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Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis 1 – 3 . Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets 4 , 5 . The natural compound darobactin targets the bacterial insertase BamA 6 —the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins 7 – 13 . BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid β-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel. Structural studies resolve how the antibiotic darobactin inhibits the bacterial BAM insertase.

The folding and insertion of integral β-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect β-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize amonoclonal antibody that selectively inhibits an essential component of the Escherichia coli β-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its β-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outermembrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying β-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors. Multidrug efflux pumps actively expel a wide range of toxic substrates from bacteria and play a major role in drug resistance. Here authors show the in situ structure of the efflux pump AcrAB-TolC obtained by electron cryo-tomography and subtomogram averaging.

Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump. Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, the authors identify pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through allosteric inhibition of its primary RND transporter.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens 1 , 2 . These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds 3 , 4 . As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s 2 . We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics. Bacterial symbionts of animals may contain antibiotics that are particularly suitable for development into therapeutics; one such compound, darobactin, is active against important Gram-negative pathogens both in vitro and in animal models of infection.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K . BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Outer membrane proteins (OMPs) produced by Gram-negative bacteria contain a cylindrical amphipathic β-sheet (“β-barrel”) that functions as a membrane spanning domain. The assembly (folding and membrane insertion) of OMPs is mediated by the heterooligomeric β- b arrel a ssembly m achine (BAM). The central BAM subunit (BamA) is an attractive antibacterial target because its structure and cell surface localization are conserved, it catalyzes an essential reaction, and potent bactericidal compounds that inhibit its activity have been described. Here we utilize mRNA display to discover cyclic peptides that bind to Escherichia coli BamA with high affinity. We describe three peptides that arrest the growth of BAM deficient E. coli strains, inhibit OMP assembly in live cells and in vitro, and bind to unique sites within the BamA β-barrel lumen. Remarkably, we find that if the peptides are added to cultures after a slowly assembling OMP mutant binds to BamA, they accelerate its biogenesis. The data strongly suggest that the peptides trap BamA in conformations that block the initiation of OMP assembly but favor a later assembly step. Molecular dynamics simulations provide further evidence that the peptides bind stably to BamA and function by a previously undescribed mechanism. Here the authors use mRNA display to discover peptide inhibitors of BamA, an essential factor that catalyzes the membrane insertion of bacterial outer membrane proteins. They show that three peptides are antibacterial and inhibit BamA activity by a unique mechanism.

The structure of a bacterial ribosome–RelA complex reveals that RelA, a protein recruited to the ribosome in the case of scarce amino acids, binds in a different location to translation factors, and that this binding event suppresses auto-inhibition to activate synthesis of the (p)ppGpp secondary messenger, thus initiating stringent control. How the starved ribosome exerts control When bacteria are starved of nutrients, they initiate a program known as stringent response, or stringent control, in which the transcriptional pattern responds to the changing metabolic needs. In the case of amino acid starvation, which causes ribosome stalling, RelA protein is recruited to the ribosome. Venki Ramakrishnan and colleagues have solved the cryo-electron microscopy structure of a bacterial ribosome–RelA complex to understand how amino acid deficiency is detected. They find that RelA binds in a location different from that used by translation factors, and that this binding event releases an inhibitory state of RelA that normally prevents synthesis of the (p)ppGpp secondary messenger. This messenger initiates the stringent response. In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation 1 , 2 that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns 3 . In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated 4 . Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools 5 , and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site 6 , 7 . RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp 8 , which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3′ hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.

The first inhibitor-bound X-ray crystal structures of the bacterial multidrug efflux transporter AcrB and its homologue MexB are presented, with the inhibitor shown to bind the transporter through a narrow hydrophobic pit, thereby preventing rotation of AcrB and MexB monomers. Bacterial multidrug exporter structures Inhibitors of bacterial multidrug efflux transporters are necessary to combat bacterial multidrug resistance, but no clinically useful inhibitors are currently available. The multidrug efflux transporter AcrB and its homologues facilitate the multidrug resistance of many Gram-negative pathogens, and in this paper Akihito Yamaguchi and colleagues describe the first X-ray crystal structures of inhibitor-bound AcrB and its homologue MexB. The inhibitor, a pyridopyrimidine derivative, binds in a narrow hydrophobic 'pit' and inhibits the functional rotation of the AcrB/MexB monomers. These inhibitor-bound structures may facilitate the development of new inhibitors of this family of multidrug efflux transporters, which could be used in conjunction with existing antibiotics to help make them more effective. The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens 1 , 2 . However, despite efforts to develop efflux inhibitors 3 , clinically useful inhibitors are not available at present 4 , 5 . Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY 6 , 7 ; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa 8 , 9 , 10 . We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics 11 , 12 , 13 . Drugs were shown to be exported by a functionally rotating mechanism 12 through tandem proximal and distal multisite drug-binding pockets 13 . Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π–π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa .

Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1–dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo . Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.