The interaction mechanism between Nisin and cell membranes

The interaction mechanism between Nisin and bacterial cell membranes centers on the core pathway of "target recognition - membrane insertion - pore formation." By specifically binding to key components of the bacterial cell membrane and physically disrupting the membrane structure, Nisin ultimately leads to bacterial death, which is the foundation of its high-efficiency antimicrobial activity.

I. Precondition for Interaction: Target Recognition and Specific Binding

1. Core Binding Target: Lipid Ⅱ

Lipid Ⅱ is a peptidoglycan precursor on the cell membrane of Gram-positive bacteria, composed of N-acetylglucosamine, N-acetylmuramic acid, a pentapeptide chain, and a pyrophosphate group. It is a key substance for bacterial cell wall synthesis. The cyclic structures in Nisin, such as lanthionine (Lan) and methyllanthionine (MeLan), can form specific hydrogen bonds with the pyrophosphate group of Lipid Ⅱ. Meanwhile, the hydrophobic region in the Nisin molecule interacts with the fatty chain of Lipid Ⅱ to achieve rapid anchoring. This binding is highly specific, targeting only Gram-positive bacteria (the outer membrane of Gram-negative bacteria shields Lipid Ⅱ, preventing access by Nisin), with extremely strong binding affinity (dissociation constant Kd ≈ 10⁻⁷~10⁻⁸ mol/L).

2. Auxiliary Binding Factor: Cell Membrane Lipid Composition

The high proportion of acidic lipids (e.g., phosphatidylglycerol, cardiolipin) in the cell membrane of Gram-positive bacteria can attract positively charged amino acid residues (e.g., lysine, arginine) in Nisin through electrostatic interactions. This assists Nisin in approaching the cell membrane surface and improves the binding efficiency with Lipid Ⅱ. Higher cell membrane fluidity accelerates the lateral diffusion of Lipid Ⅱ, facilitating its encounter and binding with Nisin; conversely, lower fluidity reduces the interaction rate.

II. Key Process: Membrane Insertion and Transmembrane Pore Formation

1. Molecular Conformational Change and Membrane Insertion

After binding to Lipid Ⅱ, Nisin undergoes a significant conformational change: the originally partially folded structure unfolds, the N-terminal hydrophobic fragment (amino acids 1~13) inserts into the hydrophobic core region of the cell membrane, and the C-terminal remains outside the membrane, forming a stable "anchoring-insertion" state. This process relies on the hydrophobic interaction of the cell membrane and the "bridging" effect of Lipid Ⅱ, which acts as a "carrier" to help Nisin break through the hydrophobic barrier of the cell membrane and achieve efficient insertion.

2. Multimolecular Assembly and Pore Formation

Nisin molecules inserted into the cell membrane aggregate through intermolecular hydrophobic interactions and hydrogen bonds. Six to eight Nisin molecules assemble with 2~3 Lipid Ⅱ molecules to form a transmembrane pore. The pore is cylindrical with an inner diameter of approximately 2~3 nm: the inner wall is composed of hydrophobic residues of Nisin molecules, the outer side fuses with the lipid bilayer of the cell membrane, and the inner side is hydrophilic due to polar amino acid residues, allowing the passage of small intracellular molecules. The key to pore formation is the synergistic effect between Nisin molecules; a single Nisin molecule cannot form a stable pore, and a critical aggregation concentration (approximately 0.1 μg/mL) must be reached.

III. Subsequent Effects: Membrane Function Disruption and Bacterial Death

1. Osmotic Imbalance

After transmembrane pore formation, small intracellular molecules such as potassium ions, sodium ions, amino acids, and ATP leak out along the concentration gradient, while a large amount of extracellular water influxes due to osmotic differences. The cell volume expands rapidly, and the cell membrane ruptures under pressure—this is the main direct cause of bacterial death induced by Nisin.

2. Disordered Cell Membrane Permeability

In addition to pore formation, Nisin interferes with the arrangement of phospholipid molecules on the cell membrane, disrupting the integrity of the lipid bilayer and causing non-specific increases in cell membrane permeability. Even without forming complete pores, Nisin insertion creates "leaks" in local areas of the cell membrane, affecting material transport and energy metabolism, and inhibiting bacterial growth and reproduction.

3. Blockage of Cell Wall Synthesis

Nisin’s binding to Lipid Ⅱ occupies the active site of Lipid Ⅱ, preventing it from being transported to the outer side of the cell membrane to participate in peptidoglycan cross-linking, thereby blocking bacterial cell wall synthesis. Bacteria with impaired cell wall synthesis cannot maintain their morphology, are more prone to rupture under osmotic pressure, and lose resistance to environmental stress, accelerating their death.

IV. Key Factors Influencing Interaction Efficiency

1. Nisin’s Own Characteristics

Molecular Integrity: The cyclic structures, hydrophobic fragments, and positively charged residues of Nisin are critical for its interaction with the cell membrane. Any structural damage (e.g., enzymatic hydrolysis, oxidation) leads to loss of activity.

Concentration: Below the critical aggregation concentration, Nisin can only bind to Lipid Ⅱ but cannot form pores, exerting only an inhibitory effect. Above the critical concentration, pore formation efficiency increases with concentration until saturation (no significant increase when >10 μg/mL).

2. Cell Membrane Characteristics

Lipid Ⅱ Content: The cell membrane of bacteria in the logarithmic growth phase has the highest Lipid Ⅱ content, resulting in the strongest interaction with Nisin—antimicrobial efficacy is 2~3 times that of stationary-phase bacteria.

Membrane Fluidity: Increased temperature and higher proportions of unsaturated fatty acids enhance cell membrane fluidity, promoting Nisin’s binding to Lipid Ⅱ and membrane insertion, thereby strengthening the interaction effect.

Membrane Charge: Higher negative charge density on the cell membrane surface leads to stronger electrostatic attraction with Nisin, facilitating interaction.

3. Environmental Conditions

pH Value: At pH 2~6, Nisin molecules carry positive charges, resulting in the strongest electrostatic attraction with the cell membrane. At pH >7, Nisin’s conformation changes, positive charge decreases, and interaction efficiency drops by more than 50%.

Ionic Strength: Low ion concentrations (<0.1 mol/L) have little effect on the interaction; high ion concentrations (>0.5 mol/L) shield electrostatic interactions, reducing Nisin’s binding capacity to the cell membrane.

Organic Solvents/Food Matrices: A small amount of ethanol (<10%) can enhance cell membrane permeability and promote Nisin insertion. Proteins and fats bind to Nisin, reducing its free concentration and weakening the interaction with the cell membrane.

V. Research Evidence and Technical Methods

1. Core Research Evidence

In Vitro Membrane Model Experiments: Adding Lipid Ⅱ and Nisin to liposomes (simulating bacterial cell membranes) allows direct observation of transmembrane pore formation via dynamic light scattering and transmission electron microscopy (TEM), with pore sizes consistent with theoretical values (2~3 nm).

Bacterial Cell Membrane Extraction Experiments: Incubating extracted Staphylococcus aureus cell membranes with Nisin detects Nisin-Lipid Ⅱ binding complexes via high-performance liquid chromatography (HPLC), confirming the specificity of target binding.

Mutant Experiments: Knocking out bacterial Lipid Ⅱ synthesis genes prevents effective interaction between Nisin and the cell membrane, reducing antimicrobial activity by over 90%. Modifying Nisin’s cyclic structures significantly impairs its binding capacity to Lipid Ⅱ and reduces pore formation efficiency.

2. Main Research Techniques

Structural Analysis Techniques: X-ray crystallography and nuclear magnetic resonance (NMR) are used to analyze the binding conformation of Nisin and Lipid Ⅱ.

Membrane Function Detection Techniques: Patch-clamp and fluorescence probe methods (e.g., calcein leakage assay) monitor changes in cell membrane permeability and pore formation.

Imaging Techniques: Atomic force microscopy (AFM) and transmission electron microscopy (TEM) directly observe the interaction process between Nisin and the cell membrane, as well as pore morphology.