Effect of N-terminal modification on the antimicrobial activity of nisin

Highlights

• Activity of nisin crosslinked with genipin increased.

• Nisin activity increased after crosslinking with low concentration of chitosan.

• Less chitosan increased local number density and antimicrobial ability of nisin.

Abstract

Nisin, the only antimicrobial peptide that has GRAS (generally considered as safe) status from both WHO and FDA, has the potential to become an important natural antibiotic as bacteria become more resistant to traditional antibiotics. Many studies have focused on how modification of nisin affects antimicrobial activity. In this study, nisin and chitosan were crosslinked using genipin to modify the N-terminal of nisin. The antimicrobial experiments were carried out on Staphylococcus aureus ATCC 6538. The results showed that nisin still retained antimicrobial activity after modification. When the crosslinking ratio of nisin to chitosan increased to 200: 1, MIC decreased from 48 μg/ml to 40 μg/ml. Less chitosan increased local number density and antimicrobial activity of nisin. When the local number density was high, adjacent peptides could cooperatively penetrate into the lipid membrane. The results of this study may help to better understand the antimicrobial mechanism and provide suggestions for the further modification of nisin.

Introduction

Antimicrobial peptides (AMPs) are widely distributed in animals, plants and microorganisms. They are often an important barrier for organisms to prevent invasion from exogenous microorganisms. As more bacteria are becoming more resistant to traditional antibiotics, AMPs have the potential to become candidates to replace them because of their biodegradability and the ability to lyse the cell membrane of pathogens (Glasmästar et al., 2002, Hancock and Sahl, 2006, Mccubbin et al., 2011). Nisin, a 34 amino acid polypeptide produced by some Lactococcus lactis, is a typical AMP with high efficiency and safety (Campos et al., 2011, Millette et al., 2007, Ye et al., 2008). Its molecular structure includes unusual amino acids and thioether rings (Delves-Roughton, 2007), which are presumed to be important for its activity. Nisin is the only bacteriocin that has GRAS status from both the World Health Organization (WHO) and the U.S. Food and Drug Administration (FDA).

Nisin shows antimicrobial activity against most Gram-positive bacteria, especially spore formers. However, it is ineffective against Gram-negative bacteria (Basch et al., 2013, Bryan et al., 2000) because of the existence of an outer membrane in the biofilm, which can resist the interference of nisin. Therefore, how to improve the antimicrobial activity and bacterial range of nisin has been studied using conditions, such as freezing (Steeg, Hellemons, & Kok, 1999), heating (Murad, Anas, Osaili, Mutamed, & Reyad, 2012), lowering pH (Cabo et al., 2001, Phillips and Duggan, 2002), and treatment with EDTA (Economou et al., 2009, Stevens et al., 1991). These treatments can enhance the ability of nisin to interfere with the growth of some Gram-negative bacteria, such as Salmonella, Escherichia coli, and Pseudomonas. However, the antimicrobial activity requires a relatively higher concentration of nisin. Furthermore, nisin shows both a significant loss of activity and reduced solubility above pH 3.0, where it has been suggested that the Dha and Dhb residues of nisin become more susceptible to modifications due to the presence of nucleophiles (Liu & Hansen, 1990).

The N-terminal of nisin binds to the carbohydrate-pyrophosphate moiety of lipid II, which enables the C-terminal of nisin to penetrate into the cell membrane. Several nisin-lipid II complexes work synergistically to form a stable pore with a diameter of 2 nm on the targeted cell membrane. The pore leads to an increase in cell membrane permeability and a dissipation of the membrane potential, leading to cell leakage (Sahl, Ersfeld-Dressen, & Bierbaum, 1988). Consequently, the damaged cell is unable to maintain its metabolism and eventually dies. The C-terminal of nisin has an important role in the membrane penetration, so it seems that the N-terminal may offer opportunities to be modified.

Genipin, a product of gardenoside hydrolyzed by β-glucosidase, was used to bind nisin onto chitosan. Genipin is a good natural biological crosslinking reagent, which can crosslink chitosan, collagen, gelatin and proteins containing primary amino groups to produce blue-colored fluorescent products (Koo, Lim, Jung, & Park, 2006). The crosslinked products have not been found to be cytotoxic for animal and human cells. Due to its lower cytotoxicity and higher stability, genipin might be an ideal substitute for glutaraldehyde, and thereby be useful with foods (Delmar & Bianco-Peled, 2015). Genipin can also be used to treat liver diseases, hypotension, cataract, diabetes, periodontitis, nerve regeneration and wound repair (Muzzarelli, 2009). Chitosan, an amino-polymer obtained by the alkaline deacetylation of chitin, derived from crustacean shells, is used in the field of medicine, food, chemicals, cosmetics and biochemistry due to its good biocompatibility, high biodegradability and low toxicity (Illum, Jabbal-Gill, Hinchcliffe, Fisher, & Davis, 2001). Chitosan shows good adaptability in response to external pH changes and can be easily modified, because of the presence of amino groups and hydroxyl groups, which provides for the possibilities of adding specific compounds (Krajewska, 2004).

In this study, a method for the N-terminal modification of nisin involving the crosslinking of nisin and chitosan using genipin was developed. The purpose of the study was to explore the effect of N-terminal modification on the antimicrobial activity of nisin and possibly suggest further modifications.