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Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability.

ABSTRACT: Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in the short half-life in physiological settings. Previous mutagenesis work on mutacin 1140 has been limited to the lanthionine-forming residues, the C-terminally decarboxylated residue, and single amino acid substitutions at residues Phe1, Trp4, Dha5, and Arg13. To study the importance of the dehydrated (Dha5 and Dhb14) and protease-susceptible (Lys2 and Arg13) residues within mutacin 1140 for stability and bioactivity, each of these residues was evaluated for its impact on production and inhibitory activity. More than 15 analogs were purified, enabling direct comparison of the activities against a select panel of Gram-positive bacteria. The efficiency of the posttranslational modification (PTM) machinery of mutacin 1140 is highly restricted on its substrate. Analogs in the various intermediate stages of PTMs were observed as minor products following single point mutations at the 2nd, 5th, 13th, and 14th positions. The combination of alanine substitutions at the Dha5 and Dhb14 positions abolished mutacin 1140 production, while the production was restored by substitution of a Gly residue at one of these positions. Analogs with improved activity, productivity, and proteolytic stability were identified.IMPORTANCE Our findings show that the efficiency of mutacin 1140 PTMs is highly dependent on the core peptide sequence. Analogs in various intermediate stages of PTMs can be transported by the bacterium, which indicates that PTMs and transport are finely tuned for the native mutacin 1140 core peptide. Only certain combinations of amino acid substitutions at the Dha5 and Dhb14 dehydrated residue positions were tolerated. Observation of glutamylated core peptide analogs shows that dehydrations occur in a glutamate-dependent manner. Interestingly, mutations at positions outside rings A and B, the lipid II binding domain, would interfere with lipid II binding. Purified mutacin 1140 analogs have various activities and selectivities against different genera of bacteria, supporting the effort to generate analogs with higher specificity against pathogenic bacteria. The discovery of analogs with improved inhibitory activity against pathogenic bacteria, increased stability in the presence of protease, and higher product yields may promote the clinical development of this unique antimicrobial compound.

SUBMITTER: Geng M

PROVIDER: S-EPMC6052277 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability.

Geng Mengxin M Smith Leif L

Applied and environmental microbiology 20180717 15

Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in t ...[more]

PMID: 29776930

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Project description:Lantibiotics offer an untapped pipeline for the development of novel antibiotics to treat serious Gram-positive (+) infections including Clostridium difficile. Mutacin 1140 (MU1140) is a lantibiotic produced by Streptococcus mutans and acts via a novel mechanism of action, which may limit the development of resistance. This study sought to identify a lead compound for the treatment of C. difficile associated diarrhea (CDAD). Compounds were selected from a saturation mutagenesis library of 418 single amino acid variants of MU1140. Compounds were produced by small scale fermentation, purified, characterized and then subjected to a panel of assays aimed at identifying the best performers. The screening assays included: in vitro susceptibility testing [MIC against Micrococcus luteus, Clostridium difficile, vancomycin-resistant enterococci (VRE), Staphylococcus aureus, Streptococcus pneumonia, Mycobacterium phlei, and Pseudomonas aeruginosa; cytotoxicity screening on HepG2 hepatocytes; in vitro pharmacological profiling with the Safety Screen 44TM, metabolic and chemical stability in biologically relevant fluids (FaSSGF, FaSSIF and serum); and efficacy in vivo]. Several lantibiotic compounds had better MIC against C. difficile, compared to vancomycin, but not against other bacterial species tested. The Safety Screen 44TMin vitro pharmacological profiling assay suggested that this class of compounds has relatively low overall toxicity and that compound OG253 (MU1140, Phe1Ile) is not likely to present inadvertent off-target effects, as evidenced by a low promiscuity score. The in vitro cytotoxicity assay also indicated that this class of compounds was characterized by low toxicity; the EC50 of OG253 was 636 mg/mL on HepG2 cells. The half-life in simulated gastric fluid was >240 min. for all compound tested. The stability in simulated intestinal fluid ranged between a half-life of 5 min to >240 min, and paralleled the half-life in serum. OG253 ultimately emerged as the lead compound based on superior in vivo efficacy along with an apparent lack of relapse in a hamster model of infection. The lessons learned from this report are applicable to therapeutic lanthipeptides in general and may assist in the design of novel molecules with improved pharmacological, therapeutic and physicochemical profiles. The data presented also support the continued clinical development of OG253 as a novel antibiotic against CDAD that could prevent recurrence of the infection.

Project description:UNLABELLED:Lantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that is cleaved to yield the active antibacterial peptide. Significant advancements in molecular tools that promote the study of lantibiotic biosynthesis can be used in Streptococcus mutans. Herein, we further our understanding of leader peptide sequence and core peptide structural requirements for the biosynthesis and transport of the lantibiotic mutacin 1140. Our study on mutacin 1140 biosynthesis shows a dedicated secondary cleavage site within the leader peptide and the dependency of transport on core peptide posttranslational modifications (PTMs). The secondary cleavage site on the leader peptide is found at the -9 position, and secondary cleavage occurs before the core peptide is transported out of the cell. The coordinated cleavage at the -9 position was absent in a lanT deletion strain, suggesting that the core peptide interaction with the LanT transporter enables uniform cleavage at the -9 position. Following transport, the LanP protease was found to be tolerant to a wide variety of amino acid substitutions at the primary leader peptide cleavage site, with the exception of arginine at the -1 position. Several leader and core peptide mutations produced core peptide variants that had intermediate stages of PTM enzyme modifications, supporting the concept that PTM enzyme modifications, secondary cleavage, and transport are occurring in a highly coordinated fashion. IMPORTANCE:Mutacin 1140 belongs to the class I lantibiotic family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The biosynthesis of mutacin 1140 is a highly efficient process which does not lead to a discernible level of production of partially modified core peptide variants. The products isolated from an extensive mutagenesis study on the leader and core peptides of mutacin 1140 show that the posttranslational modifications (PTMs) on the core peptide occur under a highly coordinated dynamic process. PTMs are dictated by the distance of the core peptide modifiable residues from PTM enzyme active sites. The formation of lanthionine rings aids in the formation of successive PTMs, as was observed in a peptide variant lacking a C-terminal decarboxylation.

Project description:Mutacin 1140 belongs to the epidermin group of lantibiotics. Epidermin class lantibiotics are ribosomally synthesized and posttranslationally modified antibiotics with potent activity against Gram-positive bacteria. In particular, this class is effective at targeting drug-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium tuberculosis, and Clostridium difficile A C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) residue is derived from a decarboxylation of a terminal cysteine that is involved in lanthionine ring formation. Studies on mutacin 1140 have revealed new insight into the structural importance of the C-terminal AviCys residue. A C-terminal carboxyl analogue of mutacin 1140 was engineered. Capping the C-terminal carboxyl group with a primary amine restores bioactivity and affords a novel opportunity to synthesize new analogues. A C-terminal fluorescein-labeled mutacin 1140 analogue traps lipid II into a large lipid II lantibiotic complex, similar to that observed in vivo for the lantibiotic nisin. A C-terminal carboxyl analogue of mutacin 1140 competitively inhibits the activity of native mutacin 1140 and nisin. The presence of a C-terminal carboxyl group prevents the formation of the large lipid II lantibiotic complexes but does not prevent the binding of the lantibiotic to lipid II.IMPORTANCE This study addressed the importance of the C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) residue for antibacterial activity. We have learned that the posttranslational modification for making the AviCys residue is presumably important for the lateral assembly mechanism of activity that traps lipid II into a large complex. The C-terminal carboxyl analogue of this class of lantibiotics is agreeable to the addition of a wide variety of substrates. The addition of fluorescein enabled in vivo visualization of the epidermin class of lantibiotics in action. These results are significant because, as we demonstrate, the presence of the AviCys residue is not essential for bioactivity, but, more importantly, the removal of the carboxyl group is essential. The ability to make a C-terminal carboxyl analogue that is modifiable will facilitate the synthesis of novel analogues of the epidermin class of lantibiotics that can be developed for new applications.

Project description:Owing to extensive metagenomic studies, we now have access to numerous sequences of novel bacteriocin-like antimicrobial peptides encoded by various cultivable and noncultivable bacteria. However, relatively rarely, we even have access to these cultivable strains to examine the potency and the targets of the predicted bacteriocins. In this study, we evaluated a heterologous biosynthetic system to produce biologically active nonnative novel lantibiotics, which are modified bacteriocins. We chose Streptococcus mutans, a dental pathogen, as the host organism because it is genetically easy to manipulate and is inherently a prolific producer of various bacteriocins. We chose the S. mutans T8 strain as the host, which produces the lantibiotic mutacin II, to express 10 selected homologs of mutacin II identified from GenBank. These lantibiotic peptides either are novel or have been studied very minimally. The core regions of the selected lantibiotic peptides were fused to the leader sequence of the mutacin II peptide and integrated into the chromosome such that the core region of the native mutacin II was replaced with the new core sequences. By this approach, using the mutacin II biosynthesis machinery, we obtained one bioactive novel lantibiotic peptide with 52% different residues compared to the mutacin II core region. This unknown lantibiotic is encoded by Streptococcus agalactiae and Streptococcus ovuberis strains. Since this peptide displays some homology with nukacin ISK-1, we named it nukacin Spp. 2. This study demonstrated that the mutacin II biosynthesis machinery can be successfully used as an efficient system for the production of biologically active novel lantibiotics. IMPORTANCE In this study, we report for the first time that Streptococcus mutans can be used as a host to produce various nonnative lantibiotics. We showed that in the T8 strain, we could produce bioactive lacticin 481 and nukacin ISK-1, both of which are homologs of mutacin II, using T8's modification and secretion apparatus. Similarly, we also synthesized a novel bioactive lantibiotic, which we named nukacin Spp. 2.

Project description:Bacteriocins are ribosomally synthesized proteinaceous antibacterial peptides. They selectively interfere with the growth of other bacteria. The production and secretion of bacteriocins confer a distinct ecological advantage to the producer in competing against other bacteria that are present in the same ecological niche. Streptococcus mutans, a significant contributor to the development of dental caries, is one of the most prolific producers of bacteriocins, known as mutacins in S. mutans In this study, we characterized the locus encoding mutacin B-Ny266, a lantibiotic with a broad spectrum of activity. The chromosomal locus is composed of six predicted operon structures encoding proteins involved in regulation, antimicrobial activity, biosynthesis, modification, transport, and immunity. Mutacin B-Ny266 was purified from semisolid cultures, and two inhibitory peptides, LanA and LanA', were detected. Both peptides were highly modified. Such modifications include dehydration of serine and threonine and the formation of a C-terminal aminovinyl-cysteine (AviCys) ring. While LanA peptide alone is absolutely required for antimicrobial activity, the presence of LanA' enhanced the activity of LanA, suggesting that B-Ny266 may function as a two-peptide lantibiotic. The activation of lanAA' expression is most likely controlled by the conserved two-component system NsrRS, which is activated by LanA peptide but not by LanA'. The chromosomal locus encoding mutacin B-Ny266 was not universally conserved in all sequenced S. mutans genomes. Intriguingly, the genes encoding LanAA' peptides were restricted to the most invasive serotypes of S. mutans IMPORTANCE Although dental caries is largely preventable, it remains the most common and costly infectious disease worldwide. Caries is initiated by the presence of dental plaque biofilm that contains Streptococcus mutans, a species extensively characterized by its role in caries development and formation. S. mutans deploys an arsenal of strategies to establish itself within the oral cavity. One of them is the production of bacteriocins that confer a competitive advantage by targeting and killing closely related competitors. In this work, we found that mutacin B-Ny266 is a potent lantibiotic that is effective at killing a wide array of oral streptococci, including nearly all S. mutans strains tested. Lantibiotics produced by oral bacteria could represent a promising strategy to target caries pathogens embedded in dental plaque biofilm.

Dataset Information

Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability.

Publications

Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability.

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