Enterobacterial Outer Membrane Research Articles

PLASMINOGEN ACTIVATOR OF YERSINIA PESTIS

Plague has been the cause of three pandemics and has led to the death of millions of people. Plague is a typical zoonosis caused by Yersinia pestis that circulates in populations of wild rodents inhabiting natural plague foci on all continents except for Australia. Transmission of plague is provided by flea bites. Circulation of Y. pestis in natural plague foci is supported by a numerous of pathogenicity factors. This review explores one of them, plasminogen activator Pla. This protein is one of representatives of omptins, a family of enterobacterial outer membrane proteases that are responsible for colonization of specific organs or even infection generalization as a result of successful overcoming of the host innate immunity. The review reflects the history of its discovery and studying of its genetic control, biosynthesis, isolation and purification, physicochemical properties. Highly purified preparations of plasminogen activator are deficient in enzymatic activities but renaturation in the presence of Y. pestis lipooligosaccharide restores enzymatic properties of Pla. This pathogenicity factor is absent in representatives of the most ancient phylogenetic group of the plague pathogen, bv. caucasica, while the ancestor of other groups of Y. pestis subsp. microtus obtained in result of horizontal transfer Pla isoform with characteristics similar to properties of omptins from the less virulent enterobacteria. After that in the course of microevolution the “classic” isoform of Pla with increased protease activity was selected that is typical of all highly virulent for humans strains of Y. pestis subsp. pestis. The “classic” isoform of Pla Y. pestis is functionally similar to mammalian plasminogen activators transforming plasminogen into plasmin with the help of limited proteolysis. Pla protease activating plasminogen and also degrading the main plasmin inhibitor — α2-antiplasmin and, respectively, determining Y. pestis ability to lyse fibrin clots preventing bacteria dissemination after bites of infected fleas or subcutaneous challenge is believed to be the main Y. pestis factor responsible for generalization of infectious process. Pla-mediated ability of Y. pestis for selective binding with extracellular matrix and basal membranes may promote further hydrolysis of these structures by the host’s plasmin and overcoming tissue barriers by the pathogen. Y. pestis plasminogen activator also hydrolyses C3 complement component, human antimicrobial peptide — cathelicidin LL-37 and such cytokines as tumor necrosis factor α, interferon γ, interleukin 8 and protein 1 of monocyte chemotaxis. The main endogenic TFPI tissue factor pathway inhibitor also highly susceptible to proteolytic action of Pla, and efficiency of TFPI inactivation is much higher than efficacy of plasminogen activation. The review also debates the possibility of using Pla as a molecular target for prophylaxis and treatment of plague.

An Early Response to Environmental Stress Involves Regulation of OmpX and OmpF, Two Enterobacterial Outer Membrane Pore-Forming Proteins

Bacterial adaptation to external stresses and toxic compounds is a key step in the emergence of multidrug-resistant strains that are a serious threat to human health. Although some of the proteins and regulators involved in antibiotic resistance mechanisms have been described, no information is available to date concerning the early bacterial response to external stresses. Here we report that the expression of ompX, encoding an outer membrane protein, is increased during early exposure to drugs or environmental stresses. At the same time, the level of ompF porin expression is noticeably affected. Because of the role of these proteins in membrane permeability, these data suggest that OmpF and OmpX are involved in the control of the penetration of antibiotics such as beta-lactams and fluoroquinolones through the enterobacterial outer membrane. Consequently, the early control of ompX and ompF induced by external stresses may represent a preliminary response to antibiotics, thus triggering the initial bacterial line of defense against antibiotherapy.

Permeabilising effects of sub-inhibitory concentrations of microcystin on the growth of Escherichia coli

Microcystin, a cyanotoxin produced by Microcystis aeruginosa, lacks potent antibacterial activity. When tested in combination, in vitro, inhibitory values for a range of hydrophobic antibiotics were significantly reduced in the presence of at least 1/20×the minimum inhibitory concentration of microcystin. The degree of inhibition was equivalent to that of a well-characterised permeabilising agent, polymyxin B nonapeptide. The permeabilising ability of sub-inhibitory concentrations of microcystin to affect the envelope of Escherichia coli was demonstrated by a rapid and sustained reduction in absorbance values of lysozyme-treated cells and by enhanced uptake of crystal violet in microcystin-treated cultures. Direct effects of appropriate concentrations of microcystin on the integrity of bacterial outer and inner membranes were measured by release of specific enzyme markers. Although the exact mechanism for permeabilising E. coli with microcystin has not been elucidated, the effects were consistent with permeability changes to the enterobacterial outer membrane caused by polymyxin B nonapeptide.

Ability of cecropin B to penetrate the enterobacterial outer membrane.

The cationic amphipathic insect peptide cecropin B was almost as active on wild-type enteric bacteria as it was on their lipopolysaccharide and lipid A mutants that have very defective outer membrane. The polymyxin-resistant strains, which elaborate altered, less anionic lipopolysaccharide, were completely susceptible to cecropin B. No synergism was found between cecropin B and hydrophobic antibiotics. Throughout the study, the activity of cecropin B resembled that of quaternary detergents.

Comparative analysis of antibodies to the lipopolysaccharides and to cross-reactive enterobacterial outer membrane proteins in patients with enteritis caused by Salmonellae or Yersinia enterocolitica

Sera from patients with recent or ongoing Salmonella ( n = 25) or Yersinia enterocolitica O3 enteritis ( n = 32), were tested by ELISA for antibodies cross-reacting with outer membrane (OM) proteins from an Escherichia coli O55 strain. In addition, the sera were tested by ELISA for serotype-specific antibodies against lipopolysaccharides (LPS ELISA) from Salmonellae or Y. enterocolitica O3. The results were matched against those obtained by testing healthy controls ( n = 30). The LPS ELISAs proved their efficacy in diagnosing enteritis caused by the Salmonellae or Yersinia with positive tests in 96% and 100% respectively. Significantly elevated means of the levels of cross-reacting anti-OM protein antibodies were shown for all of the immunoglobulin classes IgA, IgG, and IgM. These antibody rises were less marked than those of the anti-LPS antibody rises. Levels of the two groups of antibodies showed no correlation in individual patients. The cross-reacting antibodies were directed against OM proteins which varied from one individual to another and which included the porin protein, OmpA, the peptidoglycan-associated lipoprotein, and Braun's lipoprotein. The findings emphasize that cross-reacting antibodies may reduce specificity of serodiagnostic test for Salmonella or Yersinia-induced enteritis unless highly purified antigens are used.

Use of the Enterobacterial Outer Membrane Protein PhoE in the Development of New Vaccines and DNA Probes

PhoE protein is a major outer membrane protein of Escherichia coli. The polypeptide spans the membrane 16 times, thereby exposing 8 regions at the cell surface. Insertions in these regions did not affect the biogenesis of the protein. Therefore, we considered the possibility of using PhoE as a vector for the exposure of foreign antigenic determinants at the cell surface, with the ultimate goal of constructing new (live oral) vaccines. Via recombinant DNA techniques, B-cell epitopes of VP1 protein of foot-and-mouth-disease virus were inserted in the exposed regions of PhoE. The inserted epitopes were antigenic and immunogenic in the PhoE-associated conformation. Guinea pigs, immunized with such a hybrid protein were protected against viral challenge. Similarly, a T-cell epitope of the 65 kDa heat-shock protein of Mycobacterium tuberculosis remained antigenic and immunogenic in the PhoE-associated conformation, although recognition by the cells of the immune system was dependent on the amino acids, flanking the epitope. When the amino acid sequences of the PhoE proteins of different members of the family of Enterobacteriaceae are compared, the cell surface-exposed regions are hypervariable. Therefore, we considered the possibility that the DNA segments encoding these regions are species-specific. By using synthetic oligonucleotides corresponding to such DNA segments, primer couples for the specific detection and identification of different enterobacterial species, including Salmonella, by polymerase chain reactions have been developed.

The Ssc protein of enteric bacteria has significant homology to the acyltransferase Lpxa of lipid A biosynthesis, and to three acetyltransferases

The Ssc protein, a novel essential protein affecting the function of the enterobacterial outer membrane, matched in a protein homology search best with LpxA (UDP- N-acetylglucosamine 3-hydroxymyristoyl transferase), the enzyme which catalyzes the first step of lipid A biosynthesis. The corresponding genes, located O.56 kb apart, were 46.7% identical. The search also revealed homology to the bacterial acetyltransferases LacA and NodL, as well as to a hypothetical protein Yglm. The region of residues 109–149 Ssc displayed the highest homology and was also homologous with another bacterial acetyltransferase, CysE, and three other bacterial proteins, two of which are hypothetical. This region and the corresponding regions of all other proteins were found to have a peculiar repeated hexapeptide pattern. Each hexapeptide unit starts with isoleucine (or its equivalent leucine and valine). In most units, the second residue is glycine and the fifth residue either valine or alanine.

Enhancement by a serum factor of the immunoaccessibility of an enterobacterial porin protein domain

Monoclonal antibody (MAb) generated against the domain Po I on the outer membrane (OM) porin (Po) protein of an Escherichia coli 055 strain showed weak binding by the OM in ELISA. Human serum and sera from different animal species enhanced the MAb binding in ELISA when the antibody was incubated together with serum or the OM was pretreated with serum. Human serum also enhanced the MAb binding when coats were prepared by using OMs from various cross-reacting bacteria. The ability of human serum to amplify the MAb binding by OMs was similar to that of lysostaphin. Amplification by serum was not observed when MAbs against three other enterobacterial OM proteins were tested. The amplifying serum factor was destroyed by heating (100 degrees C) and by mercaptoethanol. It appeared in fractions which corresponded to an apparent molecular weight of 75,000-80,000 after gel permeation, and, after ion-exchange chromatography, in fractions which contained a protein of 60 kD when analysed by SDS-PAGE. These data support the notion that the serum-induced enhancement of the anti-Po I MAb binding was due to a previously described serum amidase (N-acetylmuramyl-L-alanine amidase) which has peptidoglycan-degrading activity. The effects of the amplifying serum factor may influence the antibody levels which are measured when OMs from Gram-negative bacteria are used as antigen in a serodiagnostic test.

Identification and sequence analysis of the gene mutated in the conditionally lethal outer membrane permeability mutant SS-C of Salmonella typhimurium.

The biosynthesis and structure-function relationships of the enterobacterial outer membrane are subjects of current intensive research. We have previously described the antibiotic supersensitive SS-C mutant (SH7622) of Salmonella typhimurium and shown that its outer membrane permeability barrier against hydrophobic antibiotics is severely defective. In this study, we show that this mutant is heat-sensitive, conditionally lethal, and carries a missense base-pair substitution in a novel gene which we have recently reported and now named the ssc gene. ssc encodes an earlier uncharacterized 36 kd protein (the Ssc protein) and the mutant expresses Ssc which has valine 291 changed to methionine in a methionine-rich region of Ssc. A plasmid containing the wild-type ssc allele completely reverts the antibiotic- and heat-sensitive phenotype of the SS-C mutant. Corresponding plasmids carrying the mutant allele, or an identical mutant allele prepared by localized mutagenesis, are inactive. The ssc gene is probably analogous to the firA locus of Escherichia coli which has earlier been implicated in a totally different function, mRNA synthesis. Furthermore, ssc apparently lies very close to the lpx genes involved in the thus far known steps of lipid A biosynthesis (the distance, approximately 560 bp). To conclude, our findings define a new essential gene involved in the generation of the outer membrane.

A conserved domain on enterobacterial porin protein analysed by monoclonal antibody

Broadly cross-reactive monoclonal antibodies (MAbs) against enterobacterial outer membrane (OM) porin (Po) protein were isolated after immunization of BALB/c mice with whole cells of E. coli 055:B5. MAbs (n = 6) of the IgG class but of four different isotypes were studied. Based on a competition ELISA, all of the MAbs were directed against one and the same Po protein domain (Po I). The MAbs cross-reacted with 72 of 74 strains from 10 different genera of the Enterobacteriaceae. One Morganella and one Salmonella strain showed no cross-reactivity. Also, nine strains of various Neisseria spp. cross-reacted while 21 strains of various other nonenteric Gram-negative bacteria showed no cross-reactivity. The Po I sites were inaccessible in intact homologous bacteria but partially accessible in the OM. Digestion of OM with lysozyme or lysostaphin affected the accessibility of the Po I sites in OMs of various enterobacteria. Lysostaphin strongly enhanced the immunoaccessibility, whereas lysozyme had lesser effects. The enzymes also affected the binding by Neisseria OMs of the anti-Po I MAb. The Po I site was immunogenic both in humans and rabbits. The data indicate that Po I is an important Po protein domain, and that the effects of peptidoglycan-degrading enzymes must be considered in studies of Po protein domains.

Significance of composition and structure of the enterobacterial outer membrane to the transport of desired and undesired substances and its inhibition

AbstractThe enterobacterial outer membrane forms a bilayer. Its outer monolyer consists of lipopolysaccharides and proteins, its inner monolayer of phospholipids and proteins. It thus represents an efficient penetration barrier against hydrophobic and anionic compounds (such as detergents or hydrophobic antibiotics) and against higher molecular substances (such as proteolytic, lipolytic, and murolylic enzymes).Some of the proteins (“porins”) form channels through the outer membrane through which neutral and cationic hydrophylic compounds up to a molecular weight of about 800 can pass. Besides the porins additional transport systems have been described. They play an important part in providing the bacteria with substances necessary for their growth, i.e., phosphate, iron ions, and others.Organic polycations are able to generate more or less severe disorganizations in the outer membrane through which they can pass the bilayer (“self‐promoted pathway”). Some of these polycations represent efficient antibiotics (polymyxin B, nourseothricin). Bacteria are able to protect themselves against the harmful action of these substances by changing the composition of the outer membrane.

Antibody response to defined domains on enterobacterial outer membrane proteins in healthy persons and patients with bacteraemia

Monoclonal antibody (MAb)-based competitive enzyme immunoassays (cELISAs) were elaborated to measure antibodies against MAb-defined domains on three different enterobacterial outer membrane (OM) proteins in sera from healthy individuals (n = 30) and in paired serum samples from patients (n = 45) with bacteraemia caused by enterobacteria or by various nonenteric bacteria (n = 15). The MAb-defined domains were Hm I and Hm II on the heat-modifiable (Hm) protein, PALp I and PALp II on the peptidoglycan-associated lipoprotein (PALp), and BLp I on Braun's lipoprotein (BLp). All MAbs have shown broad cross-reactivity with and specificity for enterobacteria. Sera from healthy individuals and from patients with infections caused by nonenteric bacteria contained low levels of MAb-blocking antibodies. Bacteraemia caused by enterobacteria resulted in generation of antibodies against the MAb-defined domains in many of the patients. Thus, 40% and 69% showed a positive BLp I cELISA with the first and second serum samples, respectively. Of the second serum samples, 20-38% showed positive Hm and PALp cELISAs. The BLp I cELISA showed higher diagnostic sensitivity than the previously described indirect ELISA for IgG antibodies against E. coli 055 OM protein antigens. Assays using the MAbs as competitors showed that the patients bacteraemic with enterobacteria, also generated antibodies against other domains on the OM proteins. The cELISAs may be useful in the diagnosis and management of patients with serious infections caused by enterobacteria. In this regard, the BLp I cELISA showed the most promising results.

Monoclonal antibodies against three different enterobacterial outer membrane proteins. Characterization, cross-reactivity, and binding to bacteria.

BALB/c mice were immunized with whole-cells of Escherichia coli 055:B5 or Proteus mirabilis NCTC 60 to produce broadly cross-reacting monoclonal antibodies (MAbs) against outer membrane (OM) proteins. A total of 10 anti-OM MAbs of the IgG class were selected. These included 5 MAbs against the heat-modifiable (Hm) protein, 3 against the peptidoglycan-associated lipoprotein (PALp), and 2 against Braun's lipoprotein (BLp). Based on competition ELISA, the MAbs defined 2 Hm protein binding sites (Hm I and Hm II), 2 PALp sites (PALp I and PALp II), and one BLp site (BLp I). The MAbs showed broad cross-reactivity against 74 strains of 10 different genera of the Enterobacteriaceae. Non-cross-reacting enteric bacilli occurred only among bacteria of the genera Salmonella, Proteus, and Providentia. The results revealed that Proteus and Providentia strains differed from other enteric bacilli with regard to BLp synthesis or specificity. A panel of 30 non-enteric Gram-negative bacteria did not cross-react. Testing of MAb binding to bacteria showed that a part of the BLp I, PALp I, and PALp II sites was immunoaccessible in intact homologous bacteria, and that the Hm I and Hm II epitopes were inaccessible. The MAbs should facilitate studies of structure and immunobiological function of enterobacterial OM proteins and should have a potential as immunodiagnostic reagents.

O-antigenic chains of lipopolysaccharide prevent binding of antibody molecules to an outer membrane pore protein in Enterobacteriaceae

The accessibility of outer membrane pore protein PhoE to antibody molecules at the cell surface of intact cells of various Enterobacteriaceae was investigated. Significant antibody binding was observed for only two of the nine strains tested. Analysis of the lipopolysaccharide by sodium dodecyl sulphate gel electrophoresis revealed a clear correlation between the presence of an O-antigenic side chain and the inability to bind PhoE protein-specific antibodies. As mutants that lack the O-antigen chain appeared to have acquired the ability to bind antibody, it must be concluded that the presence of O-antigenic chains of lipopolysaccharide prevents binding of antibodies to PhoE protein at the surface of intact cells. The relevance of this conclusion for the potential use of enterobacterial outer membrane pore proteins as vaccine components is discussed.

Polymyxin B nonapeptide complexes with lipopolysaccharide

We have recently shown that a polymyxin derivative, polymyxin B nonapeptide (PMBN), which lacks the fatty acid part, the bactericidal activity and the toxicity of polymyxin [1,2] nevertheless causes disorganization of the enterobacterial outer membrane (OM). Thus, PMBN makes the OM of smooth encapsulated Escherichia coil and smooth Salmonella permeable to hydrophobic antibiotics such as actinomycin D, novobiocin, fusidic acid, erythromycin and others, and sensitizes the bacteria to all those agents. Furthermore, PMBN sensitizes the PMBN to the attack of the complement cascade (M. Vaara and T. Vaara, manuscripts submitted for publication). Electron microscopic studies showed that fingerlike projections appear on the OM of bacteria treated with PMBN (M. Vaara and T. Vaara, submitted). These projections appear similar to those caused by polymyxin [3]. Polymyxin interacts with acidic macromolecules, including the lipopolysaccharide (LPS) present in the outer leaflet of the OM [4-8]. We have previously shown that the LPS of polymyxin-resistant pmrA mutants of Salmonella typhimurium binds less polymyxin and is less acidic (due to the nearly stoicheiometric occurrence of 4-amino-4-deoxy-arabinose in the lipid A part of the LPS) than the LPS of the polymyxin sensitive parent strains [8]. In the present communication we show that PMBN does not sensitize the pmrA strain (in contrast to the parent strain) to hydrophobic antibiotic~ Furthermore, we show that PMBN, like polymyxin, inhibits biological activities of isolated LPS (gelation of Limulus-lysate, consumption of complement). These results suggest that PMBN causes a disorganization of the OM by binding to the acidic groups of LPS, as does polymyxin.

Characterization of the cell envelope of Yersinia enterocolitica and the immune response to its components

The cell envelope was isolated from a recent patient isolate of Y. enterocolitica type 3 grown under different conditions, and a plasmid-less derivative of it. The typical enterobacterial outer membrane proteins, porin and OmpA, were identified by SDS-PAGE; they were invariant in both strains irrespective of culture conditions. By contrast, growth at 37° under Ca** limitation caused the appearance of a major new protein in the envelope of the plasmid-bearing but not the plasmid-less strain. This protein has an apparent molecular weight of 47 000; in the isolated envelope it is present in a multimeric form with an apparent molecular weight of appr. 200.000, but it is dissociated into its subunits by boiling in urea-containing buffer. The lipopolysaccharide (LPS) of both strains contained a large fraction of rough-type LPS in addition to the O-specific side chain which varies according to growth conditions. Antibodies to these separate components could be identified by immunoblotting techniques; in addition, an enzyme immunoassay to the isolated LPS was used. Sera of patients with reactive yersinia arthritis showed high titre abtibodies to the rough LPS, and often reacted also with rough LPS from other enteric bacteria. The anti-LPS antibody response often contained a strong IgA component.