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 bamA_E470K. BamA^E470K 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 BamA^E470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamA^E470K. Thus, it is the altered activity of BamA^E470K 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. Significance The spread of multidrug-resistant infections demands antibiotic development. Although new antibiotics targeting gram-positive bacteria have been developed, it has been more than 50 y since a new class of antibiotics targeting gram-negative bacteria has been approved for clinical use. The strong outer membrane permeability barrier makes it difficult for compounds to reach intracellular targets, contributing greatly to the lack of new antibiotics targeting this class of microbes. Here, we describe a compound, MRL-494, that inhibits the assembly of proteins into the outer membrane by targeting an essential member of β-barrel assembly machine. This compound appears to act at the cell surface, avoiding both the outer membrane permeability barrier and the action of multidrug-resistance efflux pumps.

The outer membrane (OM) of Gram-negative bacteria poses a barrier to antibiotic entry due to its high impermeability. Thus, there is an urgent need to study the function and biogenesis of the OM. InEnterobacterales, an order of bacteria with many pathogenic members, one of the components of the OM is enterobacterial common antigen (ECA). We have known of the presence of ECA on the cell surface ofEnterobacteralesfor many years, but its properties have only more recently begun to be unraveled.

Lipoprotein RcsF is the OM component of the Rcs envelope stress response. RcsF exists in complexes with β-barrel proteins (OMPs) allowing it to adopt a transmembrane orientation with a lipidated N-terminal domain on the cell surface and a periplasmic C-terminal domain. Here we report that mutations that remove BamE or alter a residue in the RcsF trans-lumen domain specifically prevent assembly of the interlocked complexes without inactivating either RcsF or the OMP. Using these mutations we demonstrate that these RcsF/OMP complexes are required for sensing OM outer leaflet stress. Using mutations that alter the positively charged surface-exposed domain, we show that RcsF monitors lateral interactions between lipopolysaccharide (LPS) molecules. When these interactions are disrupted by cationic antimicrobial peptides, or by the loss of negatively charged phosphate groups on the LPS molecule, this information is transduced to the RcsF C-terminal signaling domain located in the periplasm to activate the stress response.

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.

Gram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. In Enterobacteriaceae , the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughout Enterobacteriaceae , the function of which is poorly understood. Previously, we identified several genes in Escherichia coli K-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these, yhdP , a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants of yhdP with loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability from yhdP deletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore, yhdP deletion changed cellular ECA levels and yhdP was found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability with yhdP deletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found with yhdP deletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP. IMPORTANCE Enterobacterial common antigen (ECA) is a surface antigen made by all members of Enterobacteriaceae , including many clinically relevant genera (e.g., Escherichia , Klebsiella , Yersinia ). Although this surface-exposed molecule is conserved throughout Enterobacteriaceae , very few functions have been ascribed to it. Here, we have determined that the periplasmic form of ECA, cyclic ECA, plays a role in maintaining the outer membrane permeability barrier. This activity is controlled by a protein of unknown function, YhdP, and deletion of yhdP damages the OM permeability barrier in a cyclic ECA-dependent manner, allowing harmful molecules such as antibiotics into the cell. This role in maintenance of the envelope permeability barrier is the first time a phenotype has been described for cyclic ECA. As the Gram-negative envelope is generally impermeable to antibiotics, understanding the mechanisms through which the barrier is maintained and antibiotics are excluded may lead to improved antibiotic delivery.