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The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins’ key function is to prevent lethal accumulation of mislocalized lipoproteins.

Gram-negative bacteria shield themselves from antibiotics by producing an outer membrane (OM) that forms a formidable permeability barrier. Multidrug resistance among these organisms is a particularly acute problem that is exacerbated by the OM. The poor penetrance of many available antibiotics prevents their clinical use, and efforts to discover novel classes of antibiotics against Gram-negative bacteria have been unsuccessful for almost 50 years. Recent insights into how the OM is built offer new hope. Several essential multiprotein molecular machines (Bam, Lpt, and Lol) work in concert to assemble the barrier and offer a swathe of new targets for novel therapeutic development. Murepavadin has been at the vanguard of these efforts, but its recently reported phase III clinical trial toxicity has tempered the anticipation of imminent new clinical options. Nonetheless, the many concerted efforts aimed at breaking down the OM barrier provide a source of ongoing optimism for what may soon come through the development pipeline. We will review the current state of drug development against the OM assembly targets, highlighting insightful new discovery approaches and strategies.

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.

The outer membrane built by Gram-negative bacteria such as Escherichia coli forms a barrier that prevents antibiotics from entering the cell, limiting clinical options at a time of prevalent antibiotic resistance. Stress responses ensure that barrier integrity is continuously maintained. We have identified the Cpx signal transduction system as a stress response that monitors the trafficking of lipid-anchored lipoproteins to the outer membrane. These lipoproteins are needed by every machine that builds the outer membrane. Cpx monitors just one lipoprotein, NlpE, to detect the efficiency of lipoprotein trafficking in the cell. NlpE and Cpx were previously shown to play a role in resistance to copper. We show that copper blocks lipoprotein trafficking, reconciling old and new observations. Copper is an important element in innate immunity against pathogens, and our findings suggest that NlpE and Cpx help E. coli survive the assault of copper on a key outer membrane assembly pathway. Gram-negative bacteria produce lipid-anchored lipoproteins that are trafficked to their outer membrane (OM). These lipoproteins are essential components in each of the molecular machines that build the OM, including the Bam machine that assembles β-barrel proteins and the Lpt pathway that transports lipopolysaccharide. Stress responses are known to monitor Bam and Lpt function, yet no stress system has been found that oversees the fundamental process of lipoprotein trafficking. We used genetic and chemical biology approaches to induce several different lipoprotein trafficking stresses in Escherichia coli . Our results identified the Cpx two-component system as a stress response for monitoring trafficking. Cpx is activated by trafficking defects and is required to protect the cell against the consequence of the resulting stress. The OM-targeted lipoprotein NlpE acts as a sensor that allows Cpx to gauge trafficking efficiency. We reveal that NlpE signals to Cpx while it is transiting the inner membrane (IM) en route to the OM and that only a small highly conserved N-terminal domain is required for signaling. We propose that defective trafficking causes NlpE to accumulate in the IM, activating Cpx to mount a transcriptional response that protects cells. Furthermore, we reconcile this new role of NlpE in signaling trafficking defects with its previously proposed role in sensing copper (Cu) stress by demonstrating that Cu impairs acylation of lipoproteins and, consequently, their trafficking to the OM. IMPORTANCE The outer membrane built by Gram-negative bacteria such as Escherichia coli forms a barrier that prevents antibiotics from entering the cell, limiting clinical options at a time of prevalent antibiotic resistance. Stress responses ensure that barrier integrity is continuously maintained. We have identified the Cpx signal transduction system as a stress response that monitors the trafficking of lipid-anchored lipoproteins to the outer membrane. These lipoproteins are needed by every machine that builds the outer membrane. Cpx monitors just one lipoprotein, NlpE, to detect the efficiency of lipoprotein trafficking in the cell. NlpE and Cpx were previously shown to play a role in resistance to copper. We show that copper blocks lipoprotein trafficking, reconciling old and new observations. Copper is an important element in innate immunity against pathogens, and our findings suggest that NlpE and Cpx help E. coli survive the assault of copper on a key outer membrane assembly pathway.

The outer membrane (OM) of bacteria like Escherichia coli and Shigella flexneri forms a barrier that protects cells against antibiotics and immune effectors. The surface-exposed leaflet is filled by lipopolysaccharides (LPS) decorated with long “O-antigen” (O-Ag) polysaccharides. The benefit of covering the surface with O-Ag is well appreciated; these long polysaccharides shield against host assaults. Our study reveals a hidden cost to these long O-Ag polysaccharides: transporting and assembling LPS modified with O-Ag compromises integrity of the OM antibiotic barrier, rendering bacteria vulnerable to antibiotics. Cells must balance O-Ag across two parameters—protection from the host and preserving OM integrity. Our findings also present an inherent benefit to not producing O-Ag, a common feature among diverse bacterial pathogens.

Resistance to current antibiotics is increasingly common. New antibiotics that target essential processes are needed to expand clinical options. For Gram-negative bacteria, their cell surface—the outer membrane (OM)—is an essential organelle and antibiotic barrier that is an attractive target for new antibacterials. Lipoproteins are key to building the OM. The LolCDE transporter is needed to supply the OM with lipoproteins and has been a focus of recent antibiotic discovery. In vitro evidence recently proposed a two-part interaction of LolC with LolA lipoprotein chaperone (which traffics lipoproteins to the OM) via “Hook” and “Pad” regions. We show that this model does not reflect lipoprotein trafficking in vivo . Only the Hook is essential for lipoprotein trafficking and is remarkably robust to mutational changes. The Pad is non-essential for lipoprotein trafficking but plays an ancillary role, contributing to trafficking efficiency. These insights inform ongoing efforts to drug LolCDE.

The promoter most strongly induced upon activation of the Cpx two-component envelope stress response is the cpxP promoter. The 3′ untranscribed region (UTR) of the cpxP transcript is shown to produce a small RNA (sRNA), CpxQ. We investigated the role of CpxQ in combating envelope stress. Remarkably, the two effectors specified by the transcript are deployed to combat distinct stresses in different cellular compartments. CpxP acts in both a regulatory negative-feedback loop and as an effector that combats periplasmic protein misfolding. We find that CpxQ combats toxicity at the inner membrane (IM) by downregulating the synthesis of the periplasmic chaperone Skp. Our data indicate that this regulation prevents Skp from inserting β-barrel outer membrane proteins (OMPs) into the IM, a lethal event that likely collapses the proton motive force. Our findings suggest that Skp can fold and directly insert OMPs into a lipid bilayer in vivo without the aid of the Bam complex. IMPORTANCE Skp is a well-characterized periplasmic chaperone that binds unfolded OMPs. Surprisingly, we find that Skp can catalyze the folding and mistargeting of OMPs into the inner membrane without the aid of the other cellular proteins that normally assemble OMPs. Several OMPs function as diffusion pores. Accordingly, their mistargeting is lethal because it depolarizes the inner membrane. We show that the most highly expressed transcript of the Cpx stress response produces an sRNA from the 3′ UTR, CpxQ, which combats this potential toxicity by downregulating Skp production. Defects in OMP assembly trigger the σ E response to upregulate factors, including Skp, that promote OMP folding. The Cpx response downregulates σ E . Our findings reveal that this heretofore puzzling hierarchy exists to protect the inner membrane. Skp is a well-characterized periplasmic chaperone that binds unfolded OMPs. Surprisingly, we find that Skp can catalyze the folding and mistargeting of OMPs into the inner membrane without the aid of the other cellular proteins that normally assemble OMPs. Several OMPs function as diffusion pores. Accordingly, their mistargeting is lethal because it depolarizes the inner membrane. We show that the most highly expressed transcript of the Cpx stress response produces an sRNA from the 3′ UTR, CpxQ, which combats this potential toxicity by downregulating Skp production. Defects in OMP assembly trigger the σ E response to upregulate factors, including Skp, that promote OMP folding. The Cpx response downregulates σ E . Our findings reveal that this heretofore puzzling hierarchy exists to protect the inner membrane.

Gram-negative bacteria have an outer membrane, which acts as a protective barrier and excludes many antibiotics. The limited number of antibiotics active against Gram-negative bacteria, along with rising rates of antibiotic resistance, highlights the need for efficient antibiotic discovery efforts. The outer membrane (OM) of Gram-negative bacteria is an essential organelle that acts as a formidable barrier to antibiotics. Increasingly prevalent resistance to existing drugs has exacerbated the need for antibiotic discovery efforts targeting the OM. Acylated proteins, known as lipoproteins, are essential in every pathway needed to build the OM. The central role of OM lipoproteins makes their biogenesis a uniquely attractive therapeutic target, but it also complicates in vivo identification of on-pathway inhibitors, as inhibition of OM lipoprotein biogenesis broadly disrupts OM assembly. Here, we use genetics to probe the eight essential proteins involved in OM lipoprotein maturation and trafficking. We define a biological signature consisting of three simple assays that can characteristically identify OM lipoprotein biogenesis defects in vivo . We find that several known chemical inhibitors of OM lipoprotein biogenesis conform to the biological signature. We also examine MAC13243, a proposed inhibitor of OM lipoprotein biogenesis, and find that it fails to conform to the biological signature. Indeed, we demonstrate that MAC13243 activity relies entirely on a target outside of the OM lipoprotein biogenesis pathway. Hence, our signature offers simple tools to easily assess whether antibiotic lead compounds target an essential pathway that is the hub of OM assembly. IMPORTANCE Gram-negative bacteria have an outer membrane, which acts as a protective barrier and excludes many antibiotics. The limited number of antibiotics active against Gram-negative bacteria, along with rising rates of antibiotic resistance, highlights the need for efficient antibiotic discovery efforts. Unfortunately, finding the target of lead compounds, especially ones targeting outer membrane construction, remains difficult. The hub of outer membrane construction is the lipoprotein biogenesis pathway. We show that defects in this pathway result in a signature cellular response that can be used to quickly and accurately validate pathway inhibitors. Indeed, we found that MAC13243, a compound previously proposed to target outer membrane lipoprotein biogenesis, does not fit the signature, and we show that it instead targets an entirely different cellular pathway. Our findings offer a streamlined approach to the discovery and validation of lead antibiotics against a conserved and essential pathway in Gram-negative bacteria.

The β-barrel assembly machine (Bam) complex folds and inserts integral membrane proteins into the outer membrane of Gram-negative bacteria. The two essential components of the complex, BamA and BamD, both interact with substrates, but how the two coordinate with each other during assembly is not clear. To elucidate aspects of this process we slowed the assembly of an essential β-barrel substrate of the Bam complex, LptD, by changing a conserved residue near the C terminus. This defective substrate is recruited to the Bam complex via BamD but is unable to integrate into the membrane efficiently. Changes in the extracellular loops of BamA partially restore assembly kinetics, implying that BamA fails to engage this defective substrate. We conclude that substrate binding to BamD activates BamA by regulating extracellular loop interactions for folding and membrane integration.

The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in Escherichia coli that causes LPS to be modified with PG subunits, intersecting these two pathways. Synthesis of the PG-modified LPS (LPS*) requires ready access to the small PG precursor pool but does not weaken cell wall integrity, challenging models of precursor sequestration at PG assembly machinery. LPS* is efficiently transported to the cell surface without impairing OM function. Because LPS* contains the canonical vancomycin binding site, these surface-exposed molecules confer increased vancomycin-resistance by functioning as molecular decoys that titrate the antibiotic away from its intracellular target. This unexpected LPS glycosylation fuses two potent pathogen-associated molecular patterns (PAMPs). Tiny Gram-negative bacteria are one of humankind's deadliest foes, causing infections of wounds and the bloodstream that are very hard to treat. Many Gram-negative bacteria are resistant to several common antibiotics, and the few treatments available that can successfully kill the bacteria are often also toxic to the patients. Understanding how these bacteria elude antibiotics could help scientists develop better, less toxic treatments. Most bacteria are surrounded by a cell wall that helps protect the bacteria and gives them structure. Many broad-spectrum antibiotics, including penicillin and vancomycin, work by interfering with how this protective wall is built from molecules called peptidoglycans. However, Gram-negative bacteria have an outer membrane that prevents many antibiotics from reaching the cell wall, and so the antibiotics are unable to kill the bacteria. The outer membrane of Gram-negative bacteria is made up of sugars and fatty molecules called lipids. Recently, scientists discovered a mutation that interferes with the movement of the lipid and sugar molecules that make up the outer membrane, which compromises this protective layer and makes the bacteria more susceptible to antibiotics. To learn more about how this mutation interferes with the defenses of the Gram-negative bacteria Escherichia coli, Grabowicz et al. searched for compensating mutations that can counteract it and restore the antibiotic resistance of these mutant bacteria. The search revealed that a mutation in a gene called waaL increases E. coli's resistance to vancomycin, but not to other antibiotics. The gene encodes an enzyme, and the mutant form of the enzyme attaches some peptidoglycans to the surface of the outer membrane instead of incorporating them into the cell wall. The stray peptidoglycans on the cell's surface act as decoys, binding to vancomycin and keeping the drug from reaching its true target—the cell wall. The decoy strategy is similar to a mechanism used by Gram-positive bacteria—which lack a protective outer membrane—to resist vancomycin treatment, which also involves creating sites that bind the drug and keep it from its target. Vancomycin is not currently used clinically to treat E. coli or other Gram-negative infections because these bacteria are naturally quite resistant for other reasons. However, Grabowicz et al.'s findings do demonstrate how quickly bacteria can adapt and produce new defenses to antibiotics when old strategies fail.