Gram-negative bacteria are surrounded both by an inner cytoplasmic membrane and by an outer membrane that serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide 1. This large glycolipid with numerous sugars is made in the cytoplasm and a complex of proteins forms a membrane-to-membrane bridge that mediates transport from the inner membrane to the cell surface 1. The inner membrane components of the protein bridge comprise an ATP-binding cassette (ABC) transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is mysterious 2. 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. These structures, combined with biochemical and genetic experiments, identify the path for 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.

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 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.

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.

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.

Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors CD4 and CCR5 on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility. IMPORTANCE Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.