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PubMed1992

Immunogenicity and antigenic heterogeneity of a human transferrin-binding protein in Neisseria meningitidis.

Growing Neisseria meningitidis on an iron restriction medium induces the synthesis of new outer membrane proteins, some of them true iron-regulated outer membrane proteins (IROMPs) and others synthesized because of the stress produced by the iron restriction. Some of these proteins are antigenic and can be considered for the development of vaccines; this is especially desirable in the case of N. meningitidis serogroup B, for which polysaccharide vaccines are not efficient. The antigenicity of N. meningitidis 37- and 70-kDa IROMPs has been studied previously; in this work, we studied the immunogenicity and antigenic heterogeneity of another IROMP, the human transferrin-binding protein 2 (TBP2), which seems to be indispensable for meningococcal growth inside the host. Mice were inoculated with purified outer membrane vesicles (blebs) from 5 selected N. meningitidis strains, and the five serum samples obtained were analyzed for anti-TBP2 antibodies by using the homologous strain and for cross-reactivity with the TBP2 of the 4 other selected strains and another 35 heterologous N. meningitidis strains. The TBP2s of the 5 strains tested were all immunogenic in mice to various degrees depending on the strain, and all five TBP2s shared one or more epitopes with heterologous strains (as shown by the cross-reactivities of the five serum samples), although the number of cross-reacting strains was very variable, ranging from 2 for strain V002 to 35 for strain P391. This suggests that the TBP2 epitopes of different strains differ in nature or in their accessibility to the immune system. Under the iron restriction conditions used, all strains synthesized a non-TBP2 antigenic 56-kDa protein thought to be a stress protein.

Animals↗

PubMed1993

Purification and characterization of fibroblast-activating factor isolated from Porphyromonas gingivalis W50.

A 24-kDa polypeptide which activated the incorporation of [3H]thymidine into human fibroblasts was isolated from the outer membrane vesicles of Porphyromonas gingivalis W50. This polypeptide, named fibroblast activating factor (FAF), was isolated by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane-sulfonate (CHAPS) detergent extraction and purified by DEAE ion-exchange chromatography and preparative isoelectric focusing. Purified FAF (100 ng of protein per ml) caused a 400% increase in [3H]thymidine incorporation into human gingival fibroblasts (HGFs) compared with results for controls. FAF was characterized as (i) a polypeptide with molecular masses of 24 kDa when heated at 100 degrees C for 5 min and 44 kDa when unheated, (ii) heat sensitive but not affected by selected reducing reagents, and (iii) possessing slight phosphatase activity. N'-terminal sequence analysis revealed no homology with P. gingivalis peptides or with any host-derived growth factors. These data suggest that FAF functions as a significant virulence factor which in vivo is capable of modulating homeostasis in local connective tissues.

Amino Acid Sequence↗

PubMed1993

Modulation of growth and function of human gingival fibroblasts by fibroblast-activating factor derived from Porphyromonas gingivalis W50.

The effect of a 24-kDa fibroblast-activating factor (FAF) isolated from outer membrane vesicles of Porphyromonas gingivalis W50 on the modulation of [3H]thymidine uptake and cell proliferation was examined in selected fibroblast and transformed cell lines. FAF enhanced the proliferation of human gingival fibroblasts, human skin fibroblasts, and human umbilical vein endothelial cells in subconfluent and confluent cells, suggesting that FAF might be functioning as a competence factor. The transformed cell lines, U-937 and HEp-2, were not responsive. FAF and several human-derived growth factors showed a synergistic effect on proliferation. [3H]proline and [3H]leucine were rapidly incorporated into fibroblasts in the presence of FAF; however, there was no selective induction of collagen synthesis. FAF was not active in the induction of interleukin-1 and interleukin-6. It is hypothesized that FAF from P. gingivalis functions as a growth factor for human fibroblasts but is without activity for transformed cells.

Antigens, Neoplasm↗

PubMed1993

Role of Porphyromonas gingivalis-derived fibroblast-activating factor in bone resorption.

A 24-kDa protein was isolated from the outer membrane vesicles of Porphyromaonas gingivalis W50. This protein, referred to as fibroblast-activating factor (FAF), was examined for its bone-resorptive ability by the rat long-bone assay and the mouse bone marrow cell culture system. FAF resulted in a significant release of 45Ca from cultured bones, as well as the formation of tartrate-resistant acid phosphatase-positive monocytes. These cultures were compared with control cells. FAF therefore might be considered a significant bacterially expressed protein which could affect and modulate the resorption or destruction of tissue and alveolar bone in the local periodontal environment.

Animals↗

PubMed2001

Interactions of Neisseria gonorrhoeae with mature human macrophage opacity proteins influence production of proinflammatory cytokines.

The pathological features of ascending gonococcal infection suggest that proinflammatory mediators secreted by tissue-resident macrophages are important components of the host response. Challenge of fully differentiated, mature macrophages with variants of Neisseria gonorrhoeae strain P9 or purified bacterial surface components (pili, lipooligosaccharide, and outer membrane vesicles) induced the secretion of interleukin 6 (IL-6), tumor necrosis factor alpha, growth-related protein alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), and RANTES cytokines but had no effect on IL-8 production. No secretion of IL-1beta, epithelial-derived neutrophil attractant 78, granulocyte-macrophage colony-stimulating factor, IL-10, or IL-12 cytokines was observed. Notably, the P9-Opa(b) protein, in comparison to P9-Opa(a), increased the association of gonococci with macrophages and elevated the secretion of cytokines. Thus, variation in Opa protein expression by the gonococcus may be a determining factor in the severity of pelvic inflammatory disease.

Antigens, Bacterial↗

PubMed2001

Immune response induction and new effector mechanisms possibly involved in protection conferred by the Cuban anti-meningococcal BC vaccine.

This report explores the participation of some afferent mechanisms in the immune response induced by the Cuban anti-meningococcal vaccine VA-MENGOC-BC. The induction of delayed-type hypersensitivity in nursing babies and lymphocyte proliferation after immunization is demonstrated. The presence of gamma interferon IFN-gamma and interleukin-2 (IL-2) mRNAs but absence of IL-4, IL-5, and IL-10 mRNAs were observed in peripheral blood mononuclear cells from immunized subjects after in vitro challenge with outer membrane vesicles. In addition, some effector functions were also explored. The presence of opsonic activity was demonstrated in sera from vaccinees. The role of neutrophils as essential effector cells was shown. In conclusion, we have shown that, at least in the Cuban adult population, VA-MENGOC-BC induces mechanisms with a T-helper 1 pattern in the afferent and effector branches of the immune response.

Adult↗

PubMed2002

Influence of intravenous anesthesia on mucosal and systemic antibody responses to nasal vaccines.

Inhalation of antigens may stimulate the immune system by way of the upper as well as the lower airways. We have shown that at least 1,000 times more live pneumococci were recovered from pulmonary tissue after being presented as drops of a liquid suspension onto the nares of anesthetized mice compared to the number of bacteria recovered from animals that were not anesthetized in the course of the challenge. Mice that were similarly immunized intranasally by inhalation of three different nonreplicating particulate vaccine formulations, i.e., a meningococcal outer membrane vesicle (OMV) vaccine, a formalin-inactivated whole-virus influenza (INV) vaccine, and the INV vaccine with OMVs as a mucosal adjuvant, during general intravenous anesthesia developed concentrations of vaccine-specific serum immunoglobulin G (IgG) antibodies that were four to nine times higher than in mice that were fully awake during immunizations. The concentrations of IgA antibodies in serum were also higher in anesthetized than in nonanesthetized mice and correlated positively with the corresponding levels of serum IgG antibodies in the anesthetized but not in the nonanesthetized mice. In saliva and feces, however, the concentrations of IgA antibodies were equally high whether or not the animals were dormant during immunizations. The results indicate that intrapulmonary antigen presentation, as a part of an intranasal immunization strategy, is of importance for systemic but not for mucosal antibody responses. A major portion of IgA antibodies in serum may thus be derived from nonmucosal sites.

Adjuvants, Immunologic↗

PubMed2002

Cross-reactive polyclonal antibodies to the inner core of lipopolysaccharide from Neisseria meningitidis.

Sera from mice immunized with native or detergent-extracted outer membrane vesicles derived from lipopolysaccharide (LPS) mutant 44/76(Mu-4) of Neisseria meningitidis were analyzed for antibodies to LPS. The carbohydrate portion of 44/76(Mu-4) LPS consists of the complete inner core, Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->5KDO[4-->2 alpha KDO]. Immunoblot analysis revealed that some sera contained antibodies to wild-type LPS which has a fully extended carbohydrate chain of immunotype L3,7, as well as to the homologous LPS. Sera reacted only weakly to LPS from 44/76(Mu-3), which lacks the terminal glucose of the inner core. No binding to more truncated LPS was observed. Consequently, the cross-reactive epitopes are expressed mainly by the complete inner core. Dephosphorylation of wild-type LPS abolished antibody binding to LPS in all but one serum. Thus, at least two specificities of cross-reactive antibodies exist: one is dependent on phosphoethanolamine groups in LPS, and one is not. Detection of these cross-reactive antibodies strongly supports the notion that epitopes expressed by meningococcal LPS inner core are also accessible to antibodies when the carbohydrate chain is fully extended. Also, these inner core epitopes are sufficiently immunogenic to induce antibody levels detectable in polyclonal antibody responses. Meningococci can escape being killed by antibodies to LPS that bind only to a specific LPS variant, by altering the carbohydrate chain length. Cross-reactive antibodies may prevent such escape. Therefore, inner core LPS structures may be important antigens in future vaccines against meningococcal disease.

Antibodies, Bacterial↗

PubMed2003

Liposomal meningococcal B vaccination: role of dendritic cell targeting in the development of a protective immune response.

The effect of targeting strategies for improving the interaction of liposomal PorA with dendritic cells (DC) on the immunogenicity of PorA was investigated. PorA, a major antigen of Neisseria meningitidis, was purified and reconstituted in different types of (targeted) liposomes, i.e., by using mannose or phosphatidylserine as targeting moieties, or with positively charged liposomes. We studied the efficiency of liposome uptake and its effect on the maturation of and interleukin 12 (IL-12) production by murine DC. Moreover, mice were immunized subcutaneously to study the localization and immunogenicity of PorA liposomes. Uptake of liposomes by DC was significantly increased for targeted liposomes and resulted in the maturation of DC, but to various degrees. Maturation markers (i.e., CD80, CD86, major histocompatibility complex class II, and CD40) showed enhanced expression on DC incubated with targeted PorA liposomes relative to those incubated with nontargeted PorA liposomes. Moreover, only the uptake of targeted PorA liposomes induced production of IL-12 by DC, with levels similar to those produced by lipopolysaccharide (LPS)-pulsed DC. Mannose-targeted PorA liposomes administered subcutaneously had an increased localization in draining lymph nodes compared to nontargeted PorA liposomes. Liposomes in draining lymph nodes interacted preferentially with antigen-presenting cells, an effect that was enhanced with targeted PorA liposomes. Immunization studies showed an improvement of the bactericidal antibody response (i.e., increased number of responders) generated by targeted PorA liposomes compared to that generated by nontargeted ones or LPS-containing outer membrane vesicles. In conclusion, the use of targeted PorA liposomes results in an improved uptake by and activation of DC and an increased localization in draining lymph nodes. These effects correlate with an enhanced immune response toward the vaccine.

Animals↗

PubMed2006

Growth phase regulation of Vibrio cholerae RTX toxin export.

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, secretes several "accessory" toxins, including RTX toxin, which causes the cross-linking of the actin cytoskeleton. RTX toxin is exported to the extracellular milieu by an atypical type I secretion system (T1SS), and we previously noted that RTX-associated activity is detectable only in supernatant fluids from log phase cultures. Here, we investigate the mechanisms for regulating RTX toxin activity in supernatant fluids. We find that exported proteases are capable of destroying RTX activity and may therefore play a role in the growth phase regulation of toxin activity. We determined that the absence of RTX toxin in stationary-phase culture supernatant fluids is also due to a lack of toxin secretion and not attributable to solely proteolytic degradation. We ascertained that the T1SS apparatus is regulated at the transcriptional level by growth phase control that is independent of quorum sensing, unlike other virulence factors of V. cholerae. Additionally, in stationary-phase cultures, all RTX toxin activity is associated with bacterial membranes or outer membrane vesicles.

Bacterial Toxins↗

PubMed1986

Release of cell-free ice nuclei by Erwinia herbicola.

Several ice-nucleating bacterial strains, including Erwinia herbicola, Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for their ability to shed ice nuclei into the growth medium. Only E. herbicola isolates shed cell-free ice nuclei active at -2 to -10 degrees C. These cell-free nuclei exhibited a freezing spectrum similar to that of ice nuclei found on whole cells, both above and below -5 degrees C. Partially purified cell-free nuclei were examined by density gradient centrifugation, chemical and enzymatic probes, and electron microscopy. Ice-nucleating activity in these cell-free preparations was associated with outer membrane vesicles shed by cells and was sensitive to protein-modifying reagents.

Bacterial Proteins↗

PubMed1991

A gene for a new lipoprotein in the dapA-purC interval of the Escherichia coli chromosome.

Cloning and sequence analysis of the region located downstream of the dapA gene of Escherichia coli has revealed the presence of an open reading frame that is cotranscribed with dapA. This gene codes for a 344-amino-acid polypeptide with a potential signal sequence characteristic of a lipoprotein. When this gene, called nlpB, is expressed from a multicopy plasmid in bacteria grown in the presence of [3H]palmitate, a labeled 37-kDa protein is produced. A slightly larger precursor molecule is detected when minicells expressing nlpB are treated with globomycin, a specific inhibitor of lipoprotein signal peptidase. Therefore, the nlpB gene encodes a new lipoprotein, designated NlpB. This lipoprotein is detected in outer membrane vesicles prepared from osmotically lysed spheroplasts and appears to be nonessential, since a strain in which the nlpB gene is disrupted by insertion of a chloramphenicol resistance gene is still able to grow and shows no discernible NlpB phenotype. The putative transcription termination signals of the dapA-nlpB operon overlap the promoter of the adjacent purC gene.

Amino Acid Sequence↗

PubMed1994

Involvement of phospholipid end groups of group C Neisseria meningitidis and Haemophilus influenzae type b polysaccharides in association with isolated outer membranes and in immunoassays.

There are several bacterial polysaccharides (PSs) which contain a terminal lipid moiety. It has been postulated that these terminal lipid moieties anchor the PSs to the outer membrane of the bacteria. Our studies have shown that incubation of native PS from group C Neisseria meningitidis or Haemophilus influenzae type b with isolated outer membrane vesicles results in association of a portion of the PS with the vesicles. Removal of the terminal lipid from the PS by treatment with phospholipase A2 or phospholipase D eliminates this association. In other studies, it was shown that delipidated PSs are not suitable as solid-phase antigens in a currently used enzyme-linked immunosorbent assay (ELISA). Measurement of antibody units in the reference sera by using delipidated PSs as antigens in an ELISA yielded negligible absorbance compared with native PSs when methylated human serum albumin was used to coat the PSs to the plate. Nevertheless, phospholipase A2 and phospholipase D treatment did not noticeably affect antigenic epitopes, since soluble group C PS without the terminal lipid bound antibody as effectively as the native PS did, as measured by a competitive inhibition assay. Both hydrophobic and electrostatic interactions are important for the binding of group C N. meningitidis PS to the ELISA plate, while charge interactions seem to be sufficient for binding the more negatively charged H. influenzae type b PS.

Antigens, Bacterial↗

PubMed2001

Serosubtypes and PorA types of Neisseria meningitidis serogroup B isolated in Brazil during 1997--1998: overview and implications for vaccine development.

Meningococcal disease caused by N. meningitidis serogroup B (MenB) has been endemic in Brazil since 1997. In this study, we determined the prevalence of serosubtypes of MenB isolated in 10 Brazilian states and the Federal District during 1997 and 1998 and investigated the extent of PorA VR sequence variation among the most prevalent serosubtypes to evaluate the possible use of an outer membrane vesicle (OMV)-, PorA-based vaccine to prevent meningococcal disease in Brazil. During this period, a total of 8,932 cases of meningococcal disease were reported. Only 42% (n = 3,751) of the reported cases were laboratory confirmed, and about 60% (n = 2,255) of those were identified as MenB. Among 1,297 MenB strains selected for this study, the most prevalent serosubtypes were P1.19,15 (66%), P1.7,1 (11%), and P1.7,16 (4%). PorA VR typing showed that 91% of the P1.19,15 strains analyzed had VR1 and VR2 sequences identical to those of the prototype strain. No sequence variation was detected among the 40 strains representing all isolated MenB P1.7,16 strains in the three southern states, where this serosubtype accounts for 75% of the serosubtypes identified. Similarly, all P1.7,1 strains were identified by PorA typing as P1.7-1,1. Although further improvements in the reporting of cases and collection of strains in Brazil are needed, our data suggest that a trivalent OMV-based vaccine prepared with PorA types P1.19,15, P1.7-1,1, and P1.7,16 may be appropriate to control serogroup B meningococcal disease in most of the Brazilian states.

Brazil↗

PubMed2005

Stability of PorA during a meningococcal disease epidemic.

Meningococci causing New Zealand's epidemic, which began in 1991, are defined as group B, serosubtype P1.4 (subtype P1.7-2,4), belonging to the ST-41/ST-44 complex, lineage III. Of the 2,358 group B isolates obtained from disease cases from 1991 through 2003, 85.7% (2,021 of 2,358) were determined to be serosubtype P1.4. Of the remaining isolates, 156 (6.6%) were not serosubtypeable (NST). Molecular analysis of the porA gene from these B:NST meningococcal isolates was used to determine the reason. Most NST isolates (156, 88.5%) expressed a PorA that was distinct from P1.7-2,4 PorA. Fifteen isolates expressed variants of P1.7-2,4 PorA, and a further three expressed P1.7-2,4 PorA without any sequence variation. These three isolates expressed P1.7-2,4 PorA at very low levels, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and showed variation in the porA promoter region. Among the 15 meningococcal isolates expressing variants of P1.7-2,4 PorA, 11 different sequence variations were found. Compared with the P1.7-2,4 PorA sequence, the sequences of these variants contained deletions, insertions, or single-nucleotide substitutions in the VR2 region of the protein. Multilocus restriction typing was used to assess the clonal derivations of B:NST case isolates. Meningococcal isolates expressing distinct PorA proteins belonged mostly to clonal types that were unrelated to the epidemic strain, whereas all meningococcal isolates expressing variants of P1.7-2,4 PorA belonged to the ST-41/ST-44 complex, lineage III. These results, together with those obtained serologically, demonstrate that the P1.7-2,4 PorA protein of meningococci responsible for New Zealand's epidemic has remained relatively stable over 13 years and support the use of a strain-specific outer membrane vesicle vaccine to control the epidemic.

Amino Acid Sequence↗

PubMed1998

Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria.

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

Alleles↗

PubMed1975

Fine structure of the cell envelope layers of Flexibacter polymorphus.

Electron microscopy of the filamentous gliding marine bacterium Flexibacter polymorphus demonstrated that the cell envelope consists of an electron-dense intermediate layer located between two unit-type membranes: an outer membrane, presumably of lipopolysaccharide, and an inner cytoplasmic membrane. Separation of living filaments into single cells by lysozyme suggests that a peptidoglycan moiety, possibly corresponding to the intermediate layer, might be situated between the two membranes. Cell division proceeds by invagination of the cytoplasmic membrane and intermediate layer forming a transverse septum. Cells generally fail to separate after the division process, so that a common outer membrane encloses all of the cells in a single filament. There is a continuous layer of macromolecular cup-shaped elements ('goblets') attached to the outermost surface of the lipopolysaccharide membrane. Tangential thin sections, as well as negatively stained preparations of envelope fragments (produced by sonication of autolyzed cells), showed that the goblets are arranged in a close-packed hexagonal array. The presence of electron-dense structures located between the outer and inner membranes, and exhibiting the same periodicity as the goblets, suggests that some part of the goblets penetrates the outer membrane and extends across the periplasmic space to the dense intermediate layer or cytoplasmic membrane. Spontaneous autolysis in aging cultures is accompanied by the formation and release into the culture medium of large numbers of outer membrane vesicles coated with globlets. A tentative reconstruction of the envelope of F. polymorphus, based on the fine-structural data, is presented.

Bacteriolysis↗

PubMed1991

Serotypes and subtypes of Neisseria meningitidis serogroup B strains associated with meningococcal disease in Canada, 1977-1989.

Typing of Neisseria meningitidis serogroup B disease isolates was carried out using a panel of serotype-and subtype-specific monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (ELISA). Three hundred and sixty-two strains isolated from 1977 to 1986 were typed using five serotyping and seven subtyping reagents and outer membrane vesicles as antigens. Serotype 2b accounted for 30% of the disease isolates. The most common subtype was P1.2, which occurred on 18.5% of all strains or 48.6% of the serotype 2b strains. Of the 362 strains typed, 135 (37.3%) were serotyped and 122 (33.7%) were subtyped. Overall, 185 (51.1%) of the strains could be assigned a serotype and (or) subtype. Strains (221) isolated during the years 1987-1989 were typed using a panel of 6 serotyping and 12 subtyping reagents by whole-cell ELISA. Strains of serotypes 4 (21.7%) and 15 (20.8%) were the most common and carried a wide variety of subtypes. The most common subtypes were P1.2 (11.8%) and P1.16 (9.5%). Of the 221 strains analyzed, 132 (59.7%) were assigned a serotype and 123 (55.7%) a subtype and with all 18 MAbs, 192 (86.9%) of the strains were serotyped and (or) subtyped. Two different MAbs to the four epitopes 2a, 15, P1.2, and P1.16 gave discordant reactions of 0.3, 6.6, 2.6, and 2.2%, respectively, when used to analyze over 300 strains of N. meningitidis.

Canada↗