The increasing use of polymyxins1 in addition to the dissemination of plasmid-borne colistin resistance threatens to cause a serious breach in our last line of defense against multidrug resistant Gram-negative pathogens, and heralds the emergence of truly pan-resistant infections. Colistin resistance often arises through covalent modification of lipid A with cationic residues such as phosphoethanolamine (PEtN) – as is mediated by Mcr-12 – which reduce the affinity of polymyxins for lipopolysaccharide (LPS)3. Thus, new strategies are needed to address the rapidly diminishing number of treatment options for Gram-negative infections4. The difficulty in eradicating Gram-negative bacteria is largely due to a highly impermeable outer membrane, which serves as a barrier to many otherwise effective antibiotics5. Here, we describe an unconventional screening platform designed to enrich for non-lethal, outer membrane-active compounds with potential as adjuvants for conventional antibiotics. This approach identified the antiprotozoal drug pentamidine6 as an effective perturbant of the Gram-negative outer membrane through its interaction with LPS. Pentamidine displayed synergy with antibiotics typically restricted to Gram-positive bacteria, yielding effective drug combinations with activity against a wide range of Gram-negative pathogens in vitro, and against systemic Acinetobacter baumannii infections in mice. Notably, the adjuvant activity of pentamidine persisted in polymyxin resistant bacteria in vitro and in vivo. Overall, pentamidine and structural analogs represent unexploited molecules for the treatment of Gram-negative infections, particularly those having acquired polymyxin resistance determinants.

A robust and sensitive platform was developed for chemical-genomics in bacteria. Kinetic acquisitions of colony growth enable calculation of growth rates alongside conventional endpoint volume measurements, generating a wealth of chemical-genetic interactions. This kinetic platform is highly amenable to prokaryotic or eukaryotic strain collections.

Therapeutics targeting Gram-negative bacteria have the challenge of overcoming a formidable outer membrane (OM) barrier. Here, we characterize the action of SPR741, a novel polymyxin B (PMB) analogue shown to potentiate several large-scaffold antibiotics in Gram-negative pathogens. Probing the surface topology of Escherichia coli using atomic force microscopy revealed substantial OM disorder at concentrations of SPR741 that lead to antibiotic potentiation. Conversely, very little cytoplasmic membrane depolarization was observed at these same concentrations, indicating that SPR741 acts predominately on the OM. Truncating the lipopolysaccharide (LPS) core with genetic perturbations uniquely sensitized E. coli to SPR741, suggesting that LPS core residues keep SPR741 at the OM, where it can potentiate a codrug, rather than permit its entry to the cytoplasmic membrane. Further, a promoter activity assay revealed that SPR741 challenge induced the expression of RcsAB, a stress sensor for OM perturbation. Together, these results indicate that SPR741 interacts predominately with the OM, in contrast to the dual action of PMB and colistin at both the outer and cytoplasmic membranes.

Conventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo. To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli. Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms.

The outer membrane of Gram-negative bacteria is a formidable permeability barrier which allows only a small subset of chemical matter to penetrate. This outer membrane barrier can hinder the study of cellular processes and compound mechanism of action, as many compounds including antibiotics are precluded from entry despite having intracellular targets. Consequently, outer membrane permeabilizing compounds are invaluable tools in such studies. Many existing compounds known to perturb the outer membrane also impact inner membrane integrity, such as polymyxins and their derivatives, making these probes nonspecific. We performed a screen of ∼140 000 diverse synthetic compounds, for those that antagonized the growth inhibitory activity of vancomycin at 15 °C in Escherichia coli, to enrich for chemicals capable of perturbing the outer membrane. This led to the discovery that liproxstatin-1, an inhibitor of ferroptosis in human cells, and MAC-0568743, a novel cationic amphiphile, could potentiate the activity of large-scaffold antibiotics with low permeation into Gram-negative bacteria at 37 °C. Liproxstatin-1 and MAC-0568743 were found to physically disrupt the integrity of the outer membrane through interactions with lipopolysaccharide in the outer leaflet of the outer membrane. We showed that these compounds selectively disrupt the outer membrane while minimally impacting inner membrane integrity, particularly at the concentrations needed to potentiate Gram-positive-targeting antibiotics. Further exploration of these molecules and their structural analogues is a promising avenue for the development of outer membrane specific probes.