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.

Combining Mutations That Inhibit Two Distinct Steps of the ATP Hydrolysis Cycle Restores Wild-Type Function in the Lipopolysaccharide Transporter and Shows that ATP Binding Triggers Transport.

ATP-binding cassette (ABC) transporters constitute a large family of proteins present in all domains of life. They are powered by dynamic ATPases that harness energy from binding and hydrolyzing ATP through a cycle that involves the closing and reopening of their two ATP-binding domains. The LptB2FGC exporter is an essential ABC transporter that assembles lipopolysaccharides (LPS) on the surface of Gram-negative bacteria to form a permeability barrier against many antibiotics. LptB2FGC extracts newly synthesized LPS molecules from the inner membrane and powers their transport across the periplasm and through the outer membrane. How LptB2FGC functions remains poorly understood. Here, we show that the C-terminal domain of the dimeric LptB ATPase is essential for LPS transport in Escherichia coli Specific changes in the C-terminal domain of LptB cause LPS transport defects that can be repaired by intragenic suppressors altering the ATP-binding domains. Surprisingly, we found that each of two lethal changes in the ATP-binding and C-terminal domains of LptB, when present in combined form, suppressed the defects associated with the other to restore LPS transport to wild-type levels both in vivo and in vitro We present biochemical evidence explaining the effect that each of these mutations has on LptB function and how the observed cosuppression results from the opposing lethal effects these changes have on the dimerization state of the LptB ATPase. We therefore propose that these sites modulate the closing and reopening of the LptB dimer, providing insight into how the LptB2FGC transporter cycles to export LPS to the cell surface and how to inhibit this essential envelope biogenesis process.IMPORTANCE Gram-negative bacteria are naturally resistant to many antibiotics because their surface is covered by the glycolipid LPS. Newly synthesized LPS is transported across the cell envelope by the multiprotein Lpt machinery, which includes LptB2FGC, an unusual ABC transporter that extracts LPS from the inner membrane. Like in other ABC transporters, the LptB2FGC transport cycle is driven by the cyclical conformational changes that a cytoplasmic, dimeric ATPase, LptB, undergoes when binding and hydrolyzing ATP. How these conformational changes are controlled in ABC transporters is poorly understood. Here, we identified two lethal changes in LptB that, when combined, remarkably restore wild-type transport function. Biochemical studies revealed that the two changes affect different steps in the transport cycle, having opposing, lethal effects on LptB's dimerization cycle. Our work provides mechanistic details about the LptB2FGC extractor that could be used to develop Lpt inhibitors that would overcome the innate antibiotic resistance of Gram-negative bacteria.

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.

Novobiocin Enhances Polymyxin Activity by Stimulating Lipopolysaccharide Transport.

Gram-negative bacteria are challenging to kill with antibiotics due to their impenetrable outer membrane containing lipopolysaccharide (LPS). The polymyxins, including colistin, are the drugs of last resort for treating Gram-negative infections. These drugs bind LPS and disrupt the outer membrane; however, their toxicity limits their usefulness. Polymyxin has been shown to synergize with many antibiotics including novobiocin, which inhibits DNA gyrase, by facilitating transport of these antibiotics across the outer membrane. Recently, we have shown that novobiocin not only inhibits DNA gyrase but also binds and stimulates LptB, the ATPase that powers LPS transport. Here, we report the synthesis of novobiocin derivatives that separate these two activities. One analog retains LptB-stimulatory activity but is unable to inhibit DNA gyrase. This analog, which is not toxic on its own, nevertheless enhances the lethality of polymyxin by binding LptB and stimulating LPS transport. Therefore, LPS transport agonism contributes substantially to novobiocin-polymyxin synergy. We also report other novobiocin analogs that inhibit DNA gyrase better than or equal to novobiocin, but bind better to LptB and therefore have even greater LptB stimulatory activity. These compounds are more potent than novobiocin when used in combination with polymyxin. Novobiocin analogs optimized for both gyrase inhibition and LPS transport agonism may allow the use of lower doses of polymyxin, increasing its efficacy and safety.

Cell-based screen for discovering lipopolysaccharide biogenesis inhibitors.

New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway in Acinetobacter We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.