Facile Halogenation of Antimicrobial Peptides As Demonstrated by Producing Bromotryptophan-Labeled Nisin Variants with Enhanced Antimicrobial Activity.

Antimicrobial peptides (AMPs) have raised significant interest, forming a potential new class of antibiotics in the fight against multi-drug-resistant bacteria. Various AMPs are ribosomally synthesized and post-translationally modified peptides (RiPPs). One post-translational modification found in AMPs is the halogenation of Trp residues. This modification has, for example, been shown to be critical for the activity of the potent AMP NAI-107 from Actinoallomurus. Due to the importance of organohalogens, establishing methods for facile and selective halogen atom installation into AMPs is highly desirable. In this study, we introduce an expression system utilizing the food-grade strain Lactococcus lactis, facilitating the efficient incorporation of bromo-Trp (BrTrp) into (modified) peptides, exemplified by the lantibiotic nisin with a single Trp residue or analogue incorporated at position 1. This provides an alternative to the challenges posed by halogenase enzymes, such as poor substrate selectivity. Our method yields expression levels comparable to that of wild-type nisin, while BrTrp incorporation does not interfere with the post-translational modifications of nisin (dehydration and cyclization). One brominated nisin variant exhibits a 2-fold improvement in antimicrobial activity against two tested pathogens, including a WHO priority pathogen, while maintaining the same lipid II binding and bactericidal activity as wild-type nisin. The work presented here demonstrates the potential of this methodology for peptide halogenation, offering a new avenue for the development of diverse antimicrobial products labeled with BrTrp.

Engineering hybrid lantibiotics yields the highly stable and bacteriocidal peptide cerocin V.

Antimicrobial peptides (AMPs) show promise as alternatives to traditional antibiotics for treating drug-resistant infections. Their adaptability and diverse sequence possibilities allow for rational design by modulating physicochemical determinants to achieve desired biological properties, transforming them into peptides for potential new therapies. Nisin, one of the best-studied AMPs, is believed to have potential to be used as a therapeutic, particularly against antibiotic-resistant bacteria. However, its instability in physiological conditions limits its use in clinical applications and pharmaceutical development. Exploration of new natural variants of nisin has uncovered diverse properties using different domains. Shuffling peptide modules can fine-tune the chemical properties of these molecules, potentially enhancing stability while maintaining or improving antimicrobial activity. In this study, hybrid AMPs were created by combining domains from three unique nisin variants, i.e. nisin A, cesin and rombocin, leading to the identification of a promising variant, named cerocin A, which harbours only 25 amino acids compared to the typical 31-35 amino acid length of nisin. Cerocin A demonstrates potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), approaching that of nisin itself. Cerocin A's mode of action involves a dual mechanism through the combination of two domains, consisting of a small ring/domain (6 amino acids) from the C-terminal end of rombocin attached to the preceding peptide of cesin, changing it from a bacteriostatic to a bactericidal peptide. Further mutation studies identified a new variant, cerocin V, with significantly improved resistance against trypsin degradation, while maintaining high potency. Importantly, cerocin V showed no undesired toxic effects on human red blood cells and remained stable in human plasma. In conclusion, we demonstrate that peptide construction using domain engineering is an effective strategy for manipulating both biological and physicochemical aspects, leading to the creation of novel bioactive molecules with desired properties. These constructs are appealing candidates for further optimization and development as novel antibiotics.

Altering Specificity and Enhancing Stability of the Antimicrobial Peptides Nisin and Rombocin through Dehydrated Amino Acid Residue Engineering.

Nisin serves as the prototype within the lantibiotic group of antimicrobial peptides, exhibiting a broad-spectrum inhibition against Gram-positive bacteria, including important food-borne pathogens and clinically relevant antibiotic-resistant strains. The gene-encoded nature of nisin allows for gene-based bioengineering, enabling the generation of novel derivatives. It has been demonstrated that nisin mutants can be produced with improved functional properties. Here, we particularly focus on the uncommon amino acid residues dehydroalanine (Dha) and dehydrobutyrin (Dhb), whose functions are not yet fully elucidated. Prior to this study, we developed a new expression system that utilizes the nisin modification machinery NisBTC to advance expression, resulting in enhanced peptide dehydration efficiency. Through this approach, we discovered that the dehydrated amino acid Dhb at position 18 in the peptide rombocin, a short variant of nisin, displayed four times higher activity compared to the non-dehydrated peptide against the strain Lactococcus lactis. Furthermore, we observed that in the peptides nisin and rombocin, the dehydrated amino acid Dha at residue positon 18 exhibited superior activity compared to the dehydrated amino acid Dhb. Upon purifying the wild-type nisin and its variant nisinG18/Dha to homogeneity, the minimum inhibitory concentration (MIC) indicated that the variant exhibited activity similar to that of wild-type nisin in inhibiting the growth of Bacillus cereus but showed twice the MIC values against the other four tested Gram-positive strains. Further stability tests demonstrated that the dehydrated peptide exhibited properties similar to wild-type nisin under different temperatures but displayed higher resistance to proteolytic enzymes compared to wild-type nisin.

Rombocin, a Short Stable Natural Nisin Variant, Displays Selective Antimicrobial Activity against Listeria monocytogenes and Employs a Dual Mode of Action to Kill Target Bacterial Strains.

Nisin, with its unique mode of action and potent antimicrobial activity, serves as a remarkable inspiration for the design of novel antibiotics. However, peptides possess inherent weaknesses, particularly their susceptibility to proteolytic degradation, such as by trypsin, which limits their broader applications. This led us to speculate that natural variants of nisin produced by underexplored bacterial species can potentially overcome these limitations. We carried out genome mining of two Romboutsia sedimentorum strains, RC001 and RC002, leading to the discovery of rombocin A, which is a 25 amino acid residue short nisin variant that is predicted to have only four macrocycles compared to the known 31-35 amino acids long nisin variants with five macrocycles. Using the nisin-controlled expression system, we heterologously expressed fully modified and functional rombocin A in Lactococcus lactis and demonstrated its selective antimicrobial activity against Listeria monocytogenes. Rombocin A uses a dual mode of action involving lipid II binding activity and dissipation of the membrane potential to kill target bacteria. Stability tests confirmed its high stability at different pH values, temperatures, and in particular, against enzymatic degradation. With its gene-encoded characteristic, rombocin A is amenable to bioengineering to generate novel derivatives. Further mutation studies led to the identification of rombocin K, a mutant with enhanced bioactivity against L. monocytogenes. Our findings suggest that rombocin A and its bioengineered variant, rombocin K, are promising candidates for development as food preservatives or antibiotics against L. monocytogenes.