Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter.

The outer membrane (OM) is an essential organelle of Gram-negative bacteria. Lipoproteins are key to building the OM, performing essential functions in several OM assembly machines. Lipoproteins mature in the inner membrane (IM) and are then trafficked to the OM. In Escherichia coli, the LolCDE transporter is needed to extract lipoproteins from the IM to begin trafficking. Lipoproteins are then transferred from LolCDE to the periplasmic chaperone LolA which ferries them to the OM for insertion by LolB. LolA recruitment by LolC is an essential trafficking step. Structural and biochemical studies suggested that two regions (termed Hook and Pad) within a periplasmic loop of LolC worked in tandem to recruit LolA, leading to a bipartite model for recruitment. Here, we genetically examine the LolC periplasmic loop in vivo using E. coli. Our findings challenge the bipartite interaction model. We show that while the Hook is essential for lipoprotein trafficking in vivo, lipoproteins are still efficiently trafficked when the Pad residues are inactivated. We show with AlphaFold2 multimer modeling that Hook:LolA interactions are likely universal among diverse Gram-negative bacteria. Conversely, Pad:LolA interactions vary across phyla. Our in vivo data redefine LolC:LolA recruitment into a hierarchical interaction model. We propose that the Hook is the major player in LolA recruitment, while the Pad plays an ancillary role that is important for efficiency but is ultimately dispensable. Our findings expand the understanding of a fundamental step in essential lipoprotein trafficking and have implications for efforts to develop new antibacterials that target LolCDE.IMPORTANCEResistance 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.

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter.

The outer membrane (OM) is an essential organelle of Gram-negative bacteria. Lipoproteins are key to building the OM, performing essential functions in several OM assembly machines. Lipoproteins mature in the inner membrane (IM) and are then trafficked to the OM. In Escherichia coli, the LolCDE transporter is needed to extract lipoproteins from the IM to begin trafficking. Lipoproteins are then transferred from LolCDE to the periplasmic chaperone LolA which ferries them to the OM for insertion by LolB. LolA recruitment by LolC is an essential trafficking step. Structural and biochemical studies suggested that two regions (termed Hook and Pad) within a periplasmic loop of LolC worked in tandem to recruit LolA, leading to a bipartite model for recruitment. Here, we genetically examine the LolC periplasmic loop in vivo using E. coli. Our findings challenge the bipartite interaction model. We show that while the Hook is essential for lipoprotein trafficking in vivo, lipoproteins are still efficiently trafficked when the Pad residues are inactivated. We show with AlphaFold2 multimer modeling that Hook:LolA interactions are likely universal among diverse Gram-negative bacteria. Conversely, Pad:LolA interactions vary across phyla. Our in vivo data redefine LolC:LolA recruitment into a hierarchical interaction model. We propose that the Hook is the major player in LolA recruitment, while the Pad plays an ancillary role that is important for efficiency but is ultimately dispensable. Our findings expand the understanding of a fundamental step in essential lipoprotein trafficking and have implications for efforts to develop new antibacterials that target LolCDE.

Molecular insights into Escherichia coli Cpx envelope stress response activation by the sensor lipoprotein NlpE.

Bacterial two-component signal transduction systems provide sensory inputs for appropriately adapting gene expression. These systems rely on a histidine kinase that phosphorylates a response regulator which alters gene expression. Several two-component systems include additional sensory components that can activate the histidine kinase. In Escherichia coli, the lipoprotein NlpE was identified as a sensor for the Cpx cell envelope stress response. It has remained unclear how NlpE signals to Cpx in the periplasm. In this study, we used a combination of genetics, biochemistry, and AlphaFold2 complex modeling to uncover the molecular details of how NlpE triggers the Cpx response through an interaction with the CpxA histidine kinase. Remarkably, only a short loop of NlpE is required to activate the Cpx response. A single substitution in this loop inactivates NlpE signaling to Cpx and abolishes an in vivo biochemical NlpE:CpxA interaction. An independent AlphaFold multimer prediction supported a role for the loop and predicted an interaction interface at CpxA. Mutations in this CpxA region specifically blind the histidine kinase to NlpE activation but preserve the ability to respond to other cell envelope stressors. Hence, our work additionally reveals a previously unrecognized complexity in signal integration by the CpxA periplasmic sensor domain.

Teasing apart the evolution of lipoprotein trafficking in gram-negative bacteria reveals a bifunctional LolA.

The outer membrane (OM) is the defining feature of gram-negative bacteria and is an essential organelle. Accordingly, OM assembly pathways and their essential protein components are conserved throughout all gram-negative species. Lipoprotein trafficking lies at the heart of OM assembly since it supplies several different biogenesis machines with essential lipoproteins. The Escherichia coli Lol trafficking pathway relies on an inner membrane LolCDE transporter that transfers newly made lipoproteins to the chaperone LolA, which rapidly traffics lipoproteins across the periplasm to LolB for insertion into the OM. Strikingly, many gram-negative species (like Caulobacter vibrioides) do not produce LolB, yet essential lipoproteins are still trafficked to the OM. How the final step of trafficking occurs in these organisms has remained a long-standing mystery. We demonstrate that LolA from C. vibrioides can complement the deletion of both LolA and LolB in E. coli, revealing that this protein possesses both chaperone and insertion activities. Moreover, we define the region of C. vibrioides LolA that is responsible for its bifunctionality. This knowledge enabled us to convert E. coli LolA into a similarly bifunctional protein, capable of chaperone and insertion activities. We propose that a bifunctional LolA eliminates the need for LolB. Our findings provide an explanation for why some gram-negative species have retained an essential LolA yet completely lack a dedicated LolB protein.

A Biological Signature for the Inhibition of Outer Membrane Lipoprotein Biogenesis.

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.

Mucoidy, a general mechanism for maintaining lytic phage in populations of bacteria.

We present evidence that phage resistance resulting from overproduction of exopolysaccharides, mucoidy, provides a general answer to the longstanding question of how lytic viruses are maintained in populations dominated by bacteria upon which they cannot replicate. In serial transfer culture, populations of mucoid Escherichia coli MG1655 that are resistant to lytic phages with different receptors, and thereby requiring independent mutations for surface resistance, are capable of maintaining these phages with little effect on their total density. Based on the results of our analysis of a mathematical model, we postulate that the maintenance of phage in populations dominated by mucoid cells can be attributed primarily to high rates of transition from the resistant mucoid states to susceptible non-mucoid states. Our tests with both population dynamic and single cell experiments as well as genomic analysis are consistent with this hypothesis. We discuss reasons for the generalized resistance of these mucoid E. coli, and the genetic and molecular mechanisms responsible for the high rate of transition from mucoid to sensitive states responsible for the maintenance of lytic phage in mucoid populations of E. coli.

A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier.

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 ß-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.

Countering Gram-Negative Antibiotic Resistance: Recent Progress in Disrupting the Outer Membrane with Novel Therapeutics.

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.

A Stress Response Monitoring Lipoprotein Trafficking to the Outer Membrane.

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 ß-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.

Lipoproteins and Their Trafficking to the Outer Membrane.

Lipoproteins are produced by both Gram-positive and Gram-negative bacteria. Once secreted, lipoproteins are quickly acylated, anchoring them into the plasma membrane. Recent work has shown that Gram-positive bacteria are able to generate considerable diversity in the acylation of their lipoproteins, though the mechanisms involved are only just beginning to emerge. In Gram-negative organisms, most lipoproteins are subsequently trafficked to the outer membrane (OM). Lipoprotein trafficking is an essential pathway in these bacteria. At least one OM lipoprotein component is required by each of the essential machines that assemble the OM (such as the Bam and Lpt machines) and build the peptidoglycan cell wall (Lpo-penicillin-binding protein complexes). The Lol pathway has been the paradigm for OM lipoprotein trafficking: a complex of LolCDE extracts lipoproteins from the plasma membrane, LolA shuttles them through the periplasmic space, and LolB anchors them into the OM. The peptide signals responsible for OM-targeting via LolCDE have long been known for Escherichia coli. Remarkably, production of novel lipoprotein acyl forms in E. coli has reinforced the idea that lipid signals also contribute to OM targeting via LolCDE. Moreover, recent work has shown that lipoprotein trafficking can occur in E. coli without either LolA or LolB. Therefore, current evidence suggests that at least one additional, LolAB-independent route for OM lipoprotein trafficking exists. This chapter reviews the posttranslocation modifications of all lipoproteins, with a focus on the trafficking of lipoproteins to the OM of Gram-negative bacteria.

Fine-Tuning of σE Activation Suppresses Multiple Assembly-Defective Mutations in Escherichia coli.

The Gram-negative outer membrane (OM) is a selectively permeable asymmetric bilayer that allows vital nutrients to diffuse into the cell but prevents toxins and hydrophobic molecules from entering. Functionally and structurally diverse ß-barrel outer membrane proteins (OMPs) build and maintain the permeability barrier, making the assembly of OMPs crucial for cell viability. In this work, we characterize an assembly-defective mutant of the maltoporin LamB, LamBG439D We show that the folding defect of LamBG439D results in an accumulation of unfolded substrate that is toxic to the cell when the periplasmic protease DegP is removed. Selection for suppressors of this toxicity identified the novel mutant degSA323E allele. The mutant DegSA323E protein contains an amino acid substitution at the PDZ/protease domain interface that results in a partially activated conformation of this protein. This activation increases basal levels of downstream σE stress response signaling. Furthermore, the enhanced σE activity of DegSA323E suppresses a number of other assembly-defective conditions without exhibiting the toxicity associated with high levels of σE activity. We propose that the increased basal levels of σE signaling primes the cell to respond to envelope stress before OMP assembly defects threaten cell viability. This finding addresses the importance of envelope stress responses in monitoring the OMP assembly process and underpins the critical balance between envelope defects and stress response activation.IMPORTANCE Gram-negative bacteria, such as Escherichia coli, inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the σE stress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival.

Inhibitor of intramembrane protease RseP blocks the σE response causing lethal accumulation of unfolded outer membrane proteins.

The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a ß-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.

Lipoprotein Transport: Greasing the Machines of Outer Membrane Biogenesis: Re-Examining Lipoprotein Transport Mechanisms Among Diverse Gram-Negative Bacteria While Exploring New Discoveries and Questions.

The Gram-negative outer membrane (OM) is a potent permeability barrier against antibiotics, limiting clinical options amid mounting rates of resistance. The Lol transport pathway delivers lipoproteins to the OM. All the OM assembly machines require one or more OM lipoprotein to function, making the Lol pathway central for all aspects of OM biogenesis. The Lol pathways of many medically important species clearly deviate from the Escherichia coli paradigm, perhaps with implications for efforts to develop novel antibiotics. Moreover, recent work reveals the existence of an undiscovered alternate route for bringing lipoproteins to the OM. Here, lipoprotein transport mechanisms, and the quality control systems that underpin them, is re-examined in context of their diversity.

Substrate binding to BamD triggers a conformational change in BamA to control membrane insertion.

The ß-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 ß-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.

Redefining the essential trafficking pathway for outer membrane lipoproteins.

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.

Envelope Stress Responses: An Interconnected Safety Net.

The Escherichia coli cell envelope is a protective barrier at the frontline of interaction with the environment. Fidelity of envelope biogenesis must be monitored to establish and maintain a contiguous barrier. Indeed, the envelope must also be repaired and modified in response to environmental assaults. Envelope stress responses (ESRs) sense envelope damage or defects and alter the transcriptome to mitigate stress. Here, we review recent insights into the stress-sensing mechanisms of the σE and Cpx systems and the interaction of these ESRs. Small RNAs (sRNAs) are increasingly prominent regulators of the transcriptional response to stress. These fast-acting regulators also provide avenues for inter-ESR regulation that could be important when cells face multiple contemporaneous stresses, as is the case during infection.

Characterization of a stalled complex on the ß-barrel assembly machine.

The assembly of ß-barrel proteins into membranes is mediated by an evolutionarily conserved machine. This process is poorly understood because no stable partially folded barrel substrates have been characterized. Here, we slowed the folding of the Escherichia coli ß-barrel protein, LptD, with its lipoprotein plug, LptE. We identified a late-stage intermediate in which LptD is folded around LptE, and both components interact with the two essential ß-barrel assembly machine (Bam) components, BamA and BamD. We propose a model in which BamA and BamD act in concert to catalyze folding, with the final step in the process involving closure of the ends of the barrel with release from the Bam components. Because BamD and LptE are both soluble proteins, the simplest model consistent with these findings is that barrel folding by the Bam complex begins in the periplasm at the membrane interface.

The CpxQ sRNA Negatively Regulates Skp To Prevent Mistargeting of ß-Barrel Outer Membrane Proteins into the Cytoplasmic Membrane.

UNLABELLED: 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 ß-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.