Potent activity of polymyxin B is associated with long-lived super-stoichiometric accumulation mediated by weak-affinity binding to lipid A.

Polymyxins are gram-negative antibiotics that target lipid A, the conserved membrane anchor of lipopolysaccharide in the outer membrane. Despite their clinical importance, the molecular mechanisms underpinning polymyxin activity remain unresolved. Here, we use surface plasmon resonance to kinetically interrogate interactions between polymyxins and lipid A and derive a phenomenological model. Our analyses suggest a lipid A-catalyzed, three-state mechanism for polymyxins: transient binding, membrane insertion, and super-stoichiometric cluster accumulation with a long residence time. Accumulation also occurs for brevicidine, another lipid A-targeting antibacterial molecule. Lipid A modifications that impart polymyxin resistance and a non-bactericidal polymyxin derivative exhibit binding that does not evolve into long-lived species. We propose that transient binding to lipid A permeabilizes the outer membrane and cluster accumulation enables the bactericidal activity of polymyxins. These findings could establish a blueprint for discovery of lipid A-targeting antibiotics and provide a generalizable approach to study interactions with the gram-negative outer membrane.

Toxigenic Clostridium perfringens isolated from at-risk pediatric inflammatory bowel disease patients.

BACKGROUND AND AIMS: The goal was to identify microbial drivers of IBD, by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from pediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying C. perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in 8 of 9 patients' mucosal biopsies, correlating with hemolytic activity, while not in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults (18.7-27.1%) versus healthy (5.1%). In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial, neuroblasts, and neutrophils, while impact on epithelial cells was less pronounced, suggesting C. perfringens may be damaging particularly when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed PFO toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Alternative therapeutic strategies to treat antibiotic-resistant pathogens.

Resistance threatens to render antibiotics - which are essential for modern medicine - ineffective, thus posing a threat to human health. The discovery of novel classes of antibiotics able to overcome resistance has been stalled for decades, with the developmental pipeline relying almost entirely on variations of existing chemical scaffolds. Unfortunately, this approach has been unable to keep pace with resistance evolution, necessitating new therapeutic strategies. In this Review, we highlight recent efforts to discover non-traditional antimicrobials, specifically describing the advantages and limitations of antimicrobial peptides and macrocycles, antibodies, bacteriophages and antisense oligonucleotides. These approaches have the potential to stem the tide of resistance by expanding the physicochemical property space and target spectrum occupied by currently approved antibiotics.

Returning to Nature for the Next Generation of Antimicrobial Therapeutics.

Antibiotics found in and inspired by nature are life-saving cures for bacterial infections and have enabled modern medicine. However, the rise in resistance necessitates the discovery and development of novel antibiotics and alternative treatment strategies to prevent the return to a pre-antibiotic era. Once again, nature can serve as a source for new therapies in the form of natural product antibiotics and microbiota-based therapies. Screening of soil bacteria, particularly actinomycetes, identified most of the antibiotics used in the clinic today, but the rediscovery of existing molecules prompted a shift away from natural product discovery. Next-generation sequencing technologies and bioinformatics advances have revealed the untapped metabolic potential harbored within the genomes of environmental microbes. In this review, we first highlight current strategies for mining this untapped chemical space, including approaches to activate silent biosynthetic gene clusters and in situ culturing methods. Next, we describe how using live microbes in microbiota-based therapies can simultaneously leverage many of the diverse antimicrobial mechanisms found in nature to treat disease and the impressive efficacy of fecal microbiome transplantation and bacterial consortia on infection. Nature-provided antibiotics are some of the most important drugs in human history, and new technologies and approaches show that nature will continue to offer valuable inspiration for the next generation of antibacterial therapeutics.

Physical properties of the bacterial outer membrane.

It has long been appreciated that the Gram-negative outer membrane acts as a permeability barrier, but recent studies have uncovered a more expansive and versatile role for the outer membrane in cellular physiology and viability. Owing to recent developments in microfluidics and microscopy, the structural, rheological and mechanical properties of the outer membrane are becoming apparent across multiple scales. In this Review, we discuss experimental and computational studies that have revealed key molecular factors and interactions that give rise to the spatial organization, limited diffusivity and stress-bearing capacity of the outer membrane. These physical properties suggest broad connections between cellular structure and physiology, and we explore future prospects for further elucidation of the implications of outer membrane construction for cellular fitness and survival.

The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding.

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun's Lipoprotein.

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.

Border Control: Regulating LPS Biogenesis.

The outer membrane (OM) is a defining feature of Gram-negative bacteria that serves as a permeability barrier and provides rigidity to the cell. Critical to OM function is establishing and maintaining an asymmetrical bilayer structure with phospholipids in the inner leaflet and the complex glycolipid lipopolysaccharide (LPS) in the outer leaflet. Cells ensure this asymmetry by regulating the biogenesis of lipid A, the conserved and essential anchor of LPS. Here we review the consequences of disrupting the regulatory components that control lipid A biogenesis, focusing on the rate-limiting step performed by LpxC. Dissection of these processes provides critical insights into bacterial physiology and potential new targets for antibiotics able to overcome rapidly spreading resistance mechanisms.

Structure of the essential inner membrane lipopolysaccharide-PbgA complex.

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.

Massive antibody discovery used to probe structure-function relationships of the essential outer membrane protein LptD.

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.

The Escherichia coli ß-Barrel Assembly Machinery Is Sensitized to Perturbations under High Membrane Fluidity.

Integral ß-barrel membrane proteins are folded and inserted into the Gram-negative bacterial outer membrane by the ß-barrel assembly machine (BAM). This essential complex, composed of a ß-barrel protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE, resides in the outer membrane, a unique asymmetrical lipid bilayer that is difficult to recapitulate in vitro Thus, the probing of BAM function in living cells is critical to fully understand the mechanism of ß-barrel folding. We recently identified an anti-BamA monoclonal antibody, MAB1, that is a specific and potent inhibitor of BamA function. Here, we show that the inhibitory effect of MAB1 is enhanced when BAM function is perturbed by either lowering the level of BamA or removing the nonessential BAM lipoproteins, BamB, BamC, or BamE. The disruption of BAM reduces BamA activity, increases outer membrane (OM) fluidity, and activates the σE stress response, suggesting the OM environment and BAM function are interconnected. Consistent with this idea, an increase in the membrane fluidity through changes in the growth environment or alterations to the lipopolysaccharide in the outer membrane is sufficient to provide resistance to MAB1 and enable the BAM to tolerate these perturbations. Amino acid substitutions in BamA at positions in the outer membrane spanning region or the periplasmic space remote from the extracellular MAB1 binding site also provide resistance to the inhibitory antibody. Our data highlight that the outer membrane environment is a critical determinant in the efficient and productive folding of ß-barrel membrane proteins by BamA.IMPORTANCE BamA is an essential component of the ß-barrel assembly machine (BAM) in the outer membranes of Gram-negative bacteria. We have used a recently described inhibitory anti-BamA antibody, MAB1, to identify the molecular requirements for BAM function. Resistance to this antibody can be achieved through changes to the outer membrane or by amino acid substitutions in BamA that allosterically affect the response to MAB1. Sensitivity to MAB1 is achieved by perturbing BAM function. By using MAB1 activity and functional assays as proxies for BAM function, we link outer membrane fluidity to BamA activity, demonstrating that an increase in membrane fluidity sensitizes the cells to BAM perturbations. Thus, the search for potential inhibitors of BamA function must consider the membrane environment in which ß-barrel folding occurs.

A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies.

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

Monoclonal antibody targeting the ß-barrel assembly machine of Escherichia coli is bactericidal.

The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

A Novel Inhibitor of the LolCDE ABC Transporter Essential for Lipoprotein Trafficking in Gram-Negative Bacteria.

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral ß-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of Escherichia coli growth followed by selection of compounds that induced the extracytoplasmic σE stress response. Mutations in lolC, lolD, and lolE conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of E. coli cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolCQ258K), which confers high-level resistance to G0507 in vivo but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.

Comprehensive analysis reveals how single nucleotides contribute to noncoding RNA function in bacterial quorum sensing.

Five homologous noncoding small RNAs (sRNAs), called the Qrr1-5 sRNAs, function in the Vibrio harveyi quorum-sensing cascade to drive its operation. Qrr1-5 use four different regulatory mechanisms to control the expression of ∼ 20 mRNA targets. Little is known about the roles individual nucleotides play in mRNA target selection, in determining regulatory mechanism, or in defining Qrr potency and dynamics of target regulation. To identify the nucleotides vital for Qrr function, we developed a method we call RSort-Seq that combines saturating mutagenesis, fluorescence-activated cell sorting, high-throughput sequencing, and mutual information theory to explore the role that every nucleotide in Qrr4 plays in regulation of two mRNA targets, luxR and luxO. Companion biochemical assays allowed us to assign specific regulatory functions/underlying molecular mechanisms to each important base. This strategy yielded a regional map of nucleotides in Qrr4 vital for stability, Hfq interaction, stem-loop formation, and base pairing to both luxR and luxO, to luxR only, and to luxO only. In terms of nucleotides critical for sRNA function, the RSort-Seq analysis provided strikingly different results from those predicted by commonly used regulatory RNA-folding algorithms. This approach is applicable to any RNA-RNA interaction, including sRNAs in other bacteria and regulatory RNAs in higher organisms.

A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics.

Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.

Quorum sensing regulates the osmotic stress response in Vibrio harveyi.

Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress. Here we show that LuxR activates expression of the glycine betaine operon betIBA-proXWV, which enhances growth recovery under osmotic stress conditions. BetI, an autorepressor of the V. harveyi betIBA-proXWV operon, activates the expression of genes encoding regulatory small RNAs that control quorum-sensing transitions. Connecting quorum-sensing and glycine betaine pathways presumably enables V. harveyi to tune its execution of collective behaviors to its tolerance to stress.

Functional determinants of the quorum-sensing non-coding RNAs and their roles in target regulation.

Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1-5, control quorum-sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem-loops: the first stem-loop is crucial for base pairing with a subset of targets. This stem-loop also protects the Qrr sRNAs from RNase E-mediated degradation. The second stem-loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem-loop plays an accessory role in base pairing and stability. The fourth stem-loop functions as a rho-independent terminator. In the quorum-sensing regulon, Qrr sRNAs-controlled genes are the most rapid to respond to quorum-sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing.

Individual and combined roles of the master regulators AphA and LuxR in control of the Vibrio harveyi quorum-sensing regulon.

Bacteria use a chemical communication process called quorum sensing to control transitions between individual and group behaviors. In the Vibrio harveyi quorum-sensing circuit, two master transcription factors, AphA and LuxR, coordinate the quorum-sensing response. Here we show that AphA regulates 167 genes, LuxR regulates 625 genes, and they coregulate 77 genes. LuxR strongly controls genes at both low cell density and high cell density, suggesting that it is the major quorum-sensing regulator. In contrast, AphA is absent at high cell density and acts to fine-tune quorum-sensing gene expression at low cell density. We examined two loci as case studies of coregulation by AphA and LuxR. First, AphA and LuxR directly regulate expression of the genes encoding the quorum-regulatory small RNAs Qrr2, Qrr3, and Qrr4, the consequence of which is a specifically timed transition between the individual and the group life-styles. Second, AphA and LuxR repress type III secretion system genes but at different times and to different extents. The consequence of this regulation is that type III secretion is restricted to a peak at mid-cell density. Thus, the asymmetric production of AphA and LuxR coupled with differences in their strengths and timing of target gene regulation generate a precise temporal pattern of gene expression.

Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation.

Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoyl-phenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiled-coil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.