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.

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.

A Resistance-Evading Antibiotic for Treating Anthrax.

The antimicrobial resistance crisis (AMR) is associated with millions of deaths and undermines the franchise of medicine. Of particular concern is the threat of bioweapons, exemplified by anthrax. Introduction of novel antibiotics helps mitigate AMR, but does not address the threat of bioweapons with engineered resistance. We reasoned that teixobactin, an antibiotic with no detectable resistance, is uniquely suited to address the challenge of weaponized anthrax. Teixobactinbinds to immutable targets, precursors of cell wall polymers. Here we show that teixobactinis highly efficacious in a rabbit model of inhalation anthrax. Inhaling spores of Bacillus anthracis causes overwhelming morbidity and mortality. Treating rabbits with teixobactinafter the onset of disease rapidly eliminates the pathogen from blood and tissues, normalizes body temperature, and prevents tissue damage. Teixobactinassembles into an irreversible supramolecular structure of the surface of B. anthracis membrane, likely contributing to its unusually high potency against anthrax. Antibiotics evading resistance provide a rational solution to both AMR and engineered bioweapons.

A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance.

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthetase (FPGS), which normally promotes intracellular accumulation and activity of natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. Here, we describe a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase and downstream one-carbon metabolism. Contrary to methotrexate, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folates for interaction with dihydrofolate reductase. We show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1, whereas FPGS-high CRC organoids are more sensitive to methotrexate. Our results argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, using a vulnerability created by polyglutamylation deficiency.

A new 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, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55 PP, Lipid II, Lipid WTA ). 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 irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

Brevibacillin 2V, a Novel Antimicrobial Lipopeptide With an Exceptionally Low Hemolytic Activity.

Bacterial non-ribosomally produced peptides (NRPs) form a rich source of antibiotics, including more than 20 of these antibiotics that are used in the clinic, such as penicillin G, colistin, vancomycin, and chloramphenicol. Here we report the identification, purification, and characterization of a novel NRP, i.e., brevibacillin 2V (lipo-tridecapeptide), from Brevibacillus laterosporus DSM 25. Brevibacillin 2V has a strong antimicrobial activity against Gram-positive bacterial pathogens (minimum inhibitory concentration = 2 mg/L), including difficult-to-treat antibiotic-resistant Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Notably, brevibacillin 2V has a much lower hemolytic activity (HC50 > 128 mg/L) and cytotoxicity (CC50 = 45.49 ± 0.24 mg/L) to eukaryotic cells than previously reported NRPs of the lipo-tridecapeptide family, including other brevibacillins, which makes it a promising candidate for antibiotic development. In addition, our results demonstrate that brevibacillins display a synergistic action with established antibiotics against Gram-negative bacterial pathogens. Probably due to the presence of non-canonical amino acids and D-amino acids, brevibacillin 2V showed good stability in human plasma. Thus, we identified and characterized a novel and promising antimicrobial candidate (brevibacillin 2V) with low hemolytic activity and cytotoxicity, which can be used either on its own or as a template for further total synthesis and modification.

Brevibacillin 2V Exerts Its Bactericidal Activity via Binding to Lipid II and Permeabilizing Cellular Membranes.

Lipo-tridecapeptides, a class of bacterial non-ribosomally produced peptides, show strong antimicrobial activity against Gram-positive pathogens, including antibiotic-resistant Staphylococcus aureus and Enterococcus spp. However, many of these lipo-tridecapeptides have shown high hemolytic activity and cytotoxicity, which has limited their potential to be developed into antibiotics. Recently, we reported a novel antimicrobial lipo-tridecapeptide, brevibacillin 2V, which showed no hemolytic activity against human red blood cells at a high concentration of 128 mg/L, opposite to other brevibacillins and lipo-tridecapeptides. In addition, brevibacillin 2V showed much lower cytotoxicity than the other members of the brevibacillin family. In this study, we set out to elucidate the antimicrobial mode of action of brevibacillin 2V. The results show that brevibacillin 2V acts as bactericidal antimicrobial agent against S. aureus (MRSA). Further studies show that brevibacillin 2V exerts its bactericidal activity by binding to the bacterial cell wall synthesis precursor Lipid II and permeabilizing the bacterial membrane. Combined solid-state NMR, circular dichroism, and isothermal titration calorimetry assays indicate that brevibacillin 2V binds to the GlcNAc-MurNAc moiety and/or the pentapeptide of Lipid II. This study provides an insight into the antimicrobial mode of action of brevibacillin 2V. As brevibacillin 2V is a novel and promising antibiotic candidate with low hemolytic activity and cytotoxicity, the here-elucidated mode of action will help further studies to develop it as an alternative antimicrobial agent.

Mode of action of teixobactins in cellular membranes.

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.

Salmonella Effector SteA Suppresses Proinflammatory Responses of the Host by Interfering With IκB Degradation.

Salmonella enterica serovar Typhimurium is known to cause its virulence by secreting various effector proteins directly into the host cytoplasm via two distinct type III secretion systems (T3SS-1 and T3SS-2). Generally, T3SS-1-delivered effectors help Salmonella Typhimurium in the early phases of infection including invasion and immune modulation of the host cells, whereas T3SS-2 effectors mainly help in the survival of Salmonella Typhimurium within the host cells including maintenance of Salmonella-containing vacuole, replication of the bacteria, and dissemination. Some of the effectors are secreted via both T3SS-1 and T3SS-2, suggesting their role in distinct phases of infection of host cells. SteA is such an effector that is secreted by both T3SS-1 and T3SS-2. It has been shown to control the membrane dynamics of the Salmonella-containing vacuole within the host cells in the late phases of infection. In this manuscript, toward characterizing the T3SS-1 function of SteA, we found that SteA suppresses inflammatory responses of the host by interfering with the nuclear factor kappa B pathway. Our initial observation showed that the mice infected with steA-deleted Salmonella Typhimurium (ΔsteA) died earlier compared to the wild-type bacteria due to heightened immune responses, which indicated that SteA might suppress immune responses. Furthermore, our study revealed that SteA suppresses immune responses in macrophages by interfering with the degradation of IκB, the inhibitor of nuclear factor kappa B. SteA suppresses the ubiquitination and hence degradation of IκB by acting on Cullin-1 of the Skp-1, Cullin-1, F-box (SCF)-E3 ligase complex. Our study revealed that SteA suppresses a key step necessary for E3 ligase activation, i.e., neddylation of Cullin-1 by interfering with dissociation of its inhibitor Cand-1.