Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.     

Tied down: tethering redox proteins to the outer membrane in Neisseria and other genera

Typically, the redox proteins of respiratory chains in Gram-negative bacteria are localized in the cytoplasmic membrane or in the periplasm. An alternative arrangement appears to be widespread within the betaproteobacterial genus Neisseria, wherein several redox proteins are covalently associated with the outer membrane. In the present paper, we discuss the structural properties of these outer membrane redox proteins and the functional consequences of this attachment. Several tethered outer membrane redox proteins of Neisseria contain a weakly conserved repeated structure between the covalent tether and the redox protein globular domain that should enable the redox cofactor-containing domain to extend from the outer membrane, across the periplasm and towards the inner membrane. It is argued that the constraints imposed on the movement and orientation of the globular domains by these tethers favours the formation of electron-transfer complexes for entropic reasons. The attachment to the outer membrane may also affect the exposure of the host to redox proteins with a moonlighting function in the host-microbe interaction, thus affecting the host response to Neisseria infection. We identify putative outer membrane redox proteins from a number of other bacterial genera outside Neisseria, and suggest that this organizational arrangement may be more common than previously recognized.     

PapA,a peptidoglycan-associated protein,interacts with OmpC and maintains cell envelope integrity

The bacterial cell envelope is critical to support and maintain cellular life. In Gram-negative bacterial cells, the outer membrane and the peptidoglycan layer are two important parts of the cell envelope and they harbour abundant proteins. Here, we report the identification and characterization of a previously unknown p eptidoglycan-a ssociated p rotein, PapA, from the Gram-negative Comamonas testosteroni. PapA bound peptidoglycan with its C-terminal domain and interacted with the outer-membrane porin OmpC. The PapA-OmpC complex riveted the outer membrane and the peptidoglycan layer, and played a role in maintaining cell envelope integrity. When papA was disrupted, the mutant CNB-1ΔpapA apparently had an outer membrane partly separated from the peptidoglycan layer. Phenotypically, the mutant CNB-1ΔpapA lost chemotactic responses and had longer lag-phase of growth, less flagellation and higher sensitivity to harsh environments. Totally, 1093 functionally unknown PapA homologues were identified from the public NR protein database and they were mainly distributed in Burkholderiales of Betaproteobacteria. Our finding provides a clue that the PapA homologous proteins might function as a rivet to maintain cell envelope integrity in those Gram-negative bacteria.     

Backbone resonance assignment for the outer membrane lipoprotein receptor LolB from Escherichia coli

LolB, which is anchored to the outer membrane of Gram-negative bacteria, receives outer-membrane-directed lipoproteins from LolA, and incorporates them into the outer membrane. We established backbone resonance assignments of ²H/¹³C/¹⁵N labeled LolB from Escherichia coli.     

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

Summary The use of Cytophaga lysing enzymes was investigated for the liberation of poly--hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.     

Biogenesis of the Gram-negative bacterial outer membrane

Gram-negative bacteria are bounded by two membranes. The outer membrane consists of phospholipids, lipopolysaccharides, lipoproteins and integral outer membrane proteins, all of which are synthesized in the cytoplasm. Recently, much progress has been made in the elucidation of the mechanisms of transport of these molecules over the inner membrane, through the periplasm and into the outer membrane, in part by exploiting the extraordinary capacity of Neisseria meningitidis to survive without lipopolysaccharide.     

Chemical structure and function of glycosphingolipids of Sphingomonas spp and their distribution among members of the α-4 subclass of Proteobacteria

Sphingomonas spp are phylogenetically placed in the α-4 subclass of Proteobacteria. They have glycosphingolipids (GSL) in their membranes instead of lipopolysaccharide (LPS) as in other Gram-negative bacteria. S. paucimobilis, the type species of the genus, has GSL-1, which contains only glucuronic acid (GlcA) as a sugar moiety, and GSL-4A, which contains a tetrasaccharide including GlcA. GSL-1 and GSL-4A form the outer membrane of S. paucimobilis with outer membrane proteins and phospholipids. In the outer membrane, GSLs are assumed to locate and function as does the LPS of other Gram-negative bacteria. Sphingomonas spp closely related to the type species contain both GSL-1 and the oligosaccharide-type GSL such as GSL-4A, but other Sphingomonas spp and other genera in the α-4 subclass of Proteobacteria contain only GSL-1. Structural variations of fatty acids and dihydrosphingosines in the GSL-1 are presented. Received 19 April 1999/ Accepted in revised form 18 June 1999     

Use of Iodo-Gen and Iodine-125 to label the outer membrane proteins of whole cells of Neisseria gonorrhoeae

The outer membrane proteins of Neisseria gonorrhoeae are specifically labeled by use of 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodo-Gen) and ¹²⁵I under the conditions described in this report. Use of this procedure with whole cells of N. gonorrhoeae produces a clear labeling pattern which can be visualized by electrophoretic separation of the proteins, followed by autoradiography. Electrophoretograms reveal some 70 polypeptide bands, while autoradiograms reveal only 5 or 6 labeled bands. The labeled polypeptide bands correspond to isolated outer membrane proteins, the most intensely labeled of which is the principal outer membrane protein. The method described in this report is both specific and gentle, as well as rapid and convenient.     

Origin of diderm (Gram-negative) bacteria: antibiotic selection pressure rather than endosymbiosis likely led to the evolution of bacterial cells with two membranes

The prokaryotic organisms can be divided into two main groups depending upon whether their cell envelopes contain one membrane (monoderms) or two membranes (diderms). It is important to understand how these and other variations that are observed in the cell envelopes of prokaryotic organisms have originated. In 2009, James Lake proposed that cells with two membranes (primarily Gram-negative bacteria) originated from an ancient endosymbiotic event involving an Actinobacteria and a Clostridia (Lake 2009). However, this Perspective argues that this proposal is based on a number of incorrect assumptions and the data presented in support of this model are also of questionable nature. Thus, there is no reliable evidence to support the endosymbiotic origin of double membrane bacteria. In contrast, many observations suggest that antibiotic selection pressure was an important selective force in prokaryotic evolution and that it likely played a central role in the evolution of diderm (Gram-negative) bacteria. Some bacterial phyla, such as Deinococcus-Thermus, which lack lipopolysaccharide (LPS) and yet contain some characteristics of the diderm bacteria, are postulated as evolutionary intermediates (simple diderms) in the transition between the monoderm bacterial taxa and the bacterial groups that have the archetypal LPS-containing outer cell membrane found in Gram-negative bacteria. It is possible to distinguish the two stages in the evolution of diderm-LPS cells (viz. monoderm bacteria → simple diderms lacking LPS → LPS containing archetypal diderm bacteria) by means of conserved inserts in the Hsp70 and Hsp60 proteins. The insert in the Hsp60 protein also distinguishes the traditional Gram-negative diderm bacterial phyla from atypical taxa of diderm bacteria (viz. Negativicutes, Fusobacteria, Synergistetes and Elusimicrobia). The Gram-negative bacterial phyla with an LPS-diderm cell envelope, as defined by the presence of the Hsp60 insert, are indicated to form a monophyletic clade and no loss of the outer membrane from any species from this group seems to have occurred. This argues against the origin of monoderm prokaryotes from diderm bacteria by loss of outer membrane.     

Sulfonolipids are localized in the outer membrane of the gliding bacterium Cytophaga johnsonae

Earlier work in our laboratory demonstrated that gliding bacteria of the Cytophaga-Flexibacter group contain, in their cell envelopes, large quantities of unusual sulfonolipids (N-fatty acyl 2-amino-3-hydroxyisoheptadecane-1-sulfonic acids). Recently, it has been shown that these lipids are necessary for the gliding motility of C. johnsonae. As one approach to determining the role of the lipids in motility, methods have now been developed for separating the inner (cytoplasmic) and outer membranes of a strain (ATCC 43786) of this Gram-negative bacterium. Sulfonolipid is at least five times as abundant in the outer membrane as in the inner. The inner membrane has properties similar to those found for other Gram-negative bacteria; it has a buoyant density of 1.14 g/ml and is highly enriched in cytochromes and succinate dehydrogenase. The outer membrane (1.18 g/ml) is enriched in bound carbohydrate and sulfonolipid, but contains little or no 2-keto-3-deoxyoctonate (such as is found in the enterobacteria). The localization of the sulfonolipids in the outer membrane permits focus on the possible roles these unusual substances may play in gliding motility.Abbreviations used IM inner membrane - OM outer membrane - KDO 2-keto-3-deoxyoctonate - EDTA ethylenediaminetetraacetic acid - SDH succinate dehydrogenase     

A sequence-specific function for the N-terminal signal-like sequence of the TonB protein

TonB is a proline-rich protein which provides a functional link between the inner and outer membranes of Gram-negative bacteria. TonB is anchored to the inner membrane via an N-terminal signal-like sequence and spans the periplasm, interacting with transport receptors in the outer membrane. We have investigated the role of the N-terminal signal-like peptide in TonB function. Replacement of the N-terminal sequence with heterologous sequences indicates that it has at least three distinct rotes in TonB function: (i) to facilitate translocation of TonB across the cytoplasmic membrane; (ii) to anchor TonB to the cytoplasmic membrane; (iii) a sequence-specific functional interaction with the ExbBD proteins.     

Expanding the paradigm for the outer membrane: Acinetobacter baumannii in the absence of endotoxin

Asymmetry in the outer membrane has long defined the cell envelope of Gram‐negative bacteria. This asymmetry, with lipopolysaccharide (LPS) or lipooligosaccharide (LOS) exclusively in the outer leaflet of the membrane, establishes an impermeable barrier that protects the cell from a number of stressors in the environment. Work done over the past 5 years has shown that Acinetobacter baumannii has the remarkable capability to survive with inactivated production of lipid A biosynthesis and the absence of LOS in its outer membrane. The implications of LOS‐deficient A. baumannii are far‐reaching – from impacts on cell envelope biogenesis and maintenance, bacterial physiology, antibiotic resistance and virulence. This review examines recent work that has contributed to our understanding of LOS‐deficiency and compares it to studies done on Neisseria meningitidis and Moraxella catarrhalis; the two other organisms with this capability.     

Isolation of the periplasm of Neisseria gonorrhoeae

The periplasm of Neisseria gonorrhoeae should be similar to other Gram-negative bacteria, but no published reports confirm this assumption. We used a periplasmic isolation procedure developed in Escherichia coli to release the periplasmic contents of N. gonorrhoeae. The resultant periplasmic extract lacked lipopolysaccharide, protein markers of inner or outer membranes, surface-radiolabelled protein components, or ribosomal proteins. The periplasmic extract contained a single haem protein believed to be a c-type cytochrome known to exist in the periplasm of other Gram-negative species, and retained significant alkaline phosphatase activity. The dominant protein species released in the periplasmic extract was the gonococcal homologue of elongation factor Tu, a major component released in similar periplasmic extracts of E. coli. These data showed that the extraction procedure selectively released periplasmic components and that the gonococcal periplasm was comparable to that of E. coli. Further analysis of the gonococcal periplasm may provide important insights into the physiology of this pathogen of humans.     

Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli

Abstract

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs.     

The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide

Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, and chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells.     

An Outer Membrane Receptor of Neisseria meningitidis Involved in Zinc Acquisition with Vaccine Potential

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.     

Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. campestris

Background  

Outer membrane vesicles (OMVs) are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli.     

Neisseria proteomics for antigen discovery and vaccine development

Neisseria meningitidis (meningococcus) is a major causative organism of meningitis and sepsis and Neisseria gonorrhoeae (gonococcus) is the causative organism of the sexually transmitted disease gonorrhea. Infections caused by meningococci are vaccine-preventable, whereas gonococcal vaccine research and development has languished for decades and the correlates of protection are still largely unknown. In the past two decades, complementary ‘omic’ platforms have been developed to interrogate Neisseria genomes and gene products. Proteomic techniques applied to whole Neisseria bacteria, outer membranes and outer membrane vesicle vaccines have generated protein maps and also allowed the examination of environmental stresses on protein expression. In particular, immuno-proteomics has identified proteins whose expression is correlated with the development of human natural immunity to meningococcal infection and colonization and following vaccination. Neisseria proteomic techniques have produced a catalog of potential vaccine antigens and investigating the functional and biological properties of these proteins could finally provide ‘universal’ Neisseria vaccines.     

Induction of Antimeningitis Immunity by Synthetic Peptides: III. Immunoactive Synthetic Fragments of the NspA Protein from Neisseria meningitidis

Four potentially immunoactive peptide fragments of the NspA protein from the outer membrane of the Neisseria meningitidis bacterium were synthesized in order to create a synthetic vaccine against meningococcal infection by the serogroup B bacterium. Mice of various lines were immunized with free peptides nonconjugated with a protein carrier. All the synthetic peptides were shown to induce the production of the antipeptide antibodies in mice. A peptide capable of inducing a decrease in the number of bacteria in blood and the protection of infected animals from death was found in the experiments on the protection of the animals infected with two strains of the Neisseria meningitidis serogroup B.     

1H, 13C and 15N assignment of the GNA1946 outer membrane lipoprotein from Neisseria meningitidis

GNA1946 (Genome-derived Neisseria Antigen 1946) is a highly conserved exposed outer membrane lipoprotein from Neisseria meningitidis bacteria of 287 amino acid length (31 kDa). Although the structure of NMB1946 has been solved recently by X-Ray crystallography, understanding the behaviour of GNA1946 in aqueuos solution is highly relevant for the discovery of the antigenic determinants of the protein that will possibly lead to a more efficient vaccine development against virulent serogroup B strain of N.meningitidis. Here we report almost complete ¹H, ¹³C and ¹⁵N resonance assignments of GNA1946 (residues 10–287) in aqueous buffer solution.