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 outer membrane (OM) of Gram-negative bacteria is the outermost layer of the cell and serves as permeability barrier against environmental toxins, including antibiotics. The OM is built by several pathways that transport and assemble lipids and proteins into the OM. Since the OM is an essential organelle for the cell, envelope stress responses (ESRs) continuously monitor its assembly to preserve viability if defects arise. While ESRs have been extensively characterized in Escherichia coli, they are generally narrowly conserved. Lipoprotein trafficking to the OM via the "Lol" pathway is a linchpin for all OM assembly pathways. In E. coli, defects in this essential process are sensed when the sensor OM lipoprotein NlpE activates the CpxAR two-component system. Distantly related Acinetobacter baumannii encodes an NlpE homolog but lacks any Cpx homolog; how OM lipoprotein stress might be sensed and mitigated in these bacteria is therefore unclear. Here, we used CRISPRi to transiently induce defects in OM lipoprotein synthesis (targeting lgt and lnt) or trafficking (targeting lolA) in A. baumannii. We defined the transcriptional response to blocks in OM lipoprotein biogenesis. After scrutinizing candidate ESRs, we identified the BfmRS two-component systems as specifically critical for preserving A. baumannii viability during stress in OM lipoprotein biogenesis. Surprisingly, A. baumannii NlpE played no role in combatting OM lipoprotein stress. Our study identifies an A. baumannii ESR for OM lipoprotein biogenesis defects that acts in a distinct mechanism, not involving the NlpE sensor lipoprotein. As the cell's surface, the outer membrane (OM) of bacteria, such as Acinetobacter baumannii, is continuously under assault from the environment or host. OM integrity is needed for cell survival, and envelope stress responses (ESRs) act to detect and repair any defects. ESRs are well-defined in Escherichia coli but are poorly conserved. We sought to identify an ESR for the essential process of OM lipoprotein biogenesis in A. baumannii. We found that the BfmRS two-component system performs this function and does so without relying on its NlpE sensor homolog, suggesting a novel mechanism of stress sensing is involved in A. baumannii. Our work identifies a key cellular role for BfmRS.
Significance In Gram-negative bacteria, most lipoproteins synthesized in the inner membrane (IM) are trafficked to the outer membrane (OM). The Lol pathway is the trafficking paradigm: LolCDE releases lipoproteins from the IM; LolA shuttles them between membranes to LolB in the OM. Several OM lipoproteins are essential for viability. In apparent concordance, the Lol proteins are each essential in wild-type cells. However, we show that Escherichia coli grows well without LolA and LolB in the absence of one nonessential substrate and appropriately engineered stress responses, revealing that LolAB do not perform truly essential mechanistic roles in trafficking. Rather, LolAB are needed to prevent toxic lipoprotein mislocalization. Our findings change the prevailing paradigm and reveal the existence of an alternate trafficking route.
Shigella species are the causative agents of human bacillary dysentery. These bacteria spread within the lining of the gut via a process termed actin-based motility whereby an actin ‘tail’ is formed at the bacterial pole. The bacterial outer membrane protein IcsA initiates this process, and crucially is precisely positioned on the bacterial polar surface. Lipopolysaccharide (LPS) O-antigen surface structure has been implicated as an augmenting factor of polarity maintenance due to the apparent dysregulation of IcsA polarity in O-antigen deficient strains. Due to Shigellae having long and short O-antigen chains on their surfaces, it has been proposed that O-antigen chain lengths are asymmetrically distributed to optimize IcsA exposure at the pole and mask exposure laterally. Additionally, it has been proposed that LPS O-antigen restricts IcsA diffusion from the pole by maintaining minimal membrane fluidity. This study utilizes minicells and quantitative microscopy providing data refuting the models of asymmetric masking and membrane diffusion, and supporting a model of symmetric masking of IcsA. We contend that IcsA surface distribution is equivalent between wild-type and O-antigen deficient strains, and that differences in cellular IcsA levels have confounded previous conclusions.
The rod-shaped enteric intracellular pathogen Shigella flexneri and other Shigella species are the causative agents of bacillary dysentery. S. flexneri are able to spread within the epithelial lining of the gut, resulting in lesion formation, cramps and bloody stools. The outer membrane protein IcsA is essential for this spreading process. IcsA is the initiator of an actin-based form of motility whereby it allows the formation of a filamentous actin 'tail' at the bacterial pole. Importantly, IcsA is specifically positioned at the bacterial pole such that this process occurs asymmetrically. The mechanism of IcsA polarity is not completely understood, but it appears to be a multifactorial process involving factors intrinsic to IcsA and other regulating factors. In this study, we further investigated IcsA polarization by its intramolecular N-terminal and central polar-targeting (PT) regions (nPT and cPT regions, respectively). The results obtained support a role in polar localization for the cPT region and contend the role of the nPT region. We identified single IcsA residues that have measurable impacts on IcsA polarity augmentation, resulting in decreased S. flexneri sprading efficiency. Intriguingly, regions and residues involved in PT clustered around a highly conserved motif which may provide a functional scaffold for polarity-augmenting residues. How these results fit with the current model of IcsA polarity determination is discussed.
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.
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.
Significance The outermost membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), and its proper placement on the cell surface is required to establish an effective permeability barrier. The presence of LPS prevents small hydrophobic molecules from entering the cell making it difficult to develop antibiotics. LPS is synthesized inside the cell and must move across three compartments to the cell surface. The final step of LPS transport (Lpt), translocation of LPS across the outer membrane, is accomplished by a two-protein complex (LptDE) that must insert LPS into its outer leaflet. We have identified a binding site within LptE critical for the proper function of the translocon. LptE binds LPS and changes its physical state to facilitate its translocation through the outer membrane.
Gram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens.
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