Pharmacological Properties Improvement by Engineering of Nisin

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1		Paweł Kafarski	--	2400	2023-09-06 18:25:01	\|
2	Reference format revised.	Lindsay Dong	-2 word(s)	2398	2023-09-08 03:29:03	\| \|
3	Reference format revised.	Lindsay Dong	Meta information modification	2398	2023-09-08 10:48:18	\|

This entry is adapted from the peer-reviewed paper 10.3390/ph16081058

Nisin is a readily available and cheap lanthipeptide and thus serves as a good model in the search for the tools to engineer lantibiotics with improved pharmacological properties. There are basically two general means to obtain nisin analogs—protein engineering and chemical functionalization of this antibiotic. Although bioengineering techniques have been well developed and enable the creation of nisin mutants of variable structures and properties, they are lacking spectacular effects so far. Chemical modifications of nisin based on utilization of the reactivity of its free amino and carboxylic moieties, as well as reactivity of the double bonds of its dehydroamino acids, are in their infancy.

antibiotic resistance nisin lantibiotics protein engineering bioengineering

1. Nisin A

Discovered in 1928 ^[1] nisin A is the prototypical and best-studied lantibiotic, secreted by some Lactococcus lactis strains. Low toxicity and safety of use in food contributed to the worldwide success of nisin as a natural food preservative (E234), especially in dairy and canned products ^[2]^[3]^[4]. Although nisin is a complex polycyclic, positively charged, molecule consisting of 34 amino acids (molecular weight 3500 Da) it is quite readily available by fermentation, so is relatively cheap.

The remarkable commercial success of this compound has positioned it at the pinnacle of bacteriocin research. These studies are concentrated not only on knowledge attained with respect to genetics, structure, mode of action and chemical properties but also for the potential of its practical applications. It is an FDA-approved, GRAS (generally regarded as safe) peptide with recognized potential for possible clinical use ^[5]. Additionally, nisin’s immunomodulatory properties, analogous to those described for many human defense peptides, suggest its possible role as a novel immunomodulatory therapeutic ^[6].

Nisin is built from five lanthione bridges which help impart a conformation that is key to its antibacterial properties. Unusual amino acids present in this antibiotic are dehydroalanine, dehydrobutyrine and β-methyl-lanthionine (Figure 1) ^[7]. Dehydroamino acids are formed via post-translational processing of a ribosomally synthesized precursor polypeptide.

Figure 1. Structure of nisin A with numbering of amino acids and indication of A–E rings. Structures of dehydroalanine (Dha) and dehydrobutyrine (Dhb) and lanthionine and β-methyl-lanthionine are shown.

It is an antibiotic of dual action, which is a consequence of the presence of two distinct structural domains located at the N- and C-termini of the peptide (Figure 2) ^[8]^[9]^[10]. The N-terminal fragment of a peptide docks to lipid II with high affinity via interaction with lipid II pyrophosphate group, finally forming a cage-like structure by intermolecular hydrogen bonds.

Figure 2. Dual mechanism of action of nisin. Nisin binds to lipid II and then undergoes conformational change forming pore in the membrane. Pore is composed of eight C-terminal fragments of nisin.

At the same time, the C-terminus of nisin flips into the membrane and enables the formation of pores, thus causing cell leakage, namely immediate efflux of nutrients and small compounds, resulting in cell death (Figure 2). The flexible, short hinge region connects the N- and C- terminal regions and is crucial for C-terminal fragment translocation through cell membrane and pore-forming activity.

2. Design of Novel Antibacterials by Modification of Nisin Structure

The structural features and mode of action make nisin suitable for its structure engineering. These modifications are performed in order to generate nisin variants with enhanced antimicrobial activity, better solubility, and ability to evade resistance. Enhancement of activity against Gram-negative bacteria is one of the major driving forces of these studies. Thus, the obtained results should allow progress to be made in overcoming the inherent shortcomings of nisin. The two major means that have been used for this purpose are bioengineering and chemical modifications of the structure of this antibiotic.

2.1. Bioengeeneering by Mutagenesis

Due to the complex nature of nisin’s structure, chemical synthesis of its analogues is challenging, especially in economic terms. This leaves a huge space for the efforts of bioengineering, especially if considering possible industrial preparation of the analogs by fermentation. Recent years have seen a growing number of studies on the use of genetic tools (engineering within the cell) and the use of synthetic biology-based (in vitro engineering) approaches ^[11]^[12]^[13]^[14], mostly in order to advance the understanding of the fundamentals of the structure–antibacterial activity relationship.

Bioengineering could be performed either by applying genome mining, design and preparation of hybrid-peptides (by coupling lanthipeptides with variable antibacterial peptides) and especially by utilization of mutation techniques. As a result, the number of identified nisin analogs have increased tremendously in the last decades ^[13].

There are at least five naturally occurring variants of nisin A, which differ by up to 10 amino acids ^[15]. The most variable ones are produced by Streptococcus agalactiae and Straptococcus uberis. Because of their gene-encoded nature they could easily undergo bioengineering procedures, which resulted in obtaining libraries of analogs suitable for elucidating structure–function relationships ^[16]. Nisin variants are usually produced by transfection of Escherichia coli with the nisin biosynthesis pathways or the expression of nisin variant genes in a wild type nisin producing strains, from which the nisin gene has been deleted.

The simplest examples are mutations of the hinge region, in which flexibility is crucial for C-terminal membrane translocation. Additionally, the three residues, which form this region, affect antibiotic stability and solubility. The first approach was concentrated on studying the replacement of one of its three amino acid (Asn, Met or Lys) by randomly introduced proteinogenic ones using site-saturation mutagenesis. These studies initially brought quite promising results, providing variants which displayed enhanced activity against Gram-negative bacteria ^[17]. The generation of nisin derivatives with enhanced activity against Gram-positive pathogens was achieved later, in 2009, using a non-targeted approach. In this instance, the use of a random mutagenesis-based approach created over 8000 derivatives among which only one variant displayed an activity higher than the parent nisin against a human pathogen—Streptococcus agalactiae ^[18].

Mutations, consisting of changes in the length of hinge region, did not dramatically affect nisin production in host bacteria ^[17]. Deletion of one amino acid or insertion of three additional ones usually led to a sharp decrease in antimicrobial activity ^[19]. Analogs of the hinge region consisting of five or six amino acids usually exhibited moderate improvement of antimicrobial effects ^[20]. These results show that the hinge region might still be considered as a potential target for bioengineering.

Early engineering attempts were targeted at the N-terminal ring A (amino acids 3–7). Amino acids in positions 4, 5 and 6, namely in a region that is at the border of the pyrophosphate cage ^[6], suggest large mutational freedom available and thus represent suitable targets for mutagenesis. Indeed, some of the variants displayed improved activity against several non-pathogenic indicator strains ^[21]. Notably, several natural nisin variants as well as novel nisin-like peptides (agalacticin, flavucin, moraviensicin and maddinglicin) possess a lysine at position 4. Its presence ensures the interaction of positively charged lysine side chain with negatively charged phosphate and indeed brought a positive effect ^[22]. Ring B of nisin was found to be far less amenable to amino acid substitution ^[21]^[23].

Advanced, highly sensitive solid-state NMR study on binding of nisin to lipid II present in a model membrane consisting of mixture of artificial phospholipids brought identification of Ile4, Lys12, Ser 29 and the whole hinge region as functional hotspots that are critical for the cellular adaptability of nisin ^[24].

Although nisin remains the only lantibiotic that is so extensively modified by application of mutagenesis, the full use of any variant as a therapeutic entity has not yet been fulfilled. The progress of mutagenetic studies, although quite slow and cumbersome, is clear. However, the successful clinical development of nisin analogs requires more detailed understanding of the mechanism of nisin pharmacology and creative improvement of the discovered modification procedures.

2.2. Variants Containing Non-Canonical Amino Acids

Logic extension of the studies described above was introduction of nonproteinogenic (non-canonical) amino acids into discrete positions of nisin. Generally, there are two complementary approaches for the incorporation of non-canonical amino acids into the peptide chain of interest. The first is residue-specific and exploits the translational machinery of the host, which accepts the structural analog of the amino acid as a surrogate of a classic one. The second is site-specific, which is far more complex and requires the additional presence an orthogonal aminoacyl-tRNA-synthetase-tRNA pair, which do not cross-react with the endogenous expression machinery. This orthogonal pair uses a stop codon as a coding one to incorporate a chosen non-canonical amino acid into the protein of interest. Thus, the first step to biosynthetic incorporation of nonproteinogenic amino acids into lantipeptides was the elaboration of novel molecular biology tools ^[25]^[26]^[27]^[28]^[29].

Analogs of proline and methionine have been incorporated into nisin at positions of their parent amino acids without gene manipulation (Figure 3A), by feeding nutritionally deficient strains with these analogs ^[29]^[30]^[31]^[32]. In addition, variants containing tryptophan, obtained previously by mutagenesis, were used as substrates for introduction of tryptophan-like residues (Figure 3B) ^[33].

Figure 3. Residue-specific modifications of nisin with non-canonical amino acids: (A) substitutions of lysine and methionine; (B) modifications with tryptophan analogs using previously mutated variants (nisin mutation sites with tryptophan are marked in magenta). Structures of non-canonical amino acids are also shown.

A site-specific procedure has been used for modification of the structure of nisin with analogs of phenylalanine ^[28]^[29] and derivatives of lysine (Figure 4) ^[28]^[34].

Figure 4. Residue-specific modifications of nisin variants containing lysine (black arrows, mutation sites shown in orange) and phenylalanine (red arrows, mutation site shown in green). Site of replacement by (N-chloroacetylamino)phenylalanine (blue arrow) is shown separately. Structures of introduced non-canonical amino acids are indicated.

2.3. Hybrid Molecules

Combining of nisin, or its fragments, with other antimicrobial peptides is performed on the premise of receiving potent and novel antimicrobial drug candidates of multiple modes of action. So far, only single examples of the use of bioengineering approach for that purpose have been described.

The outer membrane of Gram-negative bacteria constitutes an efficient protective barrier that prevents nisin from reaching the cellular membrane and then exerting its antimicrobial action. This inconvenience was addressed by fusion of nisin, or its N-terminal fragments, with short antimicrobial peptides that combine different functionalities. These include peptides whose designs are based on statistical analyses and natural antimicrobials produced by various organisms (crocodile, honeybee, scorpion, frogs and penis fish). The obtained hybrids are able to pass the outer membrane of Gram-negative organisms while retaining as much nisin antimicrobial function as possible at the cytoplasmic membrane, which is documented by their up to 12-fold activity if compared with nisin ^[35]^[36]^[37].

3. Chemical Modifications of Nisin

3.1. Click Reaction as a Tool for Nisin Modification

Most of the described procedures of nisin modification consider the use of nisin variants carrying acetylenic or azide moieties and are thus suitable for application as substrates in a click reaction ^[38]. The first attempt was to conjugate an N-terminal fragment of nisin (amino acids 1–12) functionalized with propargylamine with vancomycin derivatives bearing 2-amino-3-azidopropane at C-terminus (Figure 5).

Figure 5. Synthesis of nisin–vancomycin conjugate by click reaction of their derivatives.

In a similar manner nisin analogues composed of its 22-amino-acid, N-terminal fragment and of its C-terminal fragment mimics, in which ring structure is built up by the replacement of lanthionines by double bonds (Figure 6A), have been obtained.

Figure 6. Representative nisin-derived conjugates: (A) variant of nisin containing artificial C-termini; (B) variant containing C-terminal analog of polyproline; (D) nisin–polypeptidoid conjugate; (C) conjugate of nisin N-termini with pexiganan.

It is worth to note that these functionalizations are possible thanks to digestion of nisin with trypsin and chymotrypsin, which selectively generate nisin fragments composed of, correspondingly, rings AB and ABC.

Connecting the ABC fragment of nisin with hydrophobic poly(octahydroindole-2-carboxylic acid), containing additional lysines at the C-termini, provided another interesting class of nisin mimics ^[39]. Lysines were introduced because they exist in cationic forms at physiological pH and thus improve solubility of the obtained variants (Figure 6B).

In addition, nisin AB fragment was conjugated with variable linear peptidoids, which are known to have increased in vivo stability compared to the corresponding peptides ^[40]. These hybrid peptide—petidoids (representative structure shown in Figure 6C) were shown to have low micromolar activity (i.e., comparable to natural nisin) against methicillin-resistant Staphylococcus aureus. Hybrid peptides, where a hydrophilic PEG4 linker was used, showed good antibacterial activity against Micrococcus luteus. One of these peptides, a hybrid of nisin ABC fragment and a 22-amino-acid antibiotic derived from magainin—pexiganan—is shown in Figure 6D.

Functionalization of nisin with fluorescent reporter molecules was designed for studies on the mode of action of this antibiotic. They were obtained from nisin C-terminally substituted with propargylamine and a series of functionalized fluorescent probe azides (for representative structure, see Figure 7). Results of the studies show that these nisin derivatives retain both their antimicrobial activity and their membrane permeabilizing properties ^[41].

Figure 7. Structures of representative nisin-fluorescent reporter conjugates.

In addition, the possibility of the residue-specific incorporation of fluorescent dyes into certain fragments of nisin was demonstrated. This was performed by using nisin analogs containing either azidohomoalanine or homopropargylglycine available from the bioengineering approach. This resulted in a library of variants bearing fluorescent dyes at various positions of the nisin peptide chain (the representative example is shown in Figure 7) ^[42].

3.2. Late-Stage Functionalization of Nisin

Functionalization of intact nisin and its variants by chemical introduction of specific functionalities in one step has not yet received suitable attention. For direct functionalization there are available: C-terminal carboxylic moiety, four free amino groups (N-terminal one and three from side chains of lysines), and three dehydroamino acids. In addition, the possibility of modifications of two side chains of methionine could be considered (Figure 8), though there is a lack of such studies. The number of reports on direct modifications of nisin or its fragments is also limited.

Figure 8. Possible sites for last-stage modifications of nisin showing reactive entities: blue arrows—free amino groups; red arrow—carboxylate; green arrows—dehydroamino acids; purple arrows—methionines.

A palladium-mediated cross-coupling reaction of nisin with a series of phenylboronic acids gave mixtures of the Heck reaction (dehydrophenylalanines are formed) and conjugated addition to double bonds (phenylalanines are produced) (Figure 9). The reaction is quite complex, but could be steered to some extent by appropriate manipulation of reaction conditions; however, the Heck reaction always predominates ^[43].

Figure 9. Reaction of nisin C-terminal dehydroalanine with phenylboronic acids.

Reactivity of the other groups of nisin, except for synthesis of substrates using the click reaction, are scarcely used for functionalization of this antibiotic. Thus, a variety of hydrophobic amines were coupled with the C-terminal AB fragment to generate semisynthetic lipopeptides that display potent inhibition of bacterial growth ^[44]. A representative example shown in Figure 10 exhibited quite potent activity against vancomycin-resistant Enterococci.

Figure 10. Representative structure of nisin lipopeptide obtained by direct acylation of amines with AB fragment of antibiotic.

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