An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Sophisticated natural products as antibiotics.

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by ß-lactams that bind covalently to inhibit transpeptidases and ß-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed ß-strands of darobactins that target the undruggable ß-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

A classic antibiotic reimagined: Rationally designed bacitracin variants exhibit potent activity against vancomycin-resistant pathogens.

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis.

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.

The Dual Mode of Antibacterial Action of the Synthetic Small Molecule DCAP Involves Lipid II Binding.

The synthetic small molecule DCAP is a chemically well-characterized compound with antibiotic activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens. Until now, its mechanism of action was proposed to rely exclusively on targeting the bacterial membrane, thereby causing membrane depolarization, and increasing membrane permeability (Eun et al. 2012, J. Am. Chem. Soc. 134 (28), 11322-11325; Hurley et al. 2015, ACS Med. Chem. Lett. 6, 466-471). Here, we show that the antibiotic activity of DCAP results from a dual mode of action that is more targeted and multifaceted than previously anticipated. Using microbiological and biochemical assays in combination with fluorescence microscopy, we provide evidence that DCAP interacts with undecaprenyl pyrophosphate-coupled cell envelope precursors, thereby blocking peptidoglycan biosynthesis and impairing cell division site organization. Our work discloses a concise model for the mode of action of DCAP which involves the binding to a specific target molecule to exert pleiotropic effects on cell wall biosynthetic and divisome machineries.

THCz: Small molecules with antimicrobial activity that block cell wall lipid intermediates.

Emerging antibiotic resistance demands identification of novel antibacterial compound classes. A bacterial whole-cell screen based on pneumococcal autolysin-mediated lysis induction was developed to identify potential bacterial cell wall synthesis inhibitors. A hit class comprising a 1-amino substituted tetrahydrocarbazole (THCz) scaffold, containing two essential amine groups, displayed bactericidal activity against a broad range of gram-positive and selected gram-negative pathogens in the low micromolar range. Mode of action studies revealed that THCz inhibit cell envelope synthesis by targeting undecaprenyl pyrophosphate-containing lipid intermediates and thus simultaneously inhibit peptidoglycan, teichoic acid, and polysaccharide capsule biosynthesis. Resistance did not readily develop in vitro, and the ease of synthesizing and modifying these small molecules, as compared to natural lipid II-binding antibiotics, makes THCz promising scaffolds for development of cell wall-targeting antimicrobials.

Modular Synthesis of Halogenated Xanthones by a Divergent Coupling Strategy.

A versatile strategy to halogenated xanthones was developed that relies on a modular coupling of vanillin derivatives with a dibromoquinone. Depending on the reaction conditions, either the 6- or the 7-bromo heterocycles may be obtained in a divergent manner. These heterocycles may be readily further elaborated by sequential Sonogashira couplings, and the sequence may be successfully applied to substructures of the antibiotic lysolipin.

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.

Host Defense Peptides: Dual Antimicrobial and Immunomodulatory Action.

The rapid rise of multidrug-resistant (MDR) bacteria has once again caused bacterial infections to become a global health concern. Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), offer a viable solution to these pathogens due to their diverse mechanisms of actions, which include direct killing as well as immunomodulatory properties (e.g., anti-inflammatory activity). HDPs may hence provide a more robust treatment of bacterial infections. In this review, the advent of and the mechanisms that lead to antibiotic resistance will be described. HDP mechanisms of antibacterial and immunomodulatory action will be presented, with specific examples of how the HDP aurein 2.2 and a few of its derivatives, namely peptide 73 and cG4L73, function. Finally, resistance that may arise from a broader use of HDPs in a clinical setting and methods to improve biocompatibility will be briefly discussed.

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.

Modular Total Synthesis of Farnesyl Analogues of Cell Wall Precursors Lipid I and II Containing the Staphylococcus aureus Pentaglycine Bridge Modification.

A scalable and modular total synthesis of 3-lipid I and 3-lipid II was accomplished by a novel route involving an efficient solid phase synthesis of the peptide fragment and an effective chemoenzymatic attachment of the second sugar moiety. The generality of this route was further documented by the synthesis of an analogue bearing the pentaglycine interpeptidic bridge modification characteristic for the human pathogen Staphylococcus aureus.

Cacaoidin, First Member of the New Lanthidin RiPP Family.

Lantibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d-amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N-dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram-positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.

Radiomics of Brain MRI: Utility in Prediction of Metastatic Tumor Type.

Purpose To investigate the feasibility of tumor type prediction with MRI radiomic image features of different brain metastases in a multiclass machine learning approach for patients with unknown primary lesion at the time of diagnosis. Materials and methods This single-center retrospective analysis included radiomic features of 658 brain metastases from T1-weighted contrast material-enhanced, T1-weighted nonenhanced, and fluid-attenuated inversion recovery (FLAIR) images in 189 patients (101 women, 88 men; mean age, 61 years; age range, 32-85 years). Images were acquired over a 9-year period (from September 2007 through December 2016) with different MRI units, reflecting heterogeneous image data. Included metastases originated from breast cancer (n = 143), small cell lung cancer (n = 151), non-small cell lung cancer (n = 225), gastrointestinal cancer (n = 50), and melanoma (n = 89). A total of 1423 quantitative image features and basic clinical data were evaluated by using random forest machine learning algorithms. Validation was performed with model-external fivefold cross validation. Comparative analysis of 10 randomly drawn cross-validation sets verified the stability of the results. The classifier performance was compared with predictions from a respective conventional reading by two radiologists. Results Areas under the receiver operating characteristic curve of the five-class problem ranged between 0.64 (for non-small cell lung cancer) and 0.82 (for melanoma); all P values were less than .01. Prediction performance of the classifier was superior to the radiologists' readings. Highest differences were observed for melanoma, with a 17-percentage-point gain in sensitivity compared with the sensitivity of both readers; P values were less than .02. Conclusion Quantitative features of routine brain MR images used in a machine learning classifier provided high discriminatory accuracy in predicting the tumor type of brain metastases. © RSNA, 2018 Online supplemental material is available for this article.

Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains.

Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro ß-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the ß-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore ß-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

Differential daptomycin resistance development in Staphylococcus aureus strains with active and mutated gra regulatory systems.

The first-in-class lipopeptide antibiotic daptomycin (DAP) is highly active against Gram-positive pathogens including ß-lactam and glycopeptide resistant strains. Its molecular mode of action remains enigmatic, since a defined target has not been identified so far and multiple effects, primarily on the cell envelope have been observed. Reduced DAP susceptibility has been described in S. aureus and enterococci after prolonged treatment courses. In line with its pleiotropic antibiotic activities, a unique, defined molecular mechanism of resistance has not emerged, instead non-susceptibility appears often accompanied by alterations in membrane composition and changes in cell wall homeostasis. We compared S. aureus strains HG001 and SG511, which differ primarily in the functionality of the histidine kinase GraS, to evaluate the impact of the GraRS regulatory system on the development of DAP non-susceptibility. After extensive serial passing, both DAPR variants reached a minimal inhibitory concentration of 31 µg/ml and shared some phenotypic characteristics (e.g. thicker cell wall, reduced autolysis). However, based on comprehensive analysis of the underlying genetic, transcriptomic and proteomic changes, we found that both strains took different routes to achieve DAP resistance. Our study highlights the impressive genetic and physiological capacity of S. aureus to counteract pleiotropic activities of cell wall- and membrane-active compounds even when a major cell wall regulatory system is dysfunctional.

Antimicrobial Dialkylresorcins from Marine-Derived Microorganisms: Insights into Their Mode of Action and Putative Ecological Relevance.

Zobellia galactanivorans has been reported as a seaweed-associated or marine-derived species with largely unknown secondary metabolites. The combination of bioinformatic analysis and MS- and bioactivity guided separation led to the isolation of a new antibiotically active dialkylresorcin from the marine bacterium Z. galactanivorans. The antibiotic profile of the new dialkylresorcin zobelliphol (1: ) was investigated and compared with related and naturally occurring dialkyresorcins (i.e., stemphol (2: ) and 4-butyl-3,5-dihydroxybenzoic acid (3: )) from the marine-derived fungus Stemphylium globuliferum. Bacterial reporter strain assays provided insights into the mode of action of this antibiotic compound class. We identified an interference with bacterial DNA biosynthesis for the dialkylresorcin derivative 1: . In addition, the putative biosynthetic gene cluster corresponding to production of 1: was identified and a biosynthetic hypothesis was deduced.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway.

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.

Targeting a cell wall biosynthesis hot spot.

Covering: up to 2017History points to the bacterial cell wall biosynthetic network as a very effective target for antibiotic intervention, and numerous natural product inhibitors have been discovered. In addition to the inhibition of enzymes involved in the multistep synthesis of the macromolecular layer, in particular, interference with membrane-bound substrates and intermediates essential for the biosynthetic reactions has proven a valuable antibacterial strategy. A prominent target within the peptidoglycan biosynthetic pathway is lipid II, which represents a particular "Achilles' heel" for antibiotic attack, as it is readily accessible on the outside of the cytoplasmic membrane. Lipid II is a unique non-protein target that is one of the structurally most conserved molecules in bacterial cells. Notably, lipid II is more than just a target molecule, since sequestration of the cell wall precursor may be combined with additional antibiotic activities, such as the disruption of membrane integrity or disintegration of membrane-bound multi-enzyme machineries. Within the membrane bilayer lipid II is likely organized in specific anionic phospholipid patches that form a particular "landing platform" for antibiotics. Nature has invented a variety of different "lipid II binders" of at least 5 chemical classes, and their antibiotic activities can vary substantially depending on the compounds' physicochemical properties, such as amphiphilicity and charge, and thus trigger diverse cellular effects that are decisive for antibiotic activity.

The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus.

The assembly of the bacterial cell wall requires synchronization of a multitude of biosynthetic machineries and regulatory networks. The eukaryotic-like serine/threonine kinase PknB has been implicated in coordinating cross-wall formation, autolysis and cell division in Staphylococcus aureus. However, the signal molecule sensed by this kinase remained elusive so far. Here, we provide compelling biochemical evidence that PknB interacts with the ultimate cell wall precursor lipid II, triggering kinase activity. Moreover, we observed crosstalk of PknB with the two component system WalKR and identified the early cell division protein FtsZ as another PknB phosphorylation substrate in S. aureus. In agreement with the implied role in regulation of cell envelope metabolism, we found PknB to preferentially localize to the septum of S. aureus and the PASTA domains to be crucial for recruitment to this site. The data provide a model for the contribution of PknB to control cell wall metabolism and cell division.