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.

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.

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.

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.

Chloride Ions Are Required for Thermosipho africanus MurJ Function.

Most bacteria have a peptidoglycan cell wall that determines their cell shape and helps them resist osmotic lysis. Peptidoglycan synthesis depends on the translocation of the lipid-linked precursor lipid II across the cytoplasmic membrane by the MurJ flippase. Structure-function analyses of MurJ from Thermosipho africanus (MurJTa) and Escherichia coli (MurJEc) have revealed that MurJ adopts multiple conformations and utilizes an alternating-access mechanism to flip lipid II. MurJEc activity relies on membrane potential, but the specific counterion has not been identified. Crystal structures of MurJTa revealed a chloride ion bound to the N-lobe of the flippase and a sodium ion in its C-lobe, but the role of these ions in transport is unknown. Here, we investigated the effect of various ions on the function of MurJTa and MurJEc in vivo. We found that chloride, and not sodium, ions are necessary for MurJTa function, but neither ion is required for MurJEc function. We also showed that murJTa alleles encoding changes at the crystallographically identified sodium-binding site still complement the loss of native murJEc, although they decreased protein stability and/or function. Based on our data and previous work, we propose that chloride ions are necessary for the conformational change that resets MurJTa after lipid II translocation and suggest that MurJ orthologs may function similarly but differ in their requirements for counterions. IMPORTANCE The biosynthetic pathway of the peptidoglycan cell wall is one of the most favorable targets for antibiotic development. Lipid II, the lipid-linked PG precursor, is made in the inner leaflet of the cytoplasmic membrane and then transported by the MurJ flippase so that it can be used to build the peptidoglycan cell wall. MurJ functions using an alternating-access mechanism thought to depend on a yet-to-be-identified counterion. This study fills a gap in our understanding of MurJ's energy-coupling mechanism by showing that chloride ions are required for MurJ in some, but not all, organisms. Based on our data and prior studies, we propose that, while the general transport mechanism of MurJ may be conserved, its specific mechanistic details may differ across bacteria, as is common in transporters. These findings are important to understand MurJ function and its development as an antibiotic target.

Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of many Gram-negative bacteria, providing a barrier against the entry of toxic molecules. In Escherichia coli, LPS is exported to the cell surface by seven essential proteins (LptA-G) that form a transenvelope complex. At the inner membrane, the ATP-binding cassette (ABC) transporter LptB2FG associates with LptC to power LPS extraction from the membrane and transfer to the periplasmic LptA protein, which is in complex with the OM translocon LptDE. LptC interacts both with LptB2FG and LptADE to mediate the formation of the transenvelope bridge and regulates the ATPase activity of LptB2FG. A genetic screen has previously identified suppressor mutants at a residue (R212) of LptF that are viable in the absence of LptC. Here, we present in vivo evidence that the LptF R212G mutant assembles a six-protein transenvelope complex in which LptA mediates interactions with LptF and LptD in the absence of LptC. Furthermore, we present in vitro evidence that the mutant LptB2FG complexes restore the regulation of ATP hydrolysis as it occurs in the LptB2FGC complex to achieve wild-type efficient coupling of ATP hydrolysis and LPS movement. We also show the suppressor mutations restore the wild-type levels of LPS transport both in vivo and in vitro, but remarkably, without restoring the affinity of the inner membrane complex for LptA. Based on the sensitivity of lptF suppressor mutants to selected stress conditions relative to wild-type cells, we show that there are additional regulatory functions of LptF and LptC that had not been identified. IMPORTANCE The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli, by seven essential proteins. LptC is the unconventional regulatory subunit of the LptB2FGC ABC transporter, involved in coordinating energy production and LPS transport. Surprisingly, despite being essential for bacterial growth, LptC can be deleted, provided that a specific residue in the periplasmic domain of LptF is mutated and LptA is overexpressed. Here, we apply biochemical techniques to investigate the suppression mechanism. The data produced in this work disclose an unknown regulatory function of LptF in the transporter that not only expands the knowledge about the Lpt complex but can also be targeted by novel LPS biogenesis inhibitors.

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.

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.

Efficient and flexible synthesis of new photoactivatable propofol analogs.

Propofol is a widely used general anesthetic, which acts by binding to and modulating several neuronal ion channels. We describe the synthesis of photoactivatable propofol analogs functionalized with an alkyne handle for bioorthogonal chemistry. Such tools are useful for detecting and isolating photolabeled proteins. We designed expedient and flexible synthetic routes to three new diazirine-based crosslinkable propofol derivatives, two of which have alkyne handles. As a proof of principle, we show that these compounds activate heterologously expressed Transient Receptor Potential Ankyrin 1 (TRPA1), a key ion channel of the pain pathway, with a similar potency as propofol in fluorescence-based functional assays. This work demonstrates that installation of the crosslinkable and clickable group on a short nonpolar spacer at the para position of propofol does not affect TRPA1 activation, supporting the utility of these chemical tools in identifying and characterizing potentially druggable binding sites in propofol-interacting proteins.

The assembly of ß-barrel outer membrane proteins.

The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts contain ß-barrel integral membrane proteins. In bacteria, the five-protein ß-barrel assembly machine (Bam) accelerates the folding and membrane integration of these proteins. The central component of the machine, BamA, contains a ß-barrel domain that can adopt a lateral-open state with its N-terminal and C-terminal ß-strands unpaired. Recently, strategies have been developed to capture ß-barrel folding intermediates on the Bam complex. Biochemical and structural studies provide support for a model in which substrates assemble at the lateral opening of BamA. In this model, the N-terminal ß-strand of BamA captures the C-terminal ß-strand of substrates by hydrogen bonding to allow their directional folding and subsequent release into the membrane.

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.

Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization.

Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan. As lipid II is polymerized, nascent polymers remain membrane-anchored at one end, and the other end becomes cross-linked to the matrix2-4. How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question. Here, we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A structure of the complex at a resolution of 2.6 Å shows that the membrane protein scaffolds the hydrolase to orient its active site for cleaving the glycan strand. We propose that this complex functions to detach newly synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix.

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.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

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.

Detection of Transport Intermediates in the Peptidoglycan Flippase MurJ Identifies Residues Essential for Conformational Cycling.

Bacterial cell wall synthesis is an essential process in bacteria and one of the best targets for antibiotics. A critical step on this pathway is the export of the lipid-linked cell wall monomer, Lipid II, by its transporter MurJ. The mechanism by which MurJ mediates the transbilayer movement of Lipid II is not understood because intermediate states of this process have not been observed. Here we demonstrate a method to capture and detect interactions between MurJ and its substrate Lipid II by photo-cross-linking and subsequent biotin-tagging. We show that this method can be used to covalently capture intermediate transport states of Lipid II on MurJ in living cells. Using this strategy we probed several lethal arginine mutants and found that they retain appreciable substrate-binding ability despite being defective in Lipid II transport. We propose that Lipid II binding to these residues during transport induces a conformational change in MurJ required to proceed through the Lipid II transport cycle. The methods described to detect intermediate transport states of MurJ will be useful for characterizing mechanisms of inhibitors.

Structural coordination of polymerization and crosslinking by a SEDS-bPBP peptidoglycan synthase complex.

The shape, elongation, division and sporulation (SEDS) proteins are a highly conserved family of transmembrane glycosyltransferases that work in concert with class B penicillin-binding proteins (bPBPs) to build the bacterial peptidoglycan cell wall1-6. How these proteins coordinate polymerization of new glycan strands with their crosslinking to the existing peptidoglycan meshwork is unclear. Here, we report the crystal structure of the prototypical SEDS protein RodA from Thermus thermophilus in complex with its cognate bPBP at 3.3 Å resolution. The structure reveals a 1:1 stoichiometric complex with two extensive interaction interfaces between the proteins: one in the membrane plane and the other at the extracytoplasmic surface. When in complex with a bPBP, RodA shows an approximately 10 Å shift of transmembrane helix 7 that exposes a large membrane-accessible cavity. Negative-stain electron microscopy reveals that the complex can adopt a variety of different conformations. These data define the bPBP pedestal domain as the key allosteric activator of RodA both in vitro and in vivo, explaining how a SEDS-bPBP complex can coordinate its dual enzymatic activities of peptidoglycan polymerization and crosslinking to build the cell wall.

Staphylococcus aureus cell growth and division are regulated by an amidase that trims peptides from uncrosslinked peptidoglycan.

Bacteria are protected by a polymer of peptidoglycan that serves as an exoskeleton1. In Staphylococcus aureus, the peptidoglycan assembly enzymes relocate during the cell cycle from the periphery, where they are active during growth, to the division site where they build the partition between daughter cells2-4. But how peptidoglycan synthesis is regulated throughout the cell cycle is poorly understood5,6. Here, we used a transposon screen to identify a membrane protein complex that spatially regulates S. aureus peptidoglycan synthesis. This complex consists of an amidase that removes stem peptides from uncrosslinked peptidoglycan and a partner protein that controls its activity. Amidases typically hydrolyse crosslinked peptidoglycan between daughter cells so that they can separate7. However, this amidase controls cell growth. In its absence, peptidoglycan synthesis becomes spatially dysregulated, which causes cells to grow so large that cell division is defective. We show that the cell growth and division defects due to loss of this amidase can be mitigated by attenuating the polymerase activity of the major S. aureus peptidoglycan synthase. Our findings lead to a model wherein the amidase complex regulates the density of peptidoglycan assembly sites to control peptidoglycan synthase activity at a given subcellular location. Removal of stem peptides from peptidoglycan at the cell periphery promotes peptidoglycan synthase relocation to midcell during cell division. This mechanism ensures that cell expansion is properly coordinated with cell division.

Formation of a ß-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA.

The ß-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane ß-barrel proteins. BamA, an essential component of the complex, is itself a ß-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control ß-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA ß-barrel. Importantly, we identify contacts between the assembling ß-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze ß-barrel assembly.