Teixobactin kills bacteria by a two-pronged attack on the cell envelope.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

Synthesis and Stereochemical Determination of the Peptide Antibiotic Novo29.

This paper describes the synthesis and stereochemical determination of Novo29 (clovibactin), a new peptide antibiotic that is related to teixobactin and is active against Gram-positive bacteria. Novo29 is an eight-residue depsipeptide that contains the noncanonical amino acid hydroxyasparagine of hitherto undetermined stereochemistry in a macrolactone ring. The amino acid building blocks Fmoc-(2R,3R)-hydroxyasparagine-OH and Fmoc-(2R,3S)-hydroxyasparagine-OH were synthesized from (R,R)- and (S,S)-diethyl tartrate. Novo29 and epi-Novo29 were then prepared by solid-phase peptide synthesis using these building blocks. Correlation with an authentic sample of Novo29 through 1H NMR spectroscopy, LC-MS, and in vitro antibiotic activity established that Novo29 contains (2R,3R)-hydroxyasparagine. X-ray crystallography reveals that epi-Novo29 adopts an amphiphilic conformation, with a hydrophobic surface and a hydrophilic surface. Four sets of epi-Novo29 molecules pack in the crystal lattice to form a hydrophobic core. The macrolactone ring adopts a conformation in which the main-chain amide NH groups converge to create a cavity, which binds ordered water and acetate anion. The amphiphilic conformation of epi-Novo29 is reminiscent of the amphiphilic conformation adopted by the related antibiotic teixobactin and its derivatives, which contains a hydrophobic surface that interacts with the lipids of the bacterial cell membrane and a hydrophilic surface that interacts with the aqueous environment. Molecular modeling suggests that Novo29 can adopt an amphiphilic conformation similar to teixobactin, suggesting that Novo29 may interact with bacteria in a similar fashion to teixobactin.

A new antibiotic kills pathogens without detectable resistance.

Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Most antibiotics were produced by screening soil microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approximately 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.

Biosynthesis and Mechanism of Action of the Cell Wall Targeting Antibiotic Hypeptin.

Hypeptin is a cyclodepsipeptide antibiotic produced by Lysobacter sp. K5869, isolated from an environmental sample by the iChip technology, dedicated to the cultivation of previously uncultured microorganisms. Hypeptin shares structural features with teixobactin and exhibits potent activity against a broad spectrum of gram-positive pathogens. Using comprehensive in vivo and in vitro analyses, we show that hypeptin blocks bacterial cell wall biosynthesis by binding to multiple undecaprenyl pyrophosphate-containing biosynthesis intermediates, forming a stoichiometric 2:1 complex. Resistance to hypeptin did not readily develop in vitro. Analysis of the hypeptin biosynthetic gene cluster (BGC) supported a model for the synthesis of the octapeptide. Within the BGC, two hydroxylases were identified and characterized, responsible for the stereoselective ß-hydroxylation of four building blocks when bound to peptidyl carrier proteins. In vitro hydroxylation assays corroborate the biosynthetic hypothesis and lead to the proposal of a refined structure for hypeptin.

Crystal structure of the N-terminal domain of MtClpC1 in complex with the anti-mycobacterial natural peptide Lassomycin.

Antibiotics form our frontline therapy against disease-causing bacteria. Unfortunately, antibiotic resistance is becoming more common, threatening a future where these medications can no longer cure infections. Furthermore, the emergence of multidrug-resistant (MDR), totally drug-resistant (TDR), and extensively drug-resistant (XDR) tuberculosis has increased the urgency of discovering new therapeutic leads with unique modes of action. Some natural peptides derived from actinomycetes, such as Cyclomarin A, Lassomycin, Rufomycin I, and Ecumicin, have potent and specific bactericidal activity against Mycobacterium tuberculosis, with the specificity owing to the fact that these peptides target the ClpC1 ATPase, an essential enzyme in mycobacteria, and inhibit/activate the proteolytic activity of the ClpC1/P1/P2 complex that participates in protein homeostasis. Here, we report the high-resolution crystal structure of the N-terminal domain of ClpC1 (ClpC1 NTD) in complex with Lassomycin, showing the specific binding mode of Lassomycin. In addition, the work also compares the Lassomycin complex structure with the previously known structures of ClpC1 NTD in complex with other natural peptides such as Cyclomarin A, Rufomycin I, and Ecumicin.

Teixobactin Provides Protection against Inhalation Anthrax in the Rabbit Model.

The use of antibiotics is a vital means of treating infections caused by the bacteria Bacillus (B.) anthracis. Importantly, with the potential future use of multidrug-resistant strains of B. anthracis as bioweapons, new antibiotics are needed as alternative therapeutics. In this blinded study, we assessed the protective efficacy of teixobactin, a recently discovered antibiotic, against inhalation anthrax infection in the adult rabbit model. New Zealand White rabbits were infected with a lethal dose of B. anthracis Ames spores via the inhalation route, and blood samples were collected at various times to assess antigenemia, bacteremia, tissue bacterial load, and antibody production. Treatments were administered upon detection of B. anthracis protective antigen in the animals' sera. For comparison, a fully protective dose of levofloxacin was used as a positive control. Rabbits treated with teixobactin showed 100% survival following infection, and the bacteremia was completely resolved by 24-48 h post-treatment. In addition, the bacterial/spore loads in tissues of the animals treated with teixobactin were either zero or dramatically less relative to that of the negative control animals. Moreover, microscopic evaluation of the tissues revealed decreased pathology following treatment with teixobactin. Overall, these results show that teixobactin was protective against inhalation anthrax infection in the rabbit model, and they indicate the potential of teixobactin as a therapeutic for the disease.

Isofuranonaphthoquinone produced by an Actinoplanes isolate.

A new isofuranonaphthoquinone, 7,8-dihydroxy-1-methylnaphtho[2,3-c]furan-4,9-dione, was isolated from cultures of an Actinoplanes isolate obtained using an in situ diffusion technology that facilitates the isolation of soil microorganisms. This compound was demonstrated to have the ability to complex Fe(III). The structure was determined on the basis of spectroscopic data.

Neocitreamicins I and II, novel antibiotics with activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci.

Two novel antibiotics, neocitreamicins I and II, were isolated from a fermentation broth of a Nocardia strain. This producing strain was obtained using an in situ diffusion chamber that facilitates the cultivation of soil microorganisms. The structures of neocitreamicins I and II were elucidated using UV, MS, and NMR data, and found to be related to the polycyclic xanthone antibiotics of the citreamicin class. The neocitreamicins showed in vitro activity against Gram-positive bacteria including strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis.

In situ cultivation of previously uncultivable microorganisms using the ichip.

Most microbial species remain uncultivated, and modifying artificial nutrient media brings only an incremental increase in cultivability. We reasoned that an alternative way to cultivate species with unknown requirements is to use naturally occurring combinations of growth factors. To achieve this, we moved cultivation into the microbes' natural habitat by placing cells taken from varying environmental samples into diffusion chambers, which are then returned to nature for incubation. By miniaturizing the chambers and placing only one to several cells into each chamber, we can grow and isolate microorganisms in axenic culture in one step. We call this cultivation platform the 'isolation chip', or 'ichip'. This platform has been shown to increase microbial recovery from 5- to 300-fold, depending on the study. Furthermore, it provides access to a unique set of microbes that are inaccessible by standard cultivation. Here we provide a simple protocol for building and applying ichips for environmental cultivation of soil bacteria as an example; the protocol consists of (i) preparing the ichip; (ii) collecting an environmental sample; (iii) serially diluting cells and loading them into the ichip; (iv) returning the ichip to the environment for incubation; (v) retrieving the ichip and harvesting grown material; and (vi) domestication of the ichip-derived colonies for growth in the laboratory. The ichip's full assembly and deployment is a relatively simple procedure that, with experience, takes ∼2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take ∼1-4 h, depending on which specific procedures are used.

Uncultured microorganisms as a source of secondary metabolites.

The vast majority of microbial species are 'uncultured' and do not grow under laboratory conditions. This has led to the development of a number of methods to culture these organisms in a simulated natural environment. Approaches include placing cells in chambers that allow diffusion of compounds from the natural environment, traps enclosed with porous membranes that specifically capture organisms forming hyphae--actinobacteria and microfungi, and growth in the presence of cultivable helper species. Repeated cultivation in situ produces domesticated variants that can grow on regular media in vitro, and can be scaled up for secondary metabolite production. The co-culture approach has led to the identification of the first class of growth factors for uncultured bacteria, iron-chelating siderophores. It appears that many uncultured organisms from diverse taxonomical groups have lost the ability to produce siderophores, and depend on neighboring species for growth. The new cultivation approaches allow for the exploitation of the secondary metabolite potential of the previously inaccessible microorganisms.

Microbial pathogen genomes - new strategies for identifying therapeutic and vaccine targets.

Efficient mining of genomic sequence information from multiple pathogens for therapeutic and vaccine targets requires efficient tools. Fortunately, robust methods applicable to whole genomes have been developed and applied in the past few years to identify genes essential for growth or virulence and to detect potential vaccine targets. Successful approaches to identify potential therapeutic targets include a variety of ingenious uses of nearly random transposon insertions, more directed methods such as antisense and insertion-duplication mutagenesis, and expression profiling facilitated by microarrays. Vaccine targets have been identified by gene fusion and expression experiments to discover gene products that are immunogenic in humans or animal models. All genome-wide methods require focused secondary assays to validate the findings, but these genomic methods excel at reducing to a manageable number the genes to be examined further. This editorial reviews the latest developments in genome-wide target identification tools.

Identification and characterization of inhibitors of bacterial enoyl-acyl carrier protein reductase.

Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Escherichia coli ENR yielded four structurally distinct classes of hits. Several members of one of these, the 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles ("thiopyridines"), inhibited both purified ENR (50% inhibitory concentration [IC(50)] = 3 to 25 micro M) and the growth of Staphylococcus aureus and Bacillus subtilis (MIC = 1 to 64 micro g/ml). The effect on cell growth is due in part to inhibition of fatty acid biosynthesis as judged by inhibition of incorporation of [(14)C]acetate into fatty acids and by the increased sensitivity of cells that underexpress an ENR-encoding gene (four- to eightfold MIC shift). Synthesis of a variety of compounds in this chemical series revealed a correlation between IC(50) and MIC, and the results provided initial structure-activity relationships. Preliminary structure-activity relationships, potency on purified ENR, and activity on bacterial cells indicate that members of the thiopyridine chemical series are effective fatty acid biosynthesis inhibitors suitable for further antibacterial development.