The outer membrane (OM) of the diderm “gram-negative” class of bacteria is an essential organelle and a robust permeability barrier that prevents many antibiotics from reaching their intracellular targets (1). The OM is a unique asymmetrical lipid bilayer (Fig. 1): The inner leaflet is composed of phospholipids (PLs), and the outer leaflet consists almost exclusively of a glycolipid referred to either as lipopolysaccharide (LPS, in bacteria that attach long repeats of sugars to the glycolipid) or lipooligosaccharide (LOS, in bacteria that attach only a short oligosaccharide to cap the glycolipid) (1). Assembly of these lipids into a contiguous barrier, and how that barrier is maintained in response to damage, is a fascinating biological problem. Both PLs and LPS/LOS are synthesized inside the cell, so they must first transit the inner membrane (IM) and then traverse the hostile aqueous periplasmic environment before being assembled into an OM. Work over the past decade uncovered a protein bridge that links the IM and OM and allows LPS/LOS to flow directly into the OM outer leaflet (2). How PLs are transported to the OM remains a mystery. Understanding the pathways of OM biogenesis is a pressing goal. New antibiotics against gram-negative bacteria are urgently needed (3). Rates of antibiotic resistance continue to rise unabated, while the last truly novel antibiotic effective against gram-negative bacteria was discovered in the 1960s (3). The hope is that treatments interfering with OM biogenesis will offer new lethal therapeutics or will help permeabilize gram-negative bacteria to existing drugs. Until that promise is realized, clinicians are increasingly forced to rely on last-resort antibiotics that were once sidelined due to their unfavorable toxicity profiles, including the OM-targeting antibiotic colistin (polymyxin E) (4). In PNAS, Powers and Trent (5) provide new insights into how colistin-resistant bacteria evolve improved fitness by altering their … [↵][1]1To whom correspondence should be addressed. Email: marcin.grabowicz{at}emory.edu. [1]: #xref-corresp-1-1

Current global efforts are focused on exploring alternative pneumococcal vaccine strategies, aimed at addressing the shortcomings of existing formulations, without compromising efficacy. One such strategy involves the use of one or more pneumococcal protein antigens common to all serotypes, to provide cheap, non-serotype-dependent protection. In this study, we evaluated the protective efficacy of immunization of mice with PdB (a pneumolysin toxoid), PspA, PspC (CbpA), PhtB, and PhtE in an invasive-disease model. The antigens were administered in alum adjuvant, either alone or in various combinations. Protection against intraperitoneal challenge with virulent type 2 and 6A strains was assessed in two murine strains. Our findings show that in some situations, different individual proteins gave the best (and worst) protection. However, in many cases, a synergistic/additive effect was seen by using multiple proteins even where the individual proteins showed little value by themselves. For instance, the median survival times for mice immunized with combinations of PdB and PspA, PdB and PspC, or PspA and PspC were significantly longer than those for mice immunized with any of the single antigens. To date, the combination of PdB, PspA, and PspC offers the best protection.

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 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.

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.