Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.     

Targeting the LPS export pathway for the development of novel therapeutics

The rapid rise of multi-resistant bacteria is a global health threat. This is especially serious for Gram-negative bacteria in which the impermeable outer membrane (OM) acts as a shield against antibiotics. The development of new drugs with novel modes of actions to combat multi-drug resistant pathogens requires the selection of suitable processes to be targeted. The LPS export pathway is an excellent under exploited target for drug development. Indeed, LPS is the major determinant of the OM permeability barrier, and its biogenetic pathway is conserved in most Gram-negatives. Here we describe efforts to identify inhibitors of the multiprotein Lpt system that transports LPS to the cell surface. Despite none of these molecules has been approved for clinical use, they may represent valuable compounds for optimization. Finally, the recent discovery of a link between inhibition of LPS biogenesis and changes in peptidoglycan structure uncovers additional targets to develop novel therapeutic strategies.     

Antibacterial mechanisms of GN-2 derived peptides and peptoids against Escherichia coli

Escherichia coli is the main etiological agent of urinary trait infections, able to form biofilms in indwelling devices, resulting in chronic infections which are refractory to antibiotics treatment. In this study, we investigated the antimicrobial and anti-biofilm properties exerted against E. coli ATCC 25922, by a set of peptoids and peptides modeled upon the peptide GN-2, previously reported as a valid antimicrobial agent. The putative antimicrobials were designed to evaluate the effect of cationicity, hydrophobicity and their partitioning on the overall properties against planktonic cells and biofilms as well as on LPS binding, permeabilization of Gram-negative bacteria membranes and hemolysis. The data demonstrated that peptides are stronger antimicrobials than the analogue peptoids which in return have superior anti-biofilm properties. In this study, we present evidence that peptides antimicrobial activity correlates with enhanced LPS binding and hydrophobicity but is not affected by partitioning. The data demonstrated that the enhanced anti-biofilm properties of the peptoids are associated with decreased hydrophobicity and increased penetration of the inner membrane, compared to that of their peptide counterpart, suggesting that the characteristic flexibility of peptoids or their lack of H-bonding donors in their backbone, would play a role in their ability to penetrate bacterial membranes.     

Elucidating the mechanism by which synthetic helper peptides sensitize Pseudomonas aeruginosa to multiple antibiotics

The emergence and rapid spread of multi-drug resistant (MDR) bacteria pose a serious threat to the global healthcare. There is an urgent need for new antibacterial substances or new treatment strategies to deal with the infections by MDR bacterial pathogens, especially the Gram-negative pathogens. In this study, we show that a number of synthetic cationic peptides display strong synergistic antimicrobial effects with multiple antibiotics against the Gram-negative pathogen Pseudomonas aeruginosa. We found that an all-D amino acid containing peptide called D-11 increases membrane permeability by attaching to LPS and membrane phospholipids, thereby facilitating the uptake of antibiotics. Subsequently, the peptide can dissipate the proton motive force (PMF) (reducing ATP production and inhibiting the activity of efflux pumps), impairs the respiration chain, promotes the production of reactive oxygen species (ROS) in bacterial cells and induces intracellular antibiotics accumulation, ultimately resulting in cell death. By using a P. aeruginosa abscess infection model, we demonstrate enhanced therapeutic efficacies of the combination of D-11 with various antibiotics. In addition, we found that the combination of D-11 and azithromycin enhanced the inhibition of biofilm formation and the elimination of established biofilms. Our study provides a realistic treatment option for combining close-to-nature synthetic peptide adjuvants with existing antibiotics to combat infections caused by P. aeruginosa.     

A Forward Chemical Screen Identifies Antibiotic Adjuvants in Escherichia coli

Multi-drug-resistant infections caused by Gram-negative pathogens are rapidly increasing, highlighting the need for new chemotherapies. Unlike Gram-positive bacteria, where many different chemical classes of antibiotics show efficacy, Gram-negatives are intrinsically insensitive to many antimicrobials including the macrolides, rifamycins, and aminocoumarins, despite intracellular targets that are susceptible to these drugs. The basis for this insensitivity is the presence of the impermeant outer membrane of Gram-negative bacteria in addition to the expression of pumps and porins that reduce intracellular concentrations of many molecules. Compounds that sensitize Gram-negative cells to "Gram-positive antibiotics", antibiotic adjuvants, offer an orthogonal approach to addressing the crisis of multi-drug-resistant Gram-negative pathogens. We performed a forward chemical genetic screen of 30,000 small molecules designed to identify such antibiotic adjuvants of the aminocoumarin antibiotic novobiocin in Escherichia coli. Four compounds from this screen were shown to be synergistic with novobiocin including inhibitors of the bacterial cytoskeleton protein MreB, cell wall biosynthesis enzymes, and DNA synthesis. All of these molecules were associated with altered cell shape and small molecule permeability, suggesting a unifying mechanism for these antibiotic adjuvants. The potential exists to expand this approach as a means to develop novel combination therapies for the treatment of infections caused by Gram-negative pathogens.     

Towards Gram-negative antivirulence drugs: New inhibitors of HldE kinase

Gram-negative bacteria lacking heptoses in their lipopolysaccharide (LPS) display attenuated virulence and increased sensitivity to human serum and to some antibiotics. Thus inhibition of bacterial heptose synthesis represents an attractive target for the development of new antibacterial agents. HldE is a bifunctional enzyme involved in the synthesis of bacterial heptoses. Development of a biochemical assay suitable for high-throughput screening allowed the discovery of inhibitors 1 and 2 of HldE kinase. Study of the structure–activity relationship of this series of inhibitors led to highly potent compounds.     

Homology modeling,docking and structure-based virtual screening for new inhibitor identification of Klebsiella pneumoniae heptosyltransferase-III

Abstract

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative opportunistic pathogen commonly associated with hospital-acquired infections that are often resistant even to antibiotics. Heptosyltransferase (HEP) belongs to the family of glycosyltransferase-B (GT-B) and plays an important in the synthesis of lipopolysaccharides (LPS) essential for the formation of bacterial cell membrane. HEP-III participates in the transfer of heptose sugar to the outer surface of bacteria to synthesize LPS. LPS truncation increases the bacterial sensitivity to hydrophobic antibiotics and detergents, making the HEP as a novel drug target. In the present study, we report the 3D homology model of K. pneumoniae HEP-III and its structure validation. Active site was identified based on similarities with known structures using Dali server, and structure-based pharmacophore model was developed for the active site substrate ADP. The generated pharmacophore model was used as a 3D search query for virtual screening of the ASINEX database. The hit compounds were further filtered based on fit value, molecular docking, docking scores, molecular dynamics (MD) simulations of HEP-III complexed with hit molecules, followed by binding free energy calculations using Molecular Mechanics-Poisson–Boltzmann Surface Area (MM-PBSA). The insights obtained in this work provide the rationale for design of novel inhibitors targeting K. pneumoniae HEP-III and the mechanistic aspects of their binding.

Communicated by Ramaswamy H. Sarma     

Lipopolysaccharide as an antibiotic target

Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are amongst the highest priority drug-resistant pathogens, for which new antibiotics are urgently needed. Whilst antibiotic drug development is inherently challenging, this is particularly true for Gram-negative bacteria due to the presence of the outer membrane, a highly selective permeability barrier that prevents the ingress of several classes of antibiotic. This selectivity is largely due to an outer leaflet composed of the glycolipid lipopolysaccharide (LPS), which is essential for the viability of almost all Gram-negative bacteria. This essentiality, coupled with the conservation of the synthetic pathway across species and recent breakthroughs in our understanding of transport and membrane homeostasis has made LPS an attractive target for novel antibiotic drug development. Several different targets have been explored and small molecules developed that show promising activity in vitro. However, these endeavours have met limited success in clinical testing and the polymyxins, discovered more than 70 years ago, remain the only LPS-targeting drugs to enter the clinic thus far. In this review, we will discuss efforts to develop therapeutic inhibitors of LPS synthesis and transport and the reasons for limited success, and explore new developments in understanding polymyxin mode of action and the identification of new analogues with reduced toxicity and enhanced activity.     

Structure of the Bacterial Deacetylase LpxC Bound to the Nucleotide Reaction Product Reveals Mechanisms of Oxyanion Stabilization and Proton Transfer

The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.     

The effectiveness of antibiotics on bacteria in biofilms

Bacteria can form different types of communities, united by common notion: biofilms. The aim of the present study was to determine the capacity of different antibiotics to penetrate into biofilms and act on unrelated bacteria. The study revealed that the formation of barriers between the community and the environment on artificial biofilms occurred in all strains of unrelated Gram-positive and Gram-negative bacteria used in this investigation. The capacity of antibiotics to penetrate into biofilms varied in different strains of the same species. For certain antibiotics similarity in their penetrating capacity was found to exist with respect to biofilms of unrelated bacteria. The penetration of antibiotics into mixed biofilms depended on the strain which determined its minimal value, so that the protection of one microorganism by another was thus observed. The method for the evaluation of the effectiveness of antibiotic penetration into bacterial biofilms, suitable for use in bacteriological laboratories, is proposed.     

Designing 2-aminoimidazole alkaloids analogs with anti-biofilm activities: structure-activities relationships of polysubstituted triazoles

In order to discover novel probes that may help in the investigation and the control of bacterial biofilms, we have designed a library of triazole-based analogs of 2-aminoimidazole marine alkaloids: naamine A and isonaamine A. Twenty-two compounds were screened for their biofilm inhibitory activity against two strains of Gram-negative bacteria. Four compounds were shown to act as non-toxic inhibitors of biofilm development without effect on bacterial growth even at high concentrations (100 μM).     

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Design and synthesis of potent Gram-negative specific LpxC inhibitors

Antibiotic resistant hospital acquired infections are on the rise, creating an urgent need for novel bactericidal drugs. Enzymes involved in lipopolysaccharide (LPS) biosynthesis are attractive antibacterial targets since LPS is the major structural component of the outer membrane of Gram-negative bacteria. Lipid A is an essential hydrophobic anchor of LPS and the first committed step in lipid A biosynthesis is catalyzed by a unique zinc dependent metalloamidase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). LpxC is an attractive Gram-negative only target that has been chemically validated by potent bactericidal hydroxamate inhibitors that work by coordination of the enzyme’s catalytic zinc ion. An exploratory chemistry effort focused on expanding the SAR around hydroxamic acid zinc-binding ‘warheads’ lead to the identification of novel compounds with enzyme potency and antibacterial activity similar to CHIR-090.     

Linking dual mode of action of host defense antimicrobial peptide thanatin: Structures,lipopolysaccharide and LptAm binding of designed analogs

At present, antibiotics options to cure infections caused by drug resistant Gram-negative pathogens are highly inadequate. LPS outer membrane, proteins involved in LPS transport and biosynthesis pathways are vital targets. Thanatin, an insect derived 21-residue long antimicrobial peptide may be exploited for the development of effective antibiotics against Gram-negative bacteria. As a mode of bacterial cell killing, thanatin disrupts LPS outer membrane and inhibits LPS transport by binding to the periplasmic protein LptA_m. Here, we report structure-activity correlation of thanatin and analogs for the purpose of rational design. These analogs of thanatin are investigated, by NMR, ITC and fluorescence, to correlate structure, antibacterial activity and binding with LPS and LptA_m, a truncated monomeric variant. Our results demonstrate that an analog thanatin M21F exhibits superior antibacterial activity. In LPS interaction analyses, thanatin M21F demonstrate high affinity binding to outer membrane LPS. The atomic resolution structure of thanatin M21F in LPS micelle reveals four stranded β-sheet structure in a dimeric topology whereby the sidechain of aromatic residues Y10, F21 sustained mutual packing at the interface. Strikingly, LptA_m binding affinity of thanatin M21F has been significantly increased with an estimated K_d ~ 0.73 nM vs 13 nM for thanatin. Further, atomic resolution structures and interactions of Ala based thanatin analogs define plausible correlations with antibacterial activity and LPS, LptA_m interactions. Taken together, the current work provides a frame-work for the designing of thanatin based potent antimicrobial peptides for the treatment of drug resistance Gram-negative bacteria.     

Mutants Resistant to LpxC Inhibitors by Rebalancing Cellular Homeostasis

LpxC, the deacetylase that catalyzes the second and committed step of lipid A biosynthesis in Escherichia coli, is an essential enzyme in virtually all Gram-negative bacteria and is one of the most promising antibiotic targets for treatment of multidrug-resistant Gram-negative infections. Despite the rapid development of LpxC-targeting antibiotics, the potential mechanisms of bacterial resistance to LpxC inhibitors remain poorly understood. Here, we report the isolation and biochemical characterization of spontaneously arising E. coli mutants that are over 200-fold more resistant to LpxC inhibitors than the wild-type strain. These mutants have two chromosomal point mutations that account for resistance additively and independently; one is in fabZ, a dehydratase in fatty acid biosynthesis; the other is in thrS, the Thr-tRNA ligase. For both enzymes, the isolated mutations result in reduced enzymatic activities in vitro. Unexpectedly, we observed a decreased level of LpxC in bacterial cells harboring fabZ mutations in the absence of LpxC inhibitors, suggesting that the biosyntheses of fatty acids and lipid A are tightly regulated to maintain a balance between phospholipids and lipid A. Additionally, we show that the mutation in thrS slows protein production and cellular growth, indicating that reduced protein biosynthesis can confer a suppressive effect on inhibition of membrane biosynthesis. Altogether, our studies reveal a previously unrecognized mechanism of antibiotic resistance by rebalancing cellular homeostasis.     

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.     

Crystal structure of LpxC from Pseudomonas aeruginosa complexed with the potent BB-78485 inhibitor

The cell wall in Gram-negative bacteria is surrounded by an outer membrane comprised of charged lipopolysaccharide (LPS) molecules that prevent entry of hydrophobic agents into the cell and protect the bacterium from many antibiotics. The hydrophobic anchor of LPS is lipid A, the biosynthesis of which is essential for bacterial growth and viability. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is an essential zinc-dependant enzyme that catalyzes the conversion of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)glucosamine and acetate in the biosynthesis of lipid A, and for this reason, LpxC is an attractive target for antibacterial drug discovery. Here we disclose a 1.9 A resolution crystal structure of LpxC from Pseudomonas aeruginosa (paLpxC) in a complex with the potent BB-78485 inhibitor. To our knowledge, this is the first crystal structure of LpxC with a small-molecule inhibitor that shows antibacterial activity against a wide range of Gram-negative pathogens. Accordingly, this structure can provide important information for lead optimization and rational design of the effective small-molecule LpxC inhibitors for successful treatment of Gram-negative infections.     

Targeting LPS biosynthesis and transport in gram-negative bacteria in the era of multi-drug resistance

Gram-negative bacteria pose a major threat to human health in an era fraught with multi-drug resistant bacterial infections. Despite extensive drug discovery campaigns over the past decades, no new antibiotic target class effective against gram-negative bacteria has become available to patients since the advent of the carbapenems in 1985. Antibiotic discovery efforts against gram-negative bacteria have been hampered by limited intracellular accumulation of xenobiotics, in large part due to the impermeable cell envelope comprising lipopolysaccharide (LPS) in the outer leaflet of the outer membrane, as well as a panoply of efflux pumps. The biosynthesis and transport of LPS are essential to the viability and virulence of most gram-negative bacteria. Thus, both LPS biosynthesis and transport are attractive pathways to target therapeutically. In this review, we summarize the LPS biosynthesis and transport pathways and discuss efforts to find small molecule inhibitors against targets within these pathways.     

Design,structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria

Background

Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs.

Methods

Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR.

Results

TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix.

Conclusion

Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides.

General significance

The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.     

Heterocyclic methylsulfone hydroxamic acid LpxC inhibitors as Gram-negative antibacterial agents

The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.