Stephen A. Cochrane

Lipopeptides from Bacillus and Paenibacillus spp.: A Gold Mine of Antibiotic Candidates

The emergence of multidrug‐resistant bacteria has placed a strain on health care systems and highlighted the need for new classes of antibiotics. Bacterial lipopeptides are secondary metabolites, generally produced by nonribosomal peptide synthetases that often exhibit broad‐spectrum antimicrobial activity. Only two new structural types of antibiotics have entered the market in the last 40 years, linezolid and the bacterial lipopeptide daptomycin. A wide variety of bacteria produce lipopeptides, however Bacillus and Paenibacillus spp. in particular have yielded several potent antimicrobial lipopeptides. Many of the lipopeptides produced by these bacteria have been known for decades and represent a potential gold mine of antibiotic candidates. This list includes the polymyxins, octapeptins, polypeptins, iturins, surfactins, fengycins, fusaricidins, and tridecaptins, as well as some novel examples, including the kurstakins. These lipopeptides have a wide variety of activities, ranging from antibacterial and antifungal, to anticancer and antiviral. This review presents a reasonably comprehensive list of each class of lipopeptide and their known homologues. Emphasis has been placed on their antimicrobial activities, as well other potential applications for this interesting class of substances.

Solid Supported Chemical Syntheses of Both Components of the Lantibiotic Lacticin 3147

Lantibiotics are antimicrobial peptides produced by bacteria. Some are employed for food preservation, whereas others have therapeutic potential due to their activity against organisms resistant to current antibiotics. They are ribosomally synthesized and posttranslationally modified by dehydration of serine and threonine residues followed by attack of thiols of cysteines to form monosulfide lanthionine and methyllanthionine rings, respectively. Chemical synthesis of peptide analogues is a powerful method to verify stereochemistry and access structure-activity relationships. However, solid supported synthesis of lantibiotics has been difficult due to problems in generating lanthionines and methyllanthionines with orthogonal protection and good stereochemical control. We report the solid-phase syntheses of both peptides of a two-component lantibiotic, lacticin 3147. Both successive and interlocking ring systems were synthesized on-resin, thereby providing a general methodology for this family of natural products.

Biochemical, structural, and genetic characterization of tridecaptin A₁, an antagonist of Campylobacter jejuni.

Bacillus circulans NRRL B‐30644 (now Paenibacillus terrae) was previously reported to produce SRCAM 1580, a bacteriocin active against the food pathogen Campylobacter jejuni. We have been unable to isolate SRCAM 1580, and did not find any genetic determinants in the genome of this strain. We now report the reassignment of this activity to the lipopeptide tridecaptin A1. Structural characterization of tridecaptin A1 was achieved through NMR, MS/MS and GC‐MS studies. The structure was confirmed through the first chemical synthesis of tridecaptin A1, which also revealed the stereochemistry of the lipid chain. The impact of this stereochemistry on antimicrobial activity was examined. The biosynthetic machinery responsible for tridecaptin production was identified through bioinformatic analyses. P. terrae NRRL B‐30644 also produces paenicidin B, a novel lantibiotic active against Gram‐positive bacteria. MS/MS analyses indicate that this lantibiotic is structurally similar to paenicidin A.

Synthesis and structure-activity relationship studies of N-terminal analogues of the antimicrobial peptide tridecaptin A(1)

Chemical synthesis was used to increase the potency of the antimicrobial lipopeptide tridecaptin A1. Lipid tail modification proved to be an ideal platform for synthesizing structurally simpler analogues that are not readily accessible by isolation. The stereochemical elements of the tridecaptin A1 lipid tail are not essential for antimicrobial activity and could be replaced with hydrophobic aliphatic or aromatic groups. Some simpler analogues displayed potent antimicrobial activity against Gram-negative bacteria, including Campylobacter jejuni, Escherichia coli O157:H7, and multidrug resistant Klebsiella pneumoniae.

Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II

Significance The increasing development of antimicrobial resistance is a major global concern, and there is an urgent need for the development of new antibiotics. We show that the antimicrobial lipopeptide tridecaptin A1 selectively binds to the Gram-negative analogue of peptidoglycan precursor lipid II, disrupting the proton motive force and killing Gram-negative bacteria. We present an example of the selective targeting of Gram-negative lipid II and a binding mode to this peptidoglycan precursor. No persistent resistance develops against tridecaptin A1 in Escherichia coli cells exposed to subinhibitory concentrations of this peptide during a 1-mo period. This study showcases the excellent antibiotic properties of the tridecaptins in an age where new antibiotics that target Gram-negative bacteria are desperately needed. Tridecaptin A1 (TriA1) is a nonribosomal lipopeptide with selective antimicrobial activity against Gram-negative bacteria. Here we show that TriA1 exerts its bactericidal effect by binding to the bacterial cell-wall precursor lipid II on the inner membrane, disrupting the proton motive force. Biochemical and biophysical assays show that binding to the Gram-negative variant of lipid II is required for membrane disruption and that only the proton gradient is dispersed. The NMR solution structure of TriA1 in dodecylphosphocholine micelles with lipid II has been determined, and molecular modeling was used to provide a structural model of the TriA1–lipid II complex. These results suggest that TriA1 kills Gram-negative bacteria by a mechanism of action using a lipid-II–binding motif.

Synthesis of Tridecaptin–Antibiotic Conjugates with in Vivo Activity against Gram-Negative Bacteria

A series of tridecaptin-antibiotic conjugates were synthesized and evaluated for in vitro and in vivo activity against Gram-negative bacteria. Covalently linking unacylated tridecaptin A1 (H-TriA1) to rifampicin, vancomycin, and erythromycin enhanced their activity in vitro but not by the same magnitude as coadministration of the peptide and these antibiotics. The antimicrobial activities of the conjugates were retained in vivo, with the H-TriA1-erythromycin conjugate proving a more effective treatment of Klebseilla pneumoniae infections in mice than erythromycin alone or in combination with H-TriA1.

Unacylated tridecaptin A1 acts as an effective sensitiser of Gram-negative bacteria to other antibiotics

A derivative of the linear cationic lipopeptide tridecaptin A₁missing the N-terminal lipophilic acyl group, termed H-TriA₁, is devoid of antimicrobial activity but is extremely effective at sensitising Gram-negative bacteria to certain antibiotics. H-TriA₁has low cytotoxicity compared with the natural peptide and in low concentrations it can substantially lower the minimum inhibitory concentration (MIC) of some antibiotics against strains of Escherichia coli, Campylobacter jejuni and Klebsiella pneumoniae. In particular, the MIC of rifampicin was lowered 256-512-fold against K. pneumoniae strains using low concentrations of H-TriA₁. H-TriA₁does not exert its synergistic effect through partial membrane lysis, but does bind to model bacterial membranes in a manner akin to the natural peptide. Formation of this stable secondary structure on the outer membrane may account for the observed synergistic activity.

Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis)

Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio‐activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3‐produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (−)‐drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (−)‐drimenol synthesis suggests that drimenol synthase is likely to use a protonation‐initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (−)‐drimenol, from which useful drimane‐type terpenes can be synthesized.

Key Residues in Octyl-Tridecaptin A1 Analogues Linked to Stable Secondary Structures in the Membrane

Tridecaptin A1 is a linear antimicrobial lipopeptide comprised of 13 amino acids, including three diaminobutyric acid (Dab) residues. It displays potent activity against Gram‐negative bacteria, including multidrug‐resistant strains. Using solid‐phase peptide synthesis, we performed an alanine scan of a fully active analogue, octyl‐tridecaptin A1, to determine key residues responsible for activity. The synthetic analogues were tested against ten organisms, both Gram‐positive and Gram‐negative bacteria. Modification of D‐Dab8 abolished activity, and marked decreases were observed with substitution of D‐allo‐Ile12 and D‐Trp5. Circular dichroism showed that octyl‐tridecaptin A1 adopts a secondary structure in the presence of model phospholipid membranes, which was weakened by D‐Dab8‐D‐Ala, D‐allo‐Ile12‐D‐Ala, and D‐Trp5‐D‐Ala substitutions. The antimicrobial activity of the analogues is directly correlated to their ability to adopt a stable secondary structure in a membrane environment.

Insights into the Mechanism of Action of the Two-Peptide Lantibiotic Lacticin 3147

Lacticin 3147 is a two peptide lantibiotc (LtnA1 and LtnA2) that displays nanomolar activity against many Gram-positive bacteria. Lacticin 3147 may exert its antimicrobial effect by several mechanisms. Isothermal titration calorimetry experiments show that only LtnA1 binds to the peptidoglycan precursor lipid II, which could inhibit peptidoglycan biosynthesis. An experimentally supported model of the resulting complex suggests that the key binding partners are the C-terminus of LtnA1 and pyrophosphate of lipid II. A combination of in vivo and in vitro assays indicates that LtnA1 and LtnA2 can induce rapid membrane lysis without the need for lipid II binding. However, the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this synergistic activity.