Bacterial Ghosts Research Articles

Improved protection against lung colonization by Actinobacillus pleuropneumoniae ghosts: characterization of a genetically inactivated vaccine

Pigs immunized with Actinobacillus pleuropneumoniae ghosts or a formalin-inactivated bacterin were found to be protected against clinical disease in both vaccination groups, whereas colonization of the lungs with A. pleuropneumoniae was only prevented in ghost-vaccinated pigs. Bacterial ghosts are empty cell envelopes created by the expression of a cloned bacteriophage lysis gene and, unlike formalin-inactivated bacteria, suffer no denaturing steps during their production. This quality may lead to a superior presentation of surface antigens to the immune system. Analysis by SDS-PAGE and immunoblotting of the two vaccine preparations revealed different contents of antigenic proteins. In order to better understand the immunogenic properties of A. pleuropneumoniae ghosts and formalin-inactivated bacteria, we compared the serum antibody response induced in both treatment groups. Immune sera were tested on whole cell antigen or purified virulence factors including outer membrane protein preparations (OMPs), outer membrane lipoprotein OmlA1, transferrin binding proteins (TfbA1, TfbA7 and TfbB) and Apx toxins (ApxI, II and III). SDS-PAGE and immunoblots revealed no specific antibody response against the single virulence factors tested in any vaccinated animal. The two vaccination groups showed different recognition patterns of whole cell antigen and OMP-enriched preparations. A 100 kDa protein was recognized significantly stronger by ghost-vaccinated pigs than convalescent pigs. This unique antibody population induced by ghosts could play a determining role in the prevention of lung colonization. The same 100 kDa antigen was recognized by ghost-sera in homologous as well as heterologous serotype A. pleuropneumoniae protein preparations. Indications for a crossprotective potential in the ghost vaccine were supported by studies on rabbit hyperimmune sera.

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.

Intramuscular immunization with genetically inactivated (ghosts) Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carrier state

Bacterial ghosts are empty cell envelopes achieved by the expression of a cloned bacteriophage lysis gene and, unlike classical bacterins, suffer no denaturing steps during their production. These properties may lead to a superior presentation of surface antigens to the immune system. Currently available porcine Actinobacillus pleuropneumoniae vaccines afford only minimal protection by decreasing mortality but not morbidity. Pigs which survive infection can still be carriers of the pathogen, so a herd once infected remains infected. Carrier pigs harbour A. pleuropneumoniae in their nasal cavities, in their tonsils, or within lung lesions. A dose-defined nose-only aerosol infection model for pigs was used to study the immunogenic and protective potential of systemic immunization with ghosts made from A. pleuropneumoniae serotype 9 reference strain CVI 13261 against an homologous aerogenous challenge. Pigs were vaccinated twice intramuscularly with a dose of 5×109 CFU ghosts (GVPs) or formalin-inactivated A. pleuropneumoniae bacterins (BVPs). After 2 weeks vaccinated pigs and non-vaccinated placebo controls (PCs) were challenged with a dose of 109 CFU by aerosol. The protective efficacy of immunization was evaluated by clinical, bacteriological, serological and post-mortem examinations. Bronchoalveolar lavage in pigs was performed during the experiment to obtain lavage samples (BALF) for assessment of local antibodies. Isotype-specific antibody responses in serum and BALF were determined by ELISAs based on whole-cell antigen. Immunization with ghosts did not cause clinical side-effects. After aerosol challenge PCs developed fever and pleuropneumonia. GVPs or BVPs were found to be fully protected against clinical disease or lung lesions in both vaccination groups, whereas colonization of the respiratory tract with A. pleuropneumoniae was only prevented in GVPs. Specific immunoglobins against A. pleuropneumoniae were not detectable in BALF after immunization. A significant systemic increase of IgM, IgA, IgG(Fc′), or IgG(H+L) antibodies reactive with A. pleuropneumoniae was measured in GVPs and BVPs when compared to the non-exposed controls. BVPs reached higher titers of IgG(Fc′) and IgG(H+L) than GVPs. However, prevention of carrier state in GVPs coincided with a significant increase of serum IgA when compared to BVPs. These results suggest that immunization with ghosts, that bias antibody populations specific to non-denaturated surface antigens, may be more efficacious in protecting pigs against colonization and infection than bacterins.

Identification, characterization and purification of the lantibiotic staphylococcin T, a natural gallidermin variant.

Staphylococcin T (StT), an antibacterial agent produced by a Staphylococcus cohnii T strain, was purified to homogeneity by ammonium sulphate precipitation, gel filtration, cation exchange and fast performance liquid chromatography (FPLC). The final yield was about 20%, and over a 1000-fold increase in the specific activity was obtained. Mass determination (2166 Da), amino acid sequencing (Ile-Ala-Xaa-Lys-Phe-Leu-Xaa-Xaa-Pro-Gly-Xaa-Ala-Lys-block) and DNA sequencing demonstrated that StT is identical to gallidermin, a lanthionine-containing antimicrobial peptide. StT has a broad spectrum of bactericidal activity against Gram-positive and some Gram-negative bacteria. StT appears to damage cell membrane, and as a result causes an efflux of ions and an immediate block in macromolecular synthesis. Moreover, electron microscopic observations reveal morphological changes, with a loss of ribosomes and condensation of the nucleoid DNA. These changes are followed by a dissolution of the cell contents resulting in a bacterial ghost composed of seemingly intact cell walls with remnants of the cytoplasmic membrane and internal structure. Since StT exhibits antimicrobial activity especially against the Staphylococcus species, this compound may be of use in the treatment of staphylococcal infections.

Bacterial cell envelopes (ghosts) but not S-layers activate human endothelial cells (HUVECs) through sCD14 and LBP mechanism

Bacterial cell-envelopes (called ghosts) and surface layers (S-layers) are discussed to be used as vaccines and/or adjuvants, consequently it is necessary to find out which immunomodulatory mediators are induced in human cells. The present work focuses on the effects of ghosts ( Escherichia coli O26:B6), S-layers ( Bacillus stearothermophilus) in comparison with LPS and antibiotic-inactivated whole bacteria ( E. coli O26:B6) on human umbilical vein endothelial cells (HUVEC) with regard to the release of interleukin 6 (IL-6) and the expression of surface E-selectin and the role of lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and serum for this activation. Endothelial cells responded to ghosts, whole bacteria and LPS with IL-6 release up to 15000 pg/ml and surface E-selectin expression, while in contrast the response to S-layers with IL-6 release up to 500 pg/ml was very weak. Compared to LPS, 10–100-fold higher concentrations of bacterial ghosts and whole bacteria were required to induce the cytokine synthesis and E-selectin expression. IL-6 release and E-selectin expression of HUVECs were reduced in the absence of serum and equivalent to unstimulated samples. We have also studied the role of CD14 and LBP for the activation of endothelial cells using antiCD14 and antiLBP antibodies (Ab). AntiCD14 and antiLBP Ab both inhibited IL-6 release and E-selectin expression in a dose dependent manner after stimulation with ghosts, whole bacteria and LPS but had no effect on S-layers stimulated cells. AntiCD14 Ab inhibited more effectively than antiLBP Ab. These findings suggest that bacterial ghosts but not S-layers activate HUVECs through sCD14 and LBP dependent mechanisms.

Bacterial ghosts as drug carrier and targeting vehicles

A novel system for the packaging of drugs as well as vaccines is presented. Bacterial ghosts are intact, non-denatured bacterial envelopes that are created by lysis of bacteria through the expression of cloned phage PhiX174 gene E. Inhibition of induced E-mediated lysis by MgSO4, harvesting of cells by centrifugation, and resuspension in low-ionic-strength buffers leads to rapid, violent lysis and results in empty bacterial envelopes with large (approximately 1 μm in diameter) openings. The construction of plasmid pAV1, which encodes a streptavidin fusion protein with an N-terminal membrane anchor sequence, allows the loading of the inner side of the cytoplasmic membrane with streptavidin. The functionality and efficacy of binding of even large biotinylated compounds in such streptavidin ghosts (SA-ghosts) was assessed using the enzyme alkaline phosphatase. The successful binding of biotinylated fluorescent dextran, as well as fluorescent DNA complexed with biotinylated polylysine, was demonstrated microscopically. The display by bacterial ghosts of morphological and antigenic surface structures of their living counterparts permits their attachment to target tissues such as the mucosal surfaces of the gastrointestinal and respiratory tract, and their uptake by phagocytes and M cells. In consequence, SA-ghosts are proposed as drug carriers for site-specific drug delivery.

Extended recombinant bacterial ghost system

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new stategy for adjuvant free combination vaccines.

Pigs aerogenously immunized with genetically inactivated (ghosts) or irradiated Actinobacillus pleuropneumoniae are protected against a homologous aerosol challenge despite differing in pulmonary cellular and antibody responses

Aerosol immunization is a safe way to induce complete protection against pleuropneumonia in pigs caused by the lung pathogenic bacterium Actinobacillus pleuropneumoniae. In order to determine the local immune responses of vaccinees in concomitant with protection, lung lining fluid before and 3 weeks after immunization from pigs immunized three times with aerosols of either genetically inactivated ghosts which represent whole cell envelope preparations, or irradiated bacteria were examined following an homologous aerosol challenge. Specific antibody isotypes in the bronchoalveolar lavage were assayed by whole cell ELISAs. Total and relative numbers of cells including lymphocyte subsets were determined. In both vaccinated groups a net influx of plasma cells and lymphocytes, as well as a significant increase of specific IgG occurred. Concurrently, the CD4 +/CD8 +ratio was found to increase after aerosol immunization. The lymphocyte subsets of IgG + and IgA + cells were found significantly higher in the group immunized with irradiated bacteria when compared to pigs immunized with bacterial ghosts. The latter group showed a significant increase of IgA, IgM, and a net influx of lymphoid blasts and granulocytes in the bronchoalveolar lining fluid. Although differences between the local immune responses of both immunized groups occurred, a significant increase of specific IgG and a net influx of plasma cells and lymphocytes were found to be associated with complete protection against a homologous aerosol challenge infection.

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward λ p R promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30°C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30°C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28°C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the λ p R promoter/cI857 repressor system, allowing pathogens to grow at 37°C before induction of lysis. In this study we describe a mutation in the λ p R promoter, which allows stringent repression of gene E expression at temperatures up to 36°C, but still permits induction of cell lysis at 42°C.

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli.

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.

New strategies for combination vaccines based on the extended recombinant bacterial ghost system

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Bacterial cell envelopes (ghosts) and LPS but not bacterial S-layers induce synthesis of immune-mediators in mouse macrophages involving CD14

The synthesis of inflammatory mediators in human macrophages/monocytes seen after stimulation with lipopolysaccharide (LPS) involves the binding of CD14 to LPS complexed to lipopolysaccharide binding protein (LBP). The binding mechanisms of different LPS domains to LBP and CD14, as well as the interaction of the entire bacterial cell wall and its components with CD14 and LBP, are poorly understood. We, therefore, studied the effects of anti-mouse CD14 antibodies on the synthesis of TNFα and PGE2in RAW 264.7 mouse macrophages stimulated by bacterial cell envelopes (ghosts) of Escherichia coli 026:B6 and Salmonella typhimurium C5, LPS, lipid A, and crystalline bacterial cell surface layer (S-layer) preparations. Ghosts and S-layers, with distinct activities on the immune-system, are presently under investigation for their use as vaccines. Whereas LPS and E. coli ghosts exhibited a strong endotoxic activity in the Limulus amoebocyte lysate assay, the endotoxic activity of S-layer preparations was several orders of magnitude lower. LPS, ghosts, and bacterial S-layers all induced TNFα and PGE2synthesis as well as the accumulation of TNFα mRNA. Pre-incubation with anti-mouse CD14 antibodies resulted in a dose-dependent inhibition of TNFα and PGE2synthesis after stimulation by LPS, lipid A (30-50%) and ghosts (40-70%). The bacterial S-layer-induced mediator synthesis remained unchanged following the addition of anti-mouse CD14 antibodies. Reproducible differences could be observed for the inhibition of TNFα induced by LPS of different species by anti-CD14. Adding fetal calf serum (FCS) strongly enhanced the release of cell mediators stimulated by low doses of LPS and bacterial ghosts. These effects of the FCS may be due to the presence of LBP in the FCS. The results show that CD14 is highly relevant for the activation of mouse macrophages by bacterial cells, LPS, and lipid A. Specially defined bacterial cell wall constituents such as bacterial S-layers might act through other activation pathways.

Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines

Gram-negative bacterial ghosts produced by controlled expression of the plasmid-encoded lysis gene E offers a promising approach in non-living vaccine technology. Bacterial cell wall complex and hence the antigenic determinants of the living cells are not affected by denaturation due to cell killing. However, the endotoxin content of the Gram-negative cell wall has been discussed as a potential problem for this kind of whole cell or envelope vaccines. Here we show that bacterial ghosts prepared from Escherichia coli O26:B6 and Salmonella typhimurium C5 induce dose-dependent antibody responses against bacterial cells or their corresponding lipopolysaccharides (LPS) in doses 25 ng kg −1 when administered intravenously to rabbits in a standard immunization protocol. No differences between the immune responses of the rabbits were observed when comparing equivalent doses of bacterial ghosts and antibiotic-treated whole cells. The results indicate that the bacterial ghosts exhibit all the antigenic properties of the living cells. No significant fever responses in rabbits have been recorded in doses of <250 ng kg −1 E. coli O26:B6 ghosts and up to doses of 250 ng kg −1 S. typhimurium C5 ghosts when applying test methods recommended by the US pharmacopoeia. These findings correlate with cell culture experiments where doses 100 ng ml −1 of bacterial ghosts were needed for the release of tumour necrosis factor alpha (TNFα) and prostaglandin E 2 (PGE 2) from RAW mouse macrophage cultures. Free LPS of Salmonella abortus equi commonly used as a LPS-standard, however, stimulated TNFα and PGE 2 synthesis of RAW cells in doses of 1 ng ml −1. The endotoxic activity of our bacterial preparations analysed by a standard limulus amoebocyte lysate and 2-keto-3-deoxyoctonate assay correlated with the capacity to stimulate the release of PGE 2 and TNFα in RAW mouse macrophage cultures and the endotoxic responses in rabbits. It can be concluded that these in vitro systems can be used as easy predictive test systems for preparations of bacterial vaccines, particularly for bacterial ghosts.

Bacterial ghosts: non-living candidate vaccines

Expression of cloned PhiX174 gene E in bacteria results in lysis of bacteria. It is unique among phage lysis systems as it introduces a transmembrane tunnel structure through the cell envelope complex of Gram-negative bacteria. The resulting bacterial ghosts have intact envelope structures devoid of cytoplasmic contents. Emediated lysis has been achieved in a variety of Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Klebsiella pneumoniae, and Actinobacillus pleuropneumoniae. Such ghosts, derived from human or animal pathogens, have been proposed as nonliving candidate vaccines and represent an alternative to heat or chemically inactivated bacteria. In ‘recombinant ghosts’, foreign proteins (e.g., viral proteins) are inserted into the inner membrane via specific N-, or C-, or N-and C-terminal anchor sequences prior to lysis. Relevant advantages of (recombinant) bacterial ghosts as immunogens include: (i) inactivation procedures that denature relevant immunogenic determinants are not employed in the production of ghosts used as vaccines or as carriers of relevant antigens; (ii) the recombinant proteins are inserted into a highly immune stimulatory environment; (iii) there is no size limitation of the foreign protein moieties: multiple antigenic determinants can be presented simultaneously; (iv) bacterial ghosts can be produced inexpensively in large quantities; (v) (recombinant) ghosts are stable for long periods of time and do not require the cold chain storage system. Intraperitoneal, subcutaneous or intramuscular applications of recombinant ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and viral components. Initial aerosol vaccinations of swine with ghosts from Actinobacillus pleuropneumoniae showed that protective immunity can be established by this route of application and that the well-pre-served surface structures of ghosts obtained by Emediated lysis are able to target the mucosal immune system.

Cycling of marine dissolved organic matter. II. A model analysis

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 11:79-90 (1996) - doi:10.3354/ame011079 Cycling of marine dissolved organic matter. II. A model analysis Blackburn N, Zweifel UL, Hagström Å Nutrient pathways within the microbial food web were analysed using a mechanistic model based on a multi-stage flow-through experimental system. Good agreement with measured values reflected the model's ability to function on 3 different orders of magnitude with respect to turnover time. Important observations made from the experimental system included the production of viruses, the presence of dead bacterial cells (ghosts), and the production of detritus. This led to the concept that the bacterial community forms a continuum from live uninfected cells through infected cells, dead cells, degraded cells (detritus) and ends in dissolved organic matter. Enzymatic degradation of dissolved organic matter was balanced by its release during cell lysis caused by viruses, predation by flagellates, and degradation of detritus. This formed 3 closed loops in the model for nutrient recycling. Simulations predicted that phosphorus recycling occurred via nucleic acids released during cell lysis caused by viruses and that nitrogen was recycled via proteins released as a combined result of cell lysis caused by viruses, predation by flagellates, and degradation of detritus. This implies that for natural ecosystems, the availability of nitrogen, phosphorus, and carbon species for bacterial growth can be dominated by internal recycling, depending on the molecular species in question and the turnover time. Different nutrients are made available by different processes of recycling which potentially occur on different time scales. Dissolved organic matter recycling · Microbial food web dynamics · Nutrient recycling · Computer simulation · Bacteria · Virus · Heterotrophic nanoflagellates · Bacterial ghosts Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 11, No. 1. Publication date: August 29, 1996 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1996 Inter-Research.

Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli.

Expression of cloned gene E of bacteriophage PhiX174 induces lysis by formation of a transmembrane tunnel structure in the cell envelope of Escherichia coli. Ultrastructural studies of the location of the lysis tunnel indicate that it is preferentially located at the septum or at polar regions of the cell. Furthermore, the diameter and shape of individual tunnel structures vary greatly indicating that its structure is not rigid. Apparently, the contours of individual lysis tunnels are determined by enlarged meshes in the peptidoglycan net and the force produced at its orifice, by the outflow of cytoplasmic content. Once the tunnel is formed the driving force for the lysis process is the osmotic pressure difference between cytoplasm and medium. During the lysis process areas of the cytoplasmic membrane which are not tightly attached to the envelope are extended inward by the negative pressure produced during lysis. After cell lysis external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid bacterial ghosts.

Recombinant bacterial ghosts as vaccines

Recombinant bacterial ghosts as vaccines

Occluded Pancreatic Endoprostheses - Analysis of the Clogging Material

One of the most common late complications of transpapillary pancreatic endoprostheses is clogging of the endoprosthesis lumen. In this study we analysed the morphology and the biochemical nature of the contents of 10 clogged pancreatic endoprostheses. At the optical level the sludge presented as an organic matrix with embedded small quantities of CaCO3 crystals (and in one case CaCO3 microcalculi). Electron microscopy showed the presence of bacterial ghosts and protein threads. The characteristic pattern of proteolysed pancreatic proteins was obtained when the organic matrix was analysed by SDS-Page. The presence of trypsinogen, amylase and one of the molecular secretory forms of Pancreatic Stone Protein (PSP) was confirmed by Western-blotting. PSP was also found in association with CaCO3 crystals by immunolocalization. These results suggest that endoprosthesis clogging is due to the precipitation of whole pancreatic juice protein, probably triggered by uncontrolled proenzyme activation.

Antibacterial activity of human natural killer cells.

The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass-adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11-enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16-24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing.

Ultrastructural and biochemical alterations effected by viridin B, a bacterocin of alpha-hemolytic streptococci.

Viridin B, a bacteriocin produced by Streptococcus mitis strain 42885, was shown previously to exert a bactericidal effect against a Neisseria sicca strain but only a bacteriostatic effect against a coagulase-negative staphylococcus. In the present report, this dual effect was defined further. Viridin B caused the appearance of mesosomes in both indicator strains early in the course of exposure. Whereas mesosomes were the only noticeable alterations in the staphylococcal strain at all times of exposure to bacteriocin, progressive changes occurred in the N. sicca cells involving the nucleoid and cytoplasmic contents and resulting in the appearance of bacterial ghosts. Biochemical studies indicated a rapid and complete cessation of the incorporation of [3H]thymidine in N. sicca and in the staphylococcus. Whereas the incorporation of [3H]leucine and [3H]uridine in N. sicca was completely stopped, such incorporation was only reduced in the staphylococcus.