Lanthionine-containing Peptide Antibiotics Research Articles

An Engineered Double Lipid II Binding Motifs-Containing Lantibiotic Displays Potent and Selective Antimicrobial Activity against Enterococcus faecium

Lipid II is an essential precursor for bacterial cell wall biosynthesis and thereby an important target for various antibiotics. Several lanthionine-containing peptide antibiotics target lipid II with lanthionine-stabilized lipid II binding motifs. Here, we used the biosynthesis system of the lantibiotic nisin to synthesize a two-lipid II binding motifs-containing lantibiotic, termed TL19, which contains the N-terminal lipid II binding motif of nisin and the distinct C-terminal lipid II binding motif of one peptide of the two-component haloduracin (i.e., HalA1). Further characterization demonstrated that (i) TL19 exerts 64-fold stronger antimicrobial activity against Enterococcus faecium than nisin(1-22), which has only one lipid II binding site, and (ii) both the N- and C-terminal domains are essential for the potent antimicrobial activity of TL19, as evidenced by mutagenesis of each single and the double domains. These results show the feasibility of a new approach to synthesize potent lantibiotics with two different lipid II binding motifs to treat specific antibiotic-resistant pathogens.

Discovery and development of lantibiotics; antimicrobial agents that have significant potential for medical application

Introduction: Antimicrobial drug resistance is driving the need for novel therapeutics. Amongst the most promising antibacterial agents that are being investigated as replacements for current therapeutic antibiotics are antibacterial peptides, such as the lanthionine-containing peptide antibiotics (lantibiotics).Areas covered: This review focuses on the current methods used for discovery of potentially exploitable lantibiotics for medical applications and discusses relevant recent innovations that will have a positive impact on the discovery of useful lantibiotics.Expert opinion: Recent technological advances in a number of fields mean that increased research into the identification and characterisation of new lantibiotics is feasible. We need to increase our understanding of the various mechanisms of antibacterial action exhibited by lantibiotics and apply this knowledge to peptide engineering or novel practical applications. The advent of next-generation sequencing approaches now negate the need for extensive reverse genetics and employment of bioinformatics approaches is greatly assisting the identification of potentially useful inhibitors in the genomes of a range of clinically significant bacteria. These advances in genetic analysis and engineering will facilitate increased exploitation of lantibiotics in medical therapy.

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Insights into In Vivo Activities of Lantibiotics from Gallidermin and Epidermin Mode-of-Action Studies

The activity of lanthionine-containing peptide antibiotics (lantibiotics) is based on different killing mechanisms which may be combined in one molecule. The prototype lantibiotic nisin inhibits peptidoglycan synthesis and forms pores through specific interaction with the cell wall precursor lipid II. Gallidermin and epidermin possess the same putative lipid II binding motif as nisin; however, both peptides are considerably shorter (22 amino acids, compared to 34 in nisin). We demonstrate that in model membranes, lipid II-mediated pore formation by gallidermin depends on membrane thickness. With intact cells, pore formation was less pronounced than for nisin and occurred only in some strains. In Lactococcus lactis subsp. cremoris HP, gallidermin was not able to release K+, and a mutant peptide, [A12L]gallidermin, in which the ability to form pores was disrupted, was as potent as wild-type gallidermin, indicating that pore formation does not contribute to killing. In contrast, nisin rapidly formed pores in the L. lactis strain; however, it was approximately 10-fold less effective in killing. The superior activity of gallidermin in a cell wall biosynthesis assay may help to explain this high potency. Generally, it appears that the multiple activities of lantibiotics combine differently for individual target strains.

NisT, the Transporter of the Lantibiotic Nisin, Can Transport Fully Modified, Dehydrated, and Unmodified Prenisin and Fusions of the Leader Peptide with Non-lantibiotic Peptides

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

Evidence for a multimeric subtilin synthetase complex.

Subtilin is a lanthionine-containing peptide antibiotic (lantibiotic) produced by Bacillus subtilis. It is ribosomally synthesized as a prepeptide and modified posttranslationally. Three proteins of the subtilin gene cluster (SpaB, SpaC, and SpaT) which are probably involved in prepeptide modification and transport have been identified genetically (C. Klein, C. Kaletta, N. Schnell, and K.-D. Entian, Appl. Environ. Microbiol. 58: 132-142, 1992). Immunoblot analysis revealed that production of SpaC is strongly regulated (Z. Gutowski-Eckel, C. Klein, K. Siegers, K. Bohm, M. Hammelmann, and K.-D. Entian, Appl. Environ. Microbiol. 60:1-11, 1994). Transcription of the SpaC protein started in the late logarithmic growth phase, reaching a maximum in the early stationary growth phase. No SpaC was detectable in the early logarithmic growth phase. Deletions within the spaR and spaK genes, which act as a two-component regulatory system, resulted in failure to express SpaB and SpaC, indicating that these two genes are the regulatory targets. Western blot analysis of vesicle preparations of B. subtilis revealed that the SpaB, SpaT, and SpaC proteins are membrane bound, although some of the protein was also detectable in cell extracts. By using the yeast two-hybrid analysis system for protein interactions, we showed that a complex of at least two each of SpaT, SpaB, and SpaC is most probably associated with the substrate SpaS. These results were also confirmed by coimmunoprecipitation experiments. In these cosedimentation experiments, SpaB and SpaC were coprecipitated by antisera against SpaC, SpaB, and SpaT, as well as by a monoclonal antibody against epitope-tagged SpaS, indicating that these four proteins are associated.

Effects of the Lantibiotic Mersacidin on the Morphology of Staphylococci

Mersacidin is a lanthionine-containing peptide antibiotic (lantibiotic), able to inhibit the growth of a number of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) in a manner similar to, but distinct from, vancomycin. In order to further understand the mode of action of this lantibiotic, Staphylococcus simulans 22 cells were treated either with the antibiotics penicillin, tunicamycin or vancomycin or with mersacidin and then compared with untreated cells after electron microscopic examination. Mersacidin treatment brought about a time-dependent, generalised decrease in the thickness of the bacterial cell wall. In addition, mersacidin treatment caused a roughening of the cell wall surface layer and also reduced the thickness and frequency of formation of dividing cell septa. Reduction of cell wall thickness appears to result from inhibition of new wall biosynthesis combined with cell wall turnover. These features of mersacidin-induced effects on cell morphology confirm that it has a novel mode of action ( Brötz, H., G. Bierbaum, A. Markus, E. Molitor, and H.-G. Sahl: Antimicrob. Agents Chemother. 39 [1995] 714-719), probably directed towards a membrane-bound biosynthetic step but not towards a specific penicillin-binding-protein.

Cloning, sequencing and production of the lantibiotic mersacidin

Mersacidin is a lanthionine-containing peptide antibiotic that shows a good in vivo efficiency against methicillin-resistant Staphylococcus aureus. It is excreted during early stationary phase and could be purified from culture supernatant in a one-step procedure by reversed phase HPLC. Its structural gene was cloned from chromosomal DNA of the producer strain Bacillus subtilis HIL Y-85,54728. Sequencing revealed that pre-mersacidin consists of an unusually long 48 amino acid leader sequence and a 20 amino acid propeptide part which is modified during biosynthesis to the mature lantibiotic. The comparison of the mersacidin prepeptide with those of hitherto known lantibiotics demonstrates that mersacidin is more closely related to type B lantibiotic cinnamycin than to type A lantibiotics.

Cloning, sequencing and production of the lantibiotic mersacidin.

Mersacidin is a lanthionine-containing peptide antibiotic that shows a good in vivo efficiency against methicillin-resistant Staphylococcus aureus. It is excreted during early stationary phase and could be purified from culture supernatant in a one-step procedure by reversed phase HPLC. Its structural gene was cloned from chromosomal DNA of the producer strain Bacillus subtilis HIL Y-85,54728. Sequencing revealed that pre-mersacidin consists of an unusually long 48 amino acid leader sequence and a 20 amino acid propeptide part which is modified during biosynthesis to the mature lantibiotic. The comparison of the mersacidin prepeptide with those of hitherto known lantibiotics demonstrates that mersacidin is more closely related to type B lantibiotic cinnamycin than to type A lantibiotics.

Influence of the phosphorus and nitrogen source on nisin production in Lactococcus lactis subsp. lactis batch fermentations using a complex medium

The influence of different phosphorus and nitrogen sources on Lactococcus lactis subsp. lactis NIZO 22186 growth and nisin production was studied in batch fermentations using a complex medium. KH2PO4 was found to be the best phosphorus source for nisin production. Increasing initial phosphate concentrations from 0 to 5% KH2PO4 exerted a double effect, creating favourable pH conditions and particularly stimulating the nisin production levels, which were highest at 5% KH2PO4. Up to now, no such high initial phosphate concentrations have been reported for the production of other antibiotics or bacteriocins. Nisin, a lanthionine-containing peptide antibiotic with bacteriocin properties, clearly behaved as a primary metabolite, since its formation was linked with active growth and was not suppressed by phosphate concentrations up to 5%. A complex medium supplemented with cotton seed meal as nitrogen source also gave very high nisin yields.

Mode of action of the lanthionine-containing peptide antibiotics duramycin, duramycin B and C, and cinnamycin as indirect inhibitors of phospholipase A 2

Effects of the lanthionine-containing peptide antibiotics duramycin, duramycin B, duramycin C and cinnamycin on the activity of phospholipase A 2 from six different sources were studied, and their mode of action was investigated. The four antibiotics inhibited potently all tested phospholipases A 2, with IC 50 values of around 1 μM, using phosphatidylethanolamine or [1- 14C]oleate-labelled Escherichia coli, whose phospholipids are rich in phosphatidylethanolamine, as substrates. No inhibition was observed when the substrate was phosphatidylcholine. Binding of the antibiotics to the lipid fraction of E. coli could be demonstrated by co-sedimentation with whole, but not with lipid-depleted E. coli. In addition, preincubation of duramycin B with vesicles of phosphatidylethanolamine, but not those of phosphatidylcholine, prevented the inhibition of phospholipase A 2 activity. The interaction of duramycin B and C, but not that of the biologically inactive compounds actagardine and the duramycin B trisulphoxide, with phosphatidylethanolamine was demonstrated using circular dichroism studies. On the other hand, no interaction of duramycin B with phosphatidylcholine could be demonstrated. A strict correlation between the physico-chemical interaction of the studied lantibiotics, demonstrated by circular dichroism spectroscopy, and their inhibition of phospholipase A 2 was observed. These results suggest that lanthionine-containing peptide antibiotics inhibit phospholipase A 2 indirectly by specifically sequestering the substrate phosphatidylethanolamine. This mode of action is analogous to the one described for the protein lipocortin.

The structure of PA48009: The revised structure of duramycin.

PA48009, a lanthionine-containing peptide antibiotic was isolated from the culture broth of Streptoverticillium griseoverticillatum PA-48009, and identified as duramycin. Determination of the structure using both Edman degradations and 2D NMR spectroscopy showed the need to revise the structure of duramycin given in literature. Duramycin (PA48009) was different from lanthiopeptin (Ro 09-0198, cinnamycin) only by a Lys/Arg exchange at position 2.

Sequence determination of actagardine, a novel lantibiotic, by homonuclear 2D NMR spectroscopy.

By combination of several 1H NMR techniques, the sequence of actagardine has been elucidated. It has been shown that it is a tetracyclic 19-residue peptide antibiotic. It differs from the previously described lanthionine-containing peptide antibiotics belonging to the lantibiotic class.

Gallidermin: a new lanthionine-containing polypeptide antibiotic.

Gallidermin is a new member of the class of lanthionine-containing peptide antibiotics, which are summarized under the common name lantibiotics. The lantibiotic gallidermin is produced by Staphylococcus gallinarum (F16/P57) Tü3928, and it exhibits activities against the Propionibacteria, involved in acne disease. Gallidermin differs from the recently discovered tetracyclic 21-residue peptide antibiotic epidermin only in a Leu/Ile exchange in position 6. The isolation procedures for gallidermin included adsorption directly from the culture broth, ion-exchange chromatography of the amphiphilic and basic polypeptide followed by desalting, and final purification by reversed-phase HPLC. The structural elucidation of the polypeptide containing four thioether bridges involved mainly a combination of automated gas-phase sequencing, thermospray liquid chromatography/mass spectrometry and fast-atom-bombardment mass spectrometry.