Cell Envelope Of Gram-negative Bacteria Research Articles

New aminocoumarin antibiotics as gyrase inhibitors

The aminocoumarins novobiocin, clorobiocin and coumermycin A1 are structurally related antibiotics produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase. Their binding sites and their mode of action differ from those of fluoroquinolones such as ciprofloxacin. Novobiocin has been introduced into clinical use against Staphylococcus aureus infections, and S. aureus gyrase is particularly sensitive to inhibition by aminocoumarins, while topoisomerase IV is much less sensitive. Modern genetic techniques have allowed the engineering of the producer strains, resulting in a diverse range of new aminocoumarins, including compounds which are more active than the natural antibiotics as well as a compound which is actively imported across the cell envelope of Gram-negative bacteria. A further group of aminocoumarins are the simocyclinones which bind simultaneously to two different sites of gyrase and show a completely new mode of inhibition. Both the simocyclinones and the “classical” aminocoumarins strongly inhibit the fluoroquinolone-induced activation of RecA and thereby the SOS response in S. aureus. Therefore, a combination of aminocoumarins and fluoroquinolones strongly reduced the risk of resistance development and may offer new prospects in anti-infective therapy.

Crystal structure of the integral membrane diacylglycerol kinase

Diacylglycerol kinase (DgkA) catalyzes the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria1. For half a century, this 121-residue kinase has served as a paradigm for investigating membrane protein enzymology1,3-7, folding8,9, assembly10-13, and stability1,14. Here, we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type (WT). These reveal a homo-trimeric enzyme with three transmembrane helices and an N-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site which is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are however at variance with a published solution NMR model2 in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.

Ultrastructural Analysis of the Rugose Cell Envelope of a Member of the Pasteurellaceae Family

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wild-type and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

Kinetic Basis for the Competitive Recruitment of TolB by the Intrinsically Disordered Translocation Domain of Colicin E9

TolB and Pal are members of the Tol–Pal system that spans the cell envelope of Gram-negative bacteria and contributes to the stability and integrity of the bacterial outer membrane (OM). Lipoylated Pal is tethered to the OM and binds the β-propeller domain of periplasmic TolB, which, as recent evidence suggests, disengages TolB from its interaction with other components of the Tol system in the inner membrane. Antibacterial nuclease colicins such as colicin E9 (ColE9) also bind the β-propeller domain of TolB in order to catalyze their translocation across the bacterial OM. In contrast to Pal, however, colicin binding to TolB promotes its interaction with other components of the Tol system. Here, through a series of pre-steady-state kinetic experiments utilizing fluorescence resonance energy transfer pairs within the individual protein–protein complexes, we establish the kinetic basis for such ‘competitive recruitment’ by the TolB-binding epitope (TBE) of ColE9. Surprisingly, the 16-residue disordered ColE9 TBE associates more rapidly with TolB than Pal, a folded 13-kDa protein. Moreover, we demonstrate that calcium ions, which bind within the confines of the TolB β-propeller domain tunnel and are known to increase the affinity of the TolB–ColE9 complex, do not exert their influence through long-range electrostatic effects, as had been predicted, but through short-range effects that slow the dissociation rate of ColE9 TBE from its complex with TolB. Our study demonstrates that an intrinsically disordered protein undergoing binding-induced folding can compete effectively with a globular protein for a common target by associating more rapidly than the globular protein.

Chemical Biology Approaches Reveal Conserved Features of a C‐Terminal Processing PDZ Protease

Several proteases like the high temperature requirement A (HtrA) protein family containing internal or C-terminal PDZ domains play key roles in protein quality control in the cell envelope of Gram-negative bacteria. While several HtrA proteases have been extensively characterized, many features of C-terminal processing proteases such as tail-specific protease (Tsp) are still unknown. To fully understand these cellular control systems, individual domains need to be targeted by specific peptides acting as activators or inhibitors. Here, we describe the identification and design of potent inhibitors and activators of Tsp. Suitable synthetic substrates of Tsp were identified and served as a basis for the generation of boronic acid-based peptide inhibitors. In addition, a proteomic screen of E. coli cell envelope proteins using a synthetic peptide library was performed to identify peptides capable of amplifying Tsp's proteolytic activity. The implications of these findings for the regulation of PDZ proteases and for future mechanistic studies are discussed.

Bacterial ghosts as carriers of protein subunit and DNA-encoded antigens for vaccine applications

Bacterial ghosts (BGs) represent vaccine delivery systems gifted with outstanding natural adjuvant properties. BGs are empty cell envelopes of Gram-negative bacteria lacking cytoplasmic content yet retaining all unaltered morphological and structural features of their living counterparts. The intact surface make-up of BGs is easily recognized by professional APCs through pattern-recognition receptors, making them ideal for mucosal administration through oral, ocular, intranasal or aerogenic routes, which represent the most desirable methods of application in advanced vaccine use. BGs have been designed to be used as carriers of active substances and foreign antigens (protein and/or DNA) for vaccine development. This review highlights the salient features of the BGs’ versatile multipurpose vaccine platform for application in a wide range of human and veterinary medicines.

Protecting Bacterial Cells from Rupture: A Multiscale Simulation Study of Braun's Lipoprotein

The cell envelope of Gram-negative bacteria is composed of two membranes. The inner membrane has a relatively simple phospholipid composition, whilst the asymmetric outer membrane is a complex mixture of lipopolysaccharide (LPS) and phospholipids. The region between the two membranes, know as the periplasm, is largely composed of peptidoglycan that maintains the cell shape and protects its from rupture. Braun's lipoprotein (BLP), a coiled-coiled trimeric protein located within the periplasm, has long been known to covalently bind to the peptidoglycan and to anchor the peptidoglycan to the inner leaflet of the outer membrane. Recently, however, it has been shown that in addition to this structural role, BLP is able to adopt a trans-membrane orientation across the outer membrane.In this present study we have performed a series of molecular dynamics simulations of BLP using both atomistic and coarse-grained approaches. An atomistic model of the peptidoglycan has been developed and incorporated with our complex model of the E. coli outer membrane that contains LPS in the outer leaflet and mixtures of phospholipids in the inner leaflet. Multiple simulations of BLP, including the peptidoglycan bound and free forms, plus BLP in a trans-membrane orientation have been performed. These simulations have enabled us to explore the conformational dynamics, oligomerisation and environmental interactions of BLP in these different orientations.

Bacterial Ghosts as antigen and drug delivery system for ocular surface diseases: Effective internalization of Bacterial Ghosts by human conjunctival epithelial cells

The purpose of the presented investigation was to examine the efficiency of the novel carrier system Bacterial Ghosts (BGs), which are empty bacterial cell envelopes of Gram-negative bacteria to target human conjunctival epithelial cells, as well as to test the endocytic capacity of conjunctival cells after co-incubation with BGs generated from different bacterial species, and to foreclose potential cytotoxic effects caused by BGs. The efficiency of conjunctival cells to internalize BGs was investigated using the Chang conjunctival epithelial cell line and primary human conjunctiva-derived epithelial cells (HCDECs) as in vitro model. A high capacity of HCDECs to functionally internalize BGs was detected with the level of internalization depending on the type of species used for BGs generation. Detailed analysis showed no cytotoxic effect of BGs on HCDECs independently of the used bacterial species. Moreover, co-incubation with BGs did not enhance expression of both MHC class I and class II molecules by HCDECs, but increased expression of ICAM-1. The high rates of BG's internalization by HCDECs with no BG-mediated cytotoxic impact designate this carrier system to be a promising candidate for an ocular surface drug delivery system. BGs could be useful for future therapeutic ocular surface applications and eye-specific disease vaccine development including DNA transfer.

Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa

The Type VI secretion pathway transports proteins across the cell envelope of Gram-negative bacteria. Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen infecting humans, uses the type VI secretion pathway to export specific effector proteins crucial for its pathogenesis. The HSI-I virulence locus encodes for several proteins that has been proposed to participate in protein transport including the Hcp1 protein, which forms hexameric rings that assemble into nanotubes in vitro. Two Hcp1 paralogues have been identified in the P. aeruginosa genome, Hsp2 and Hcp3. Here, we present the structure of the Hcp3 protein from P. aeruginosa. The overall structure of the monomer resembles Hcp1 despite the lack of amino-acid sequence similarity between the two proteins. The monomers assemble into hexamers similar to Hcp1. However, instead of forming nanotubes in head-to-tail mode like Hcp1, Hcp3 stacks its rings in head-to-head mode forming double-ring structures.

In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae

The type II secretion system is a multi-protein complex that spans the cell envelope of Gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.

Assembly of outer-membrane proteins in bacteria and mitochondria

The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobic alpha-helices, integral outer-membrane proteins (OMPs) form beta-barrels. Similar beta-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How these beta-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly of beta-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrial beta-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.

Insights into the Function of the α‐Helical Tail of Haemophilus influenzae 3‐Deoxy‐D‐manno‐Octulosonate 8‐Phosphate Phosphatase

The cell envelope of Gram‐negative bacteria contains, in addition to the inner membrane and the peptidoglycan layer, a bilayered and asymmetrically organized outer membrane. The most predominant component of the outer leaflet of the outer membrane is lipopolysaccharide (LPS). LPS is an amphiphilic molecule composed of a hydrophilic heteropolysaccharide and the membrane‐embedded lipid A. The innermost 8‐carbon sugar 3‐deoxy‐D‐manno‐oct‐2‐ulosonic acid (KDO) is the only conserved structural element found in all LPS investigated to date, linking the lipid A to the carbohydrate domain of the molecule. Inhibition of KDO biosynthesis is lethal to most bacteria, making this pathway an ideal target for the development of novel antimicrobials. The KDO 8‐phosphate phosphatase (KdsC) is one of the enzymes of the KDO biosynthesis pathway and is responsible for cleavage of the phosphate group from KDO 8‐phosphate to yield KDO. In Escherichia coli, KdsC has been characterized as a homotetrameric molecule with a random‐coiled C‐terminal tail in each subunit. This tail plays a potential role in the enzyme's catalytic cycle. KdsC of Haemophilus influenzae is also a tetramer containing an α‐helical tail. In order to investigate the role of the tails in the catalytic mechanism of H. influenzae KdsC, a truncated (tail‐less) H. influenzae KdsC derivative was constructed by removal of its last C‐terminal eighteen amino acids. Both the native and the truncated H. influenzae KdsC were cloned, overexpressed, purified to apparent homogeneity, and functionally characterized.

Contribution of proteomics toward solving the fascinating mysteries of the biogenesis of the envelope of Escherichia coli

The cell envelope of Gram-negative bacteria is a complex macromolecular structure that is essential for their viability. Little is known on how the proteins which are secreted to the envelope fold into their unique three-dimensional structure. Several folding factors, including chaperones and protein folding catalysts involved in disulfide bond formation, have been identified in the periplasm. The characterization of these proteins has advanced our understanding of envelope biogenesis, although many fundamental questions remain unanswered. In particular, we still do not know how beta-barrel proteins are transported through the periplasm and inserted into the outer membrane. Here, we discuss the recent discoveries that have shed new light on the mechanisms that ensure the correct folding of envelope proteins. We have paid particular attention to the significant contribution of proteomic studies.

Novel Structure of the Conserved Gram-Negative Lipopolysaccharide Transport Protein A and Mutagenesis Analysis

Lipopolysaccharide (LPS) transport protein A (LptA) is an essential periplasmic localized transport protein that has been implicated together with MsbA, LptB, and the Imp/RlpB complex in LPS transport from the inner membrane to the outer membrane, thereby contributing to building the cell envelope in Gram-negative bacteria and maintaining its integrity. Here we present the first crystal structures of processed Escherichia coli LptA in two crystal forms, one with two molecules in the asymmetric unit and the other with eight. In both crystal forms, severe anisotropic diffraction was corrected, which facilitated model building and structural refinement. The eight-molecule form of LptA is induced when LPS or Ra-LPS (a rough chemotype of LPS) is included during crystallization. The unique LptA structure represents a novel fold, consisting of 16 consecutive antiparallel β-strands, folded to resemble a slightly twisted β-jellyroll. Each LptA molecule interacts with an adjacent LptA molecule in a head-to-tail fashion to resemble long fibers. Site-directed mutagenesis of conserved residues located within a cluster that delineate the N-terminal β-strands of LptA does not impair the function of the protein, although their overexpression appears more detrimental to LPS transport compared with wild-type LptA. Moreover, altered expression of both wild-type and mutated proteins interfered with normal LPS transport as witnessed by the production of an anomalous form of LPS. Structural analysis suggests that head-to-tail stacking of LptA molecules could be destabilized by the mutation, thereby potentially contributing to impair LPS transport.

Surface Viscoelasticity of Individual Gram-Negative Bacterial Cells Measured Using Atomic Force Microscopy

The cell envelope of gram-negative bacteria is responsible for many important biological functions: it plays a structural role, it accommodates the selective transfer of material across the cell wall, it undergoes changes made necessary by growth and division, and it transfers information about the environment into the cell. Thus, an accurate quantification of cell mechanical properties is required not only to understand physiological processes but also to help elucidate the relationship between cell surface structure and function. We have used a novel, atomic force microscopy (AFM)-based approach to probe the mechanical properties of single bacterial cells by applying a constant compressive force to the cell under fluid conditions while measuring the time-dependent displacement (creep) of the AFM tip due to the viscoelastic properties of the cell. For these experiments, we chose a representative gram-negative bacterium, Pseudomonas aeruginosa PAO1, and we used regular V-shaped AFM cantilevers with pyramid-shaped and colloidal tips. We find that the cell response is well described by a three-element mechanical model which describes an effective cell spring constant, k(1), and an effective time constant, tau, for the creep deformation. Adding glutaraldehyde, an agent that increases the covalent bonding of the cell surface, produced a significant increase in k(1) together with a significant decrease in tau. This work represents a new attempt toward the understanding of the nanomechanical properties of single bacteria while they are under fluid conditions, which could be of practical value for elucidating, for instance, the biomechanical effects of drugs (such as antibiotics) on pathogens.

The Extracytoplasmic Stress Factor, σE, Is Required to Maintain Cell Envelope Integrity in Escherichia coli

Extracytoplasmic function or ECF sigma factors are the most abundant class of alternative sigma factors in bacteria. Members of the rpoE subclass of ECF sigma factors are implicated in sensing stress in the cell envelope of Gram-negative bacteria and are required for virulence in many pathogens. The best-studied member of this family is rpoE from Escherichia coli, encoding the σE protein. σE has been well studied for its role in combating extracytoplasmic stress, and the members of its regulon have been largely defined. σE is required for viability of E. coli, yet none of the studies to date explain why σE is essential in seemingly unstressed cells. In this work we investigate the essential role of σE in E. coli by analyzing the phenotypes associated with loss of σE activity and isolating suppressors that allow cells to live in the absence of σE. We demonstrate that when σE is inhibited, cell envelope stress increases and envelope integrity is lost. Many cells lyse and some develop blebs containing cytoplasmic material along their sides. To better understand the connection between transcription by σE and cell envelope integrity, we identified two multicopy suppressors of the essentiality of σE, ptsN and yhbW. yhbW is a gene of unknown function, while ptsN is a member of the σE regulon. Overexpression of ptsN lowers the basal level of multiple envelope stress responses, but not that of a cytoplasmic stress response. Our results are consistent with a model in which overexpression of ptsN reduces stress in the cell envelope, thereby promoting survival in the absence of σE.

Biogenesis of the Gram-Negative Bacterial Outer Membrane

The cell envelope of gram-negative bacteria consists of two membranes, the inner and the outer membrane, that are separated by the periplasm. The outer membrane consists of phospholipids, lipopolysaccharides, integral membrane proteins, and lipoproteins. These components are synthesized in the cytoplasm or at the inner leaflet of the inner membrane and have to be transported across the inner membrane and through the periplasm to assemble eventually in the correct membrane. Recent studies in Neisseria meningitidis and Escherichia coli have led to the identification of several machineries implicated in these transport and assembly processes.

Mechanisms of endotoxin neutralization by synthetic cationic compounds

A basic challenge in the treatment of septic patients in critical care units is the release of bacterial pathogenicity factors such as lipopolysaccharide (LPS, endotoxin) from the cell envelope of Gram-negative bacteria due to killing by antibiotics. LPS aggregates may interact with serum and membrane proteins such as LBP (lipopolysaccharide-binding protein) and CD14 leading to the observed strong reaction of the immune system. Thus, an effective treatment of patients infected by Gram-negative bacteria must comprise beside bacterial killing the neutralization of endotoxins. Here, data are summarized for synthetic compounds indicating the stepwise development to very effective LPS-neutralizing agents. These data include synthetic peptides, based on the endotoxin-binding domains of natural binding proteins such as lactoferrin, Limulus anti-LPS factor, NK-lysin, and cathelicidins or based on LPS sequestering polyamines. Many of these compounds could be shown to act not only in vitro, but also in vivo (e.g. in animal models of sepsis), and might be useful in future clinical trials and in sepsis therapy.

Invited review: Mechanisms of endotoxin neutralization by synthetic cationic compounds

A basic challenge in the treatment of septic patients in critical care units is the release of bacterial pathogenicity factors such as lipopolysaccharide (LPS, endotoxin) from the cell envelope of Gram-negative bacteria due to killing by antibiotics. LPS aggregates may interact with serum and membrane proteins such as LBP (lipopolysaccharide-binding protein) and CD14 leading to the observed strong reaction of the immune system. Thus, an effective treatment of patients infected by Gram-negative bacteria must comprise beside bacterial killing the neutralization of endotoxins. Here, data are summarized for synthetic compounds indicating the stepwise development to very effective LPS-neutralizing agents. These data include synthetic peptides, based on the endotoxin-binding domains of natural binding proteins such as lactoferrin, Limulus anti-LPS factor, NK-lysin, and cathelicidins or based on LPS sequestering polyamines. Many of these compounds could be shown to act not only in vitro, but also in vivo (e.g . in animal models of sepsis), and might be useful in future clinical trials and in sepsis therapy.

The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O‐antigen chain length determinant

The lipopolysaccharide (LPS) is the outermost component of the cell envelope in Gram-negative bacteria. It consists of the hydrophobic lipid A, a short non-repeating core oligosaccharide and a distal polysaccharide termed O-antigen. We report here that the PmrA/PmrB and RcsC/YojN/RcsB two-component systems of Salmonella enterica serovar Typhimurium independently promote transcription of the wzzst gene, which encodes a protein that determines the chain length of the O-antigen. We show that the regulatory proteins PmrA and RcsB footprint partially overlapping regions of the wzzst promoter stimulating transcription from the same start site. Induction of the PmrA/PmrB or RcsC/YojN/RcsB systems increased the fraction of LPS molecules containing 16-35 O-antigen subunits, leading to heightened resistance to serum. The LPS of a rcsB null mutant exhibited an altered mobility in the O-antigen subunits attached to the lipid A-core region when separated on a SDS/PAGE gel, suggesting that RcsB may regulate additional LPS genes. Inactivation of the wzzst gene eliminated the enhanced swarming behaviour exhibited by the rcsB mutant. That multiple regulatory systems control wzzst expression suggests that the Wzzst protein is required under different environmental conditions.