Optimizing recombinant expression of lactocin-705 requires addressing several key challenges:

• Host selection: Expression hosts should be chosen based on their ability to correctly process the two peptide components. While E. coli systems offer high yield and well-established protocols, lactic acid bacteria hosts like Lactococcus lactis may provide more native-like processing with efficient secretion systems.

• Expression strategy for dual peptides: Since lactocin-705 requires both Lac705α and Lac705β in a specific ratio (1:4), expression systems must be designed accordingly . Options include:

  • Dual plasmid systems with different selection markers

  • Bicistronic constructs with optimized spacing

  • Separate production of each peptide followed by mixing at the optimal ratio

• Codon optimization: Adapting the coding sequences to the preferred codon usage of the expression host can significantly improve yields, especially for hosts with different GC content than the native producer.

• Fusion partners and tags: Strategic use of fusion proteins (such as SUMO, thioredoxin, or His-tags) can enhance solubility, facilitate purification, and potentially protect against degradation during expression.

• Post-expression considerations: Given the membrane-active nature of these peptides, extraction and purification protocols must be optimized to maintain their structure and activity. Ion exchange chromatography might be particularly effective due to the basic nature of both peptides (pI = 9.87 for Lac705α and pI = 8.61 for Lac705β) .

The choice of expression system should align with the specific research goals, whether focused on structural characterization, activity testing, or potential therapeutic applications.

Why does Ca2+ provide a protective effect against lactocin-705 activity?

The observation that Ca2+ ions exhibit a protective effect against lactocin-705 activity provides an interesting insight into the bacteriocin's mechanism . Several potential explanations can be proposed:

• Membrane stabilization: Ca2+ ions can bind to negatively charged phospholipid headgroups in bacterial membranes, increasing membrane rigidity and reducing fluidity. This stabilized membrane may be less susceptible to pore formation by lactocin-705.

• Electrostatic interference: Since Lac705α has a relatively high isoelectric point (pI = 9.87) , it likely carries positive charges that facilitate interaction with negatively charged bacterial membranes. Ca2+ might compete for these binding sites or shield negative charges on the membrane surface, interfering with the initial electrostatic attraction between the bacteriocin and the membrane.

• Effect on membrane potential: Ca2+ influx can influence membrane potential, potentially counteracting the bacteriocin's ability to dissipate the proton motive force. Since energized membranes are more susceptible to lactocin-705 , Ca2+-induced changes in membrane energetics might reduce sensitivity.

• Direct interaction with bacteriocin: Ca2+ might directly interact with the bacteriocin peptides, inducing conformational changes that reduce their ability to form functional pores.

Understanding this protective mechanism could provide valuable insights for optimizing lactocin-705 activity in different environmental conditions and inform potential applications in food preservation or therapeutic contexts.

What purification strategies yield the highest purity of recombinant lactocin-705?

Purifying recombinant lactocin-705 to high purity requires strategies that address the unique properties of both peptide components. A comprehensive purification workflow might include:

• Initial extraction methods:

  • For intracellular expression: Cell lysis using sonication or other mechanical methods, followed by clarification of lysate

  • For secreted expression: Ammonium sulfate precipitation from culture supernatant or acid extraction methods (pH 2-3) to exploit bacteriocin acid stability

• Chromatographic separation techniques:

  • Ion exchange chromatography: Cation exchange for both peptides, particularly effective due to their basic nature (pI = 9.87 for Lac705α and pI = 8.61 for Lac705β)

  • Hydrophobic interaction chromatography: Exploits the hydrophobic regions in bacteriocin peptides

  • Reverse phase HPLC: C18 or C8 columns for final polishing step, enabling separation of Lac705α from Lac705β for specific ratio formulation

• Two-track purification approach:

  • Purify Lac705α and Lac705β separately if expressed individually

  • Combine at 1:4 ratio (α:β) after purification for optimal activity

• Quality control assessments:

  • Tricine-SDS-PAGE for better resolution of small peptides

  • Mass spectrometry (MALDI-TOF or ESI-MS) for exact mass determination

  • Activity testing against indicator strain L. plantarum CRL691

This systematic approach allows researchers to obtain highly pure recombinant lactocin-705 peptides at the optimal ratio for antimicrobial activity studies.

How can the antimicrobial activity of recombinant lactocin-705 be quantified?

Quantifying the antimicrobial activity of recombinant lactocin-705 requires reliable and reproducible methods:

• Agar diffusion assays:

  • Create wells in agar seeded with indicator strain (e.g., L. plantarum CRL691)

  • Add samples containing recombinant lactocin-705 at the optimal 1:4 ratio of Lac705α to Lac705β

  • Measure zones of inhibition after incubation

  • Compare with standard curve of known bacteriocin concentrations

• Broth dilution assays:

  • Prepare serial dilutions of recombinant lactocin-705

  • Add standardized inoculum of indicator strain

  • Determine lowest concentration preventing visible growth (Minimum Inhibitory Concentration, MIC)

  • Express as Arbitrary Units (AU) per mL or μg/mL

• Membrane disruption assays:

  • Measure dissipation of membrane potential (ΔΨ) using fluorescent dyes

  • Monitor pH gradient (ΔpH) disruption

  • Track K+ efflux using atomic absorption spectroscopy or ion-selective electrodes

  • Quantify inorganic phosphate release

• Standardization considerations:

  • Test the peptides at the optimal 1:4 ratio (Lac705α:Lac705β)

  • Include positive controls (e.g., native lactocin-705) and negative controls

  • Test activity against energized cells (after glucose addition) for maximum sensitivity

  • Consider the protective effect of Ca2+ ions in the test medium

These methodologies provide a comprehensive framework for quantifying the antimicrobial activity of recombinant lactocin-705, enabling comparison between different production batches and with native bacteriocin preparations.

What applications of lactocin-705 in gastrointestinal health research are most promising?

While the provided search results don't specifically address lactocin-705's applications in gastrointestinal health, they do discuss the broader potential of LAB-bacteriocins in this context . These insights can be extended to lactocin-705 research:

• Pathogen inhibition: Bacteriocins from LAB have shown strong activity against gastrointestinal pathogens, including Helicobacter pylori, Clostridium species, and Listeria species . Lactocin-705's membrane-disrupting mechanism could be effective against these pathogens while potentially sparing beneficial microbiota.

• Microbiome modulation: LAB-bacteriocins can influence gut microbial communities, particularly anaerobic bacteria of Clostridium, Bacteroides, and Bifidobacterium species . Studying lactocin-705's spectrum of activity against various gut microbes could reveal its potential for selective modulation of the microbiome.

• Promotion of SCFA production: Short-chain fatty acids (SCFAs) are beneficial metabolic products that strengthen gut barrier function and regulate inflammatory responses . Research could explore whether lactocin-705 treatment indirectly promotes SCFA production by altering the gut microbiota composition.

• Therapeutic applications: LAB-bacteriocins have been investigated for treating various gastrointestinal disorders, including H. pylori infections, inflammatory bowel disease, and colon cancer . Lactocin-705's unique two-peptide nature might offer advantages in these applications.

• Delivery strategies: For gastrointestinal applications, research should address how to protect lactocin-705 from digestive enzymes and ensure it reaches its target site. Options include encapsulation technologies, biofilm-based delivery, or engineering probiotic strains to produce lactocin-705 in situ.

Methodologically, these applications would require in vitro models of gastrointestinal conditions, ex vivo tissue studies, and eventually in vivo animal models to assess efficacy and safety before human trials.

How can FTIR spectroscopy be used to study the conformational changes of lactocin-705 peptides upon membrane interaction?

FTIR spectroscopy provides valuable insights into the conformational changes of lactocin-705 peptides when they interact with membranes. A methodological approach includes:

• Sample preparation:

  • Create model membrane systems such as DPPC (dipalmitoylphosphatidylcholine) vesicles

  • Prepare pure Lac705α and Lac705β peptides individually and in combination

  • Form peptide-lipid complexes at various ratios

  • Use D₂O (deuterated water) to avoid interference in critical spectral regions

• Key spectral regions to analyze:

  • CH₂ stretching vibrations (2,800-3,000 cm⁻¹): These bands monitor lipid acyl chain order and can reveal how peptides affect membrane fluidity

  • Ester carbonyl stretching (1,700-1,750 cm⁻¹): Changes in these bands indicate alterations in interfacial hydration

  • Amide I' band (1,600-1,700 cm⁻¹): This region reveals peptide secondary structure changes

• Temperature-dependent studies:

  • Monitor CH₂ stretching frequency as a function of temperature

  • Determine lipid phase transition temperatures (Tm)

  • Compare pure lipid systems with those containing peptides

This approach has revealed important insights about lactocin-705, including:

• Lac705β modifies the Tm of DPPC from 42°C to 33°C

• Lac705β undergoes conformational reorganization upon membrane interaction, with its band shifting from 1,620 cm⁻¹ to 1,628 cm⁻¹

• Lac705α affects the hydration of polar groups in the membrane

These findings help explain the molecular basis for how these peptides form functional pore complexes in bacterial membranes.

What is the optimal peptide ratio of Lac705α to Lac705β for maximum antimicrobial activity?

Research has determined that the optimal ratio of Lac705α to Lac705β for maximum antimicrobial activity against the indicator strain Lactobacillus plantarum CRL691 is 1:4 . This non-equimolar ratio suggests that the two peptides play different but complementary roles in the pore formation process.

Several factors may explain this ratio requirement:

• Structural considerations: The transmembrane oligomer formed by both peptides likely incorporates multiple Lac705β molecules for each Lac705α peptide to create a stable and functional pore complex.

• Differential membrane interaction: FTIR data shows that Lac705β modifies membrane properties more significantly than Lac705α, decreasing the melting temperature of DPPC from 42°C to 33°C and disrupting acyl chain ordering . This suggests that multiple Lac705β molecules may be needed to sufficiently disrupt the membrane for each Lac705α.

• Conformational changes: Lac705β undergoes substantial conformational reorganization upon interaction with lipid bilayers, as evidenced by the shift of its FTIR band from 1,620 cm⁻¹ to 1,628 cm⁻¹ . This reorganization may require a higher concentration of Lac705β to ensure sufficient properly-conformed peptide is available.

For recombinant production and experimental applications, maintaining this optimal 1:4 ratio is crucial for achieving maximum antimicrobial activity. Expression systems should be designed with this ratio in mind, or the peptides should be purified separately and combined at the appropriate ratio.

What are the most significant knowledge gaps in lactocin-705 research?

Despite significant progress in understanding lactocin-705, several important knowledge gaps remain:

• Detailed three-dimensional structure: While FTIR studies have provided valuable insights into conformational changes upon membrane interaction , the precise three-dimensional structure of the Lac705α and Lac705β peptides, both individually and in their functional oligomeric complex, remains to be determined. Techniques such as NMR spectroscopy or X-ray crystallography could address this gap.

• Receptor specificity: The search results don't specify whether lactocin-705 targets specific receptors on sensitive bacteria or if its activity is solely due to direct membrane interactions. Some bacteriocins target specific receptors or intermediate molecules in cell wall biosynthesis .

• Complete spectrum of activity: While Lactobacillus plantarum CRL691 is identified as a sensitive strain , the complete spectrum of bacterial species susceptible to lactocin-705 remains undefined. A comprehensive analysis of activity against diverse bacterial species, including pathogens, would be valuable.

• In vivo efficacy and safety: The search results focus primarily on in vitro studies. The effectiveness and safety of lactocin-705 in in vivo systems remains to be thoroughly investigated, particularly for potential applications in food preservation or therapeutic use.

• Recombinant production optimization: While the search results provide insights into the peptides' properties, specific strategies for efficient recombinant production of active lactocin-705 are not detailed. Optimizing expression systems, purification protocols, and formulation methods would advance research and potential applications.

Addressing these knowledge gaps would significantly advance our understanding of lactocin-705 and expand its potential applications in research, food preservation, and potentially therapeutic contexts.

How might recombinant lactocin-705 be engineered for enhanced stability and activity?

Based on current knowledge about lactocin-705's structure and function, several engineering strategies could enhance its stability and activity:

• Terminal modifications:

  • N-terminal acetylation to prevent aminopeptidase degradation

  • C-terminal amidation to enhance stability

  • These modifications could protect against exopeptidase degradation while maintaining the peptides' ability to form functional pore complexes

• Strategic amino acid substitutions:

  • Replacing oxidation-prone residues (such as methionine in Lac705α) with more stable alternatives

  • Introducing D-amino acids at susceptible positions to enhance resistance to proteolytic degradation

  • Modifying key residues to optimize the peptides' interaction with bacterial membranes

• Formulation enhancements:

  • Developing stabilized formulations that maintain the optimal 1:4 ratio of Lac705α to Lac705β

  • Creating encapsulation systems (liposomes, nanoparticles) to protect the peptides and potentially enhance their delivery to target sites

  • Exploring synergistic combinations with other antimicrobial agents or membrane-permeabilizing compounds

• Structural fusion approaches:

  • Creating single-chain constructs where Lac705α and Lac705β are linked by a flexible peptide linker

  • This could ensure the correct stoichiometry and proximity of the two peptides

  • The linker design would need to allow proper folding and membrane interaction of both peptide components

• Rational design based on structure-function studies:

  • Using insights from FTIR spectroscopy to identify critical regions for membrane interaction

  • Enhancing regions of Lac705β that show conformational changes upon membrane binding (evidenced by the shift from 1,620 cm⁻¹ to 1,628 cm⁻¹)

  • Optimizing features that promote oligomerization and pore formation

These engineering approaches, guided by a deep understanding of the bacteriocin's mechanism of action, could lead to enhanced versions of lactocin-705 with improved stability, potency, and applicability across various research and potentially clinical contexts.