A new antibiotic traps lipopolysaccharide in its intermembrane transporter.

Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet1. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics2-6. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.

A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Single-molecule dynamics show a transient lipopolysaccharide transport bridge.

Gram-negative bacteria are surrounded by two membranes. A special feature of the outer membrane is its asymmetry. It contains lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet1-3. The proper assembly of LPS in the outer membrane is required for cell viability and provides Gram-negative bacteria intrinsic resistance to many classes of antibiotics. LPS biosynthesis is completed in the inner membrane, so the LPS must be extracted, moved across the aqueous periplasm that separates the two membranes and translocated through the outer membrane where it assembles on the cell surface4. LPS transport and assembly requires seven conserved and essential LPS transport components5 (LptA-G). This system has been proposed to form a continuous protein bridge that provides a path for LPS to reach the cell surface6,7, but this model has not been validated in living cells. Here, using single-molecule tracking, we show that Lpt protein dynamics are consistent with the bridge model. Half of the inner membrane Lpt proteins exist in a bridge state, and bridges persist for 5-10 s, showing that their organization is highly dynamic. LPS facilitates Lpt bridge formation, suggesting a mechanism by which the production of LPS can be directly coupled to its transport. Finally, the bridge decay kinetics suggest that there may be two different types of bridges, whose stability differs according to the presence (long-lived) or absence (short-lived) of LPS. Together, our data support a model in which LPS is both a substrate and a structural component of dynamic Lpt bridges that promote outer membrane assembly.

Native ß-barrel substrates pass through two shared intermediates during folding on the BAM complex.

The assembly of ß-barrel proteins into membranes is mediated by the evolutionarily conserved ß-barrel assembly machine (BAM) complex. In Escherichia coli, BAM folds numerous substrates which vary considerably in size and shape. How BAM is able to efficiently fold such a diverse array of ß-barrel substrates is not clear. Here, we develop a disulfide crosslinking method to trap native substrates in vivo as they fold on BAM. By placing a cysteine within the luminal wall of the BamA barrel as well as in the substrate ß-strands, we can compare the residence time of each substrate strand within the BamA lumen. We validated this method using two defective, slow-folding substrates. We used this method to characterize stable intermediates which occur during folding of two structurally different native substrates. Strikingly, these intermediates occur during identical stages of folding for both substrates: soon after folding has begun and just before folding is completed. We suggest that these intermediates arise due to barriers to folding that are common between ß-barrel substrates, and that the BAM catalyst is able to fold so many different substrates because it addresses these common challenges.

ß-Barrel membrane protein assembly by the Bam complex.

ß-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. ß-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts ß-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for ß-barrel assembly. Ultimately, studies of the mechanism of ß-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.

Structure of a nascent membrane protein as it folds on the BAM complex.

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a ß-barrel structure1,2. ß-Barrels are sheets of ß-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3-5. One subunit of the machines is itself a ß-barrel protein that has a central role in folding other ß-barrels. In Gram-negative bacteria, the ß-barrel assembly machine (BAM) consists of the ß-barrel protein BamA, and four lipoproteins5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid ß-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.

Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases.

Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in vivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in vivo. We further show that LpoB promotes PG synthesis by its partner PBP in vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development.

Structural basis of unidirectional export of lipopolysaccharide to the cell surface.

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide1. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown2. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.

Determination of Initial Rates of Lipopolysaccharide Transport.

Nonvesicular lipid trafficking pathways are an important process in every domain of life. The mechanisms of these processes are poorly understood in part due to the difficulty in kinetic characterization. One important class of glycolipids, lipopolysaccharides (LPS), are the primary lipidic component of the outer membrane of Gram-negative bacteria. LPS are synthesized in the inner membrane and then trafficked to the cell surface by the lipopolysaccharide transport proteins, LptB2FGCADE. By characterizing the interaction of a fluorescent probe and LPS, we establish a quantitative assay to monitor the flux of LPS between proteoliposomes on the time scale of seconds. We then incorporate photocaged ATP into this system, which allows for light-based control of the initiation of LPS transport. This control allows us to measure the initial rate of LPS transport (3.0 min-1 per LptDE). We also find that the rate of LPS transport by the Lpt complex is independent of the structure of LPS. In contrast, we find the rate of LPS transport is dependent on the proper function of the LptDE complex. Mutants of the outer membrane Lpt components, LptDE, that cause defective LPS assembly in live cells display attenuated transport rates and slower ATP hydrolysis compared to wild type proteins. Analysis of these mutants reveals that the rates of ATP hydrolysis and LPS transport are correlated such that 1.2 ± 0.2 ATP are hydrolyzed for each LPS transported. This correlation suggests a model where the outer membrane components ensure the coupling of ATP hydrolysis and LPS transport by stabilizing a transport-active state of the Lpt bridge.

The Bacterial Cell Wall: From Lipid II Flipping to Polymerization.

The peptidoglycan (PG) cell wall is an extra-cytoplasmic glycopeptide polymeric structure that protects bacteria from osmotic lysis and determines cellular shape. Since the cell wall surrounds the cytoplasmic membrane, bacteria must add new material to the PG matrix during cell elongation and division. The lipid-linked precursor for PG biogenesis, Lipid II, is synthesized in the inner leaflet of the cytoplasmic membrane and is subsequently translocated across the bilayer so that the PG building block can be polymerized and cross-linked by complex multiprotein machines. This review focuses on major discoveries that have significantly changed our understanding of PG biogenesis in the past decade. In particular, we highlight progress made toward understanding the translocation of Lipid II across the cytoplasmic membrane by the MurJ flippase, as well as the recent discovery of a novel class of PG polymerases, the SEDS (shape, elongation, division, and sporulation) glycosyltransferases RodA and FtsW. Since PG biogenesis is an effective target of antibiotics, these recent developments may lead to the discovery of much-needed new classes of antibiotics to fight bacterial resistance.

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis.

The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.

Assembly and Maintenance of Lipids at the Bacterial Outer Membrane.

The outer membrane of Gram-negative bacteria is essential for their survival in harsh environments and provides intrinsic resistance to many antibiotics. This membrane is remarkable; it is a highly asymmetric lipid bilayer. The inner leaflet of the outer membrane contains phospholipids, whereas the fatty acyl chains attached to lipopolysaccharide (LPS) comprise the hydrophobic portion of the outer leaflet. This lipid asymmetry, and in particular the exclusion of phospholipids from the outer leaflet, is key to creating an almost impenetrable barrier to hydrophobic molecules that can otherwise pass through phospholipid bilayers. It has long been known that these lipids are not made in the outer membrane. It is now believed that conserved multisubunit protein machines extract these lipids after their synthesis is completed at the inner membrane and transport them to the outer membrane. A longstanding question is how the cell builds and maintains this asymmetric lipid bilayer in coordination with the assembly of the other components of the cell envelope. This Review describes the trans-envelope lipid transport systems that have been identified to participate in outer-membrane biogenesis: LPS transport via the Lpt machine, and phospholipid transport via the Mla pathway and several recently proposed transporters.

A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW.

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.

Outer Membrane Biogenesis.

The hallmark of gram-negative bacteria and organelles such as mitochondria and chloroplasts is the presence of an outer membrane. In bacteria such as Escherichia coli, the outer membrane is a unique asymmetric lipid bilayer with lipopolysaccharide in the outer leaflet. Integral transmembrane proteins assume a ß-barrel structure, and their assembly is catalyzed by the heteropentameric Bam complex containing the outer membrane protein BamA and four lipoproteins, BamB-E. How the Bam complex assembles a great diversity of outer membrane proteins into a membrane without an obvious energy source is a particularly challenging problem, because folding intermediates are predicted to be unstable in either an aqueous or a hydrophobic environment. Two models have been put forward: the budding model, based largely on structural data, and the BamA assisted model, based on genetic and biochemical studies. Here we offer a critical discussion of the pros and cons of each.

SEDS proteins are a widespread family of bacterial cell wall polymerases.

Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.

Genetic approaches to improve clorobiocin production in Streptomyces roseochromogenes NRRL 3504.

Streptomyces roseochromogenes NRRL 3504 is best known as a producer of clorobiocin, a DNA replication inhibitor from the aminocoumarin family of antibiotics. This natural product currently draws attention as a promising adjuvant for co-application with other antibiotics against Gram-negative multidrug-resistant pathogens. Herein, we expand the genetic toolkit for NRRL 3504 by showing that a set of integrative and replicative vectors, not tested previously for this strain, could be conjugally transferred at high frequency from Escherichia coli to NRRL 3504. Using this approach, we leverage a cumate-inducible expression of cluster-situated regulatory gene novG to increase clorobiocin titers by 30-fold (up to approximately 200 mg/L). To our best knowledge, this is the highest level of clorobiocin production reported so far. Our findings set a working ground for further improvement of clorobiocin production as well as for the application of genetic methods to illuminate the cryptic secondary metabolome of NRRL 3504. Key Points • Efficient system for conjugative transfer of plasmids into NRRL 3504 was developed. • Expression of regulatory genes in NRRL 3504 led to increase in clorobiocin titer. • Secondary metabolome of NRRL 3504 becomes an accessible target for genetic manipulations using the expanded vector set and improved intergeneric conjugation protocol.

Simple Secondary Amines Inhibit Growth of Gram-Negative Bacteria through Highly Selective Binding to Phenylalanyl-tRNA Synthetase.

Antibiotics to treat drug-resistant Gram-negative infections are urgently needed but challenging to discover. Using a cell-based screen, we identified a simple secondary amine that inhibited the growth of wild-type Escherichia coli and Acinetobacter baumannii but not the growth of the Gram-positive organism Bacillus subtilis. Resistance mutations in E. coli and A. baumannii mapped exclusively to the aminoacyl-tRNA synthetase PheRS. We confirmed biochemically that the compound inhibited PheRS from these organisms and showed that it did not inhibit PheRS from B. subtilis or humans. To understand the basis for the compound's high selectivity for only some PheRS enzymes, we solved crystal structures of E. coli and A. baumannii PheRS complexed with the inhibitor. The structures showed that the compound's benzyl group mimics the benzyl of phenylalanine. The other amine substituent, a 2-(cyclohexen-1-yl)ethyl group, induces a hydrophobic pocket in which it binds. Through bioinformatic analysis and mutagenesis, we show that the ability to induce a complementary hydrophobic pocket that can accommodate the second substituent explains the high selectivity of this remarkably simple molecular scaffold for Gram-negative PheRS. Because this secondary amine scaffold is active against wild-type Gram-negative pathogens but is not cytotoxic to mammalian cells, we suggest that it may be possible to develop it for use in combination antibiotic therapy to treat Gram-negative infections.