Structure‐activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin

Abstract

The post-translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth-inhibition activity. Removal of the C-terminal five residues leads to approximately a 10-fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100-fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment ulsin t-12 is inactive itself, and specifically antagonises the growth-inhibitory action of nisin. These results are discussed in lerms of current models for the mechanism of action of nisin.Key words: Nisin; Lantibiotic; Peptide antibiotic; I'roteolysis; Structure-activity relationships mation of voltage-dependent pores in biological membranes [8][9][10], although recent evidence indicates that the inhibition of the outgrowth of spores by nisin and subtilin takes place by a different mechanism [11,12]. We have been studying the structure-activity relationships in nisin and subtilin, by both genetic and chemical modification of the structure [12][13][14][15][16][17][18][19][20], with a view to understanding their mechanism of action and to developing new derivatives with desirable properties. We now report the preparation, characterisation and anti-bacterial activity of a number of proteolytic fragments of nisin and subtilin, which allow us to define the parts of the molecule most important for biological activity. Materials and methodsNisin and subtilin were prepared as previously described [14,15]; [Ser33]-nisin, in which the serine residue at position 33 has escaped processing to a dehydroalanine residue, was isolated as a minor component of commercial nisin. All proteases were obtained from Sigma Chemical Co., Poole, Dorset, UK. ~. IntroductionThe post-translationally modified peptide antibiotics known :ts 'lantibiotics' contain cyclic structures formed by lanthioaine or 3-methyl-lanthionine residues, and often also dehydroalanine and/or dehydrobutyrine residues [1]. The first lan-!ibiotic to be characterised was nisin, produced by strains of Lactococcus lactis carrying a transposon containing genes coding for the nisin precursor and for proteins involved in aisin biosynthesis and resistance [2][3][4][5]. Nisin has been quite videly used as a food preservative, notably in cheese and ,~ther dairy products and in canned vegetables, for some 30 .,ears [6,7]. It inhibits the growth of a wide range of Gram~ositive organisms, and also inhibits the germination and/or mtgrowth of spores of Bacillus and Clostridium species [6]. Fhe growth-inhibitory activity of nisi...

Activity and Export of Engineered Nisin-(1-22) Analogs

“…The activity of nisin- (1-22) is about 10-fold higher than that reported for nisin-(1-20) [8,9]. Previously, Rink et al argued that the relatively high intrinsic antimicrobial activity of nisin-(1-22) might result from the positively charged Lys22 which counterbalances the negative charge of the C-terminal carboxyl group [8].…”

“…Studies on the activity of truncated nisin mutants showed that nisin-(1-32)-NH 2 , nisin-(1-29) and nisin- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) have respectively slightly higher, about 10-fold lower and 100-fold lower activity than nisin [9]. Rink et al found that nisin-(1-22) has only 10-fold lower activity than nisin and suggested that the positively charged Lys22 might enhance binding to the negatively-charged bacterial membrane [8].…”

Frequency of four different mutacin genes in Streptococcus mutans genotypes isolated from caries-free and caries-active individuals

“…One possible explanation is the modification of only one amino acid, which has already been shown to alter or prevent the activity of certain mutacins (Mulders et al, 1991;Rollema et al, 1995;Chan et al, 1996). The inhibition assay previously performed (Kamiya et al, 2005) could also result from the production of more than one inhibitory substance, showing that the understanding of the genetic determinants of several mutacins still needs to be improved.…”

Synthesis of Bicyclic Alkene‐/Alkane‐Bridged Nisin Mimics by Ring‐Closing Metathesis and their Biochemical Evaluation as Lipid II Binders: toward the Design of Potential Novel Antibiotics

“…[34,35] In the present study, the lipid II binding affinities of mimics 1-7 were evaluated and compared with those of the native AB and ABC fragments 41 and 42, respectively. The native AB and ABC fragments were obtained by trypsin and chymotrypsin digestion of nisin A, respectively, according to Chan et al [36] In a typical experiment, preincubation of CF-loaded DOPClipid II vesicles with AB(C) fragments 1-7, 41, and 42 should result in the occupation of nisin-binding sites of lipid II. Depending on the affinity of the synthetic AB(C) fragments toward lipid II, subsequent addition of full-length nisin will result in a reduced amount of pore formation and reduced fluorescence.…”