Recombinant Lantibiotic mutacin B-Ny266

What is the genetic organization of mutacin B-Ny266?

Mutacin B-Ny266 is encoded by a chromosomal locus composed of six predicted operon structures that encode proteins involved in regulation, antimicrobial activity, biosynthesis, modification, transport, and immunity. The core structural genes include lanA (encoding a 63-amino-acid peptide) and lanA' (encoding a 64-amino-acid peptide with 57% homology to LanA). This genetic organization supports the production and processing of both peptides that constitute the active lantibiotic .

The activation of lanAA' expression is most likely controlled by the conserved two-component system NsrRS, which is activated by the LanA peptide but not by the LanA' peptide. Interestingly, this genetic locus is not universally conserved across all sequenced S. mutans genomes, with the genes encoding LanAA' peptides appearing to be restricted to the most invasive serotypes of S. mutans .

How does mutacin B-Ny266 differ structurally from other lantibiotics?

Mutacin B-Ny266 belongs to the type A class of lantibiotics, which consists of linear, cationic monopeptides or dipeptides. The mature peptide shows structural similarity to gallidermin and epidermin produced by Staphylococcus gallinarum and Staphylococcus epidermidis, respectively .

Both LanA and LanA' peptides of mutacin B-Ny266 undergo extensive post-translational modifications, including:

• Dehydration of serine and threonine residues

• Formation of a C-terminal aminovinyl-cysteine (AviCys) ring

• Formation of lanthionine (Lan) or methyllanthionine (MeLan) bridges

The molecular structure of mutacin B-Ny266's ring B incorporates lanthionine (Lan) bridges between positions 8-11, with leucine at position 9, while related lantibiotics like nisin have different ring configurations .

What is the antimicrobial spectrum of mutacin B-Ny266?

Mutacin B-Ny266 demonstrates an exceptionally broad spectrum of antimicrobial activity, making it particularly valuable for research and potential therapeutic applications:

• Effective against a wide array of oral streptococci, including nearly all S. mutans strains tested

• Active against multiple bacterial pathogens and multiresistant strains of staphylococci, streptococci, and Neisseria

• Shows activity against certain Gram-negative pathogens such as Neisseria gonorrhoeae and Helicobacter pylori

• As effective as vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in mouse infection models

• Active against many nisin A-resistant strains including Listeria monocytogenes Scott A and Pediococcus acidilactici

• Effective against oxacillin-resistant strains (N. gonorrhoeae, Enterococcus faecalis, S. aureus, and S. epidermidis) and vancomycin-resistant strains (N. gonorrhoeae, E. faecalis)

Its wide spectrum of activity in the nanomolar range makes mutacin B-Ny266 an excellent candidate for therapeutic applications .

What is the functional relationship between the LanA and LanA' peptides in mutacin B-Ny266?

Research indicates that mutacin B-Ny266 may function as a two-peptide lantibiotic, with a unique synergistic relationship between its component peptides:

• The LanA peptide alone is absolutely required for antimicrobial activity, as demonstrated through experiments isolating the individual peptides

• While LanA can function independently, the presence of LanA' enhances the activity of LanA, suggesting a cooperative mechanism

• This enhancement effect suggests that mutacin B-Ny266 operates as a two-peptide lantibiotic system, where the second peptide amplifies the efficacy of the first

The molecular basis for this synergistic interaction likely involves coordinated targeting of the bacterial membrane and cell wall synthesis pathways, though the precise mechanism of this enhancement effect requires further investigation .

What are the experimental approaches for producing and purifying recombinant mutacin B-Ny266?

Several approaches have been employed for the production and purification of lantibiotics like mutacin B-Ny266:

• Fermentation methods:

  • Use of 10% inoculums in late log growth phase

  • Incorporation of yeast extract as a critical media component

  • Optimization has demonstrated higher yields of production compared to earlier methods

• Semi-synthetic approaches:

  • While not specifically applied to mutacin B-Ny266, semi-synthetic methods have been proven feasible for related lantibiotics like lacticin 481

  • These approaches use purified recombinant expression products of the prepropeptide and modification enzymes to demonstrate in vitro modification

• Purification from semisolid cultures:

  • Mutacin B-Ny266 has been successfully purified from semisolid cultures

  • This approach allows for isolation of both LanA and LanA' peptides for functional characterization

• Solid-phase peptide synthesis:

  • For specific structural components, such as ring structures

  • Has been successfully used to prepare individual ring structures from related lantibiotics like nisin and mutacin

It's worth noting that unlike enzymes for some lantibiotics that can be used in vitro, the post-translational modifications brought about by LanB and LanC (characteristic of Type A lantibiotics) appear to occur only when the lantibiotic synthetase complex is formed in the bacterial membrane, adding complexity to recombinant production .

What is the mechanism of action for mutacin B-Ny266?

Mutacin B-Ny266 exhibits a dual mechanism of action against sensitive bacterial cells:

• Membrane disruption:

  • Forms pores in the cellular membrane

  • Leads to dissipation of membrane potential

  • Causes efflux of small metabolites

• Cell wall synthesis inhibition:

  • Blocks peptidoglycan synthesis

  • Binding to the peptidoglycan precursor lipid II serves as the molecular basis for both mechanisms

The model proposed for mutacin B-Ny266's mechanism involves binding to the membrane via lipid II, which serves as a docking or target molecule. This interaction has been studied through molecular recognition experiments, including the synthesis and characterization of individual ring structures .

While some lantibiotics that are too short to form a pore across the bilayer membrane (such as group B mutacins) maintain their antibacterial activity through an alternative mechanism involving removal of lipid II from the septum, the specific mechanism for mutacin B-Ny266 appears to involve both membrane disruption and cell wall synthesis inhibition .

What are the comparative advantages of mutacin B-Ny266 over other lantibiotics in terms of resistance development?

A significant advantage of mutacin B-Ny266 in comparison to other lantibiotics is its apparent resistance to the development of bacterial resistance:

• Resistant mutants against mutacin B-Ny266 could not be obtained in laboratory settings, unlike nisin- and pediocin-resistant mutants which appear relatively easily

• Mutacin B-Ny266 is active against many nisin A-resistant strains, suggesting different mechanisms of action or binding sites

• The peptide demonstrates strong activity against strains that have developed resistance to conventional antibiotics like oxacillin and vancomycin

This resistance to the development of bacterial resistance may be attributed to:

• Structural features that make proteolytic degradation difficult, as seen in the related lantibiotic mutacin 1140, which has a horseshoe-like conformation that protects potentially susceptible residues from protease cleavage

• The synergistic action of the two-peptide system (LanA and LanA')

• The essential nature of its target (lipid II) in bacterial cell wall synthesis

What experimental models have been used to evaluate mutacin B-Ny266's efficacy?

Several experimental models have been employed to evaluate the efficacy of mutacin B-Ny266:

• In vitro antimicrobial assays:

  • Determination of Minimum Inhibitory Concentrations (MICs) against various bacterial strains

  • Comparison with conventional antibiotics like vancomycin and oxacillin

  • Activity testing against antibiotic-resistant strains

• Mouse infection models:

  • Demonstrated activity comparable to vancomycin against methicillin-resistant Staphylococcus aureus (MRSA)

  • Supports the potential development of mutacin B-Ny266 as a clinical antibiotic

• Cell-free supernatant assays:

  • Used for preliminary screening of bacteriocin-like inhibitory substances

  • Extraction of secreted bioactive molecules responsible for anti-S. mutans activity

• Molecular interaction studies:

  • Analysis of binding to lipid II

  • Characterization of individual ring structures and their interactions with targets

These diverse experimental approaches have established mutacin B-Ny266 as a promising candidate for further development as an antimicrobial agent with potential applications in treating dental caries and other bacterial infections.

How can researchers study the regulatory mechanisms of mutacin B-Ny266 expression?

To investigate the regulatory mechanisms controlling mutacin B-Ny266 expression, researchers can employ several methodological approaches:

• Genetic analysis of the two-component system NsrRS:

  • Construction of deletion mutants for NsrRS components

  • Reporter gene assays to monitor activation of lanAA' expression

  • Analysis of differential activation by LanA versus LanA' peptides

• Transcriptional analysis:

  • qRT-PCR to quantify expression of genes in the mutacin B-Ny266 locus under various conditions

  • Promoter mapping to identify regulatory elements

  • RNA-seq to analyze the complete transcriptional landscape

• Protein-DNA interaction studies:

  • Electrophoretic mobility shift assays (EMSAs) to identify binding of regulatory proteins to promoter regions

  • DNase footprinting to identify specific binding sequences

  • Chromatin immunoprecipitation (ChIP) to analyze in vivo interactions

Understanding these regulatory mechanisms could provide insights for optimizing production of recombinant mutacin B-Ny266 and for engineering strains with enhanced production capabilities.

What techniques are used to analyze the post-translational modifications in mutacin B-Ny266?

The extensive post-translational modifications in lantibiotics like mutacin B-Ny266 require specialized analytical techniques:

• Mass spectrometry:

  • MALDI-TOF MS for molecular weight determination of modified peptides

  • Tandem MS (MS/MS) for structural characterization and identification of specific modifications

  • LC-MS/MS for comprehensive analysis of complex peptide mixtures

• NMR spectroscopy:

  • 3D NMR structural analyses to determine the three-dimensional conformation

  • Identification of lanthionine bridges and other modifications

  • Characterization of horseshoe-like conformations that protect from protease cleavage

• Chemical derivatization:

  • Specific reagents to identify dehydrated residues

  • Alkylation reactions to detect free sulfhydryl groups

  • Acid hydrolysis followed by amino acid analysis

• Recombinant expression systems:

  • Analysis of in vitro and in vivo modification by lantibiotic biosynthetic enzymes

  • Comparison of modification patterns with mutant peptides

  • Investigation of enzyme specificity for different substrates

These techniques allow for detailed characterization of the dehydration of serine and threonine residues, formation of lanthionine bridges, and creation of aminovinyl-cysteine rings that are characteristic of lantibiotics like mutacin B-Ny266.

How does mutacin B-Ny266 compare with other lantibiotics in structure and activity?

The following table compares key characteristics of mutacin B-Ny266 with other well-studied lantibiotics:

This comparison highlights the unique characteristics of mutacin B-Ny266, particularly its two-peptide nature and resistance to the development of bacterial resistance, which distinguish it from other lantibiotics and may contribute to its therapeutic potential .

What challenges exist in scaling up production of recombinant mutacin B-Ny266?

Researchers face several challenges when attempting to scale up production of recombinant mutacin B-Ny266:

• Post-translational modification complexity:

  • The extensive modifications by LanB and LanC enzymes appear to require the formation of a lantibiotic synthetase complex in the bacterial membrane

  • Cell extracts and recombinant epidermin LanB and LanC enzymes show no in vitro activity, unlike enzymes for some other lantibiotics

• Two-peptide system:

  • The need to produce both LanA and LanA' peptides and ensure proper processing of both components

  • Optimization of the ratio between the two peptides for maximum activity

• Production optimization:

  • While fermentation methods for mutacin B-Ny266 have demonstrated higher yields, further optimization is needed

  • Key components include 10% inoculums in late log growth and yeast extract as a media component

• Purification challenges:

  • Separation of the modified peptides from culture media

  • Maintaining biological activity during purification processes

  • Achieving pharmaceutical-grade purity

• Recombinant expression systems:

  • Selection of appropriate host organisms that can correctly process the lantibiotic

  • Engineering of synthetic gene clusters that include all necessary modification enzymes

Understanding these challenges is essential for developing efficient production methods for potential therapeutic or food preservation applications of mutacin B-Ny266.

What are promising applications of mutacin B-Ny266 beyond dental caries prevention?

While mutacin B-Ny266 shows clear potential for targeting caries pathogens embedded in dental plaque biofilm, several other promising applications deserve investigation:

• Treatment of antibiotic-resistant infections:

  • Activity against MRSA comparable to vancomycin in mouse models suggests potential for treating resistant staphylococcal infections

  • Effectiveness against vancomycin-resistant enterococci and oxacillin-resistant strains indicates potential for addressing multidrug-resistant pathogens

• Food preservation:

  • Mutacin B-Ny266 is related to nisin, the only purified bacteriocin approved for use as a food additive

  • Its activity against foodborne pathogens and the difficulty in obtaining resistant mutants make it an excellent candidate for food biopreservation

  • Its nanomolar activity range suggests economic feasibility

• Targeted antimicrobial therapy:

  • The specificity of mutacin B-Ny266 could allow for targeted elimination of pathogens while preserving beneficial microbiota

  • Potential applications in microbiome management and precision antimicrobial approaches

• Biofilm disruption:

  • Beyond dental plaque, activity against biofilm-forming pathogens could address infections associated with medical devices and chronic wounds

  • The lipid II binding mechanism may be particularly effective against bacteria in biofilm states

• Peptide engineering platform:

  • The modification machinery for mutacin B-Ny266 could serve as a platform for engineering novel antimicrobial peptides with designer properties

  • The two-peptide system offers unique opportunities for creating synergistic combinations

Each of these applications builds on the fundamental understanding of mutacin B-Ny266's structure, function, and mechanism of action, highlighting the importance of continued basic research in this field.