Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Francisella tularensis, the etiologic agent of tularemia in humans, is a potential biological threat due to its low infectious dose and multiple routes of entry. F. tularensis replicates within several cell types, eventually causing cell death by inducing apoptosis. In this study, a modified Himar1 transposon (HimarFT) was used to mutagenize F. tularensis LVS. Approximately 7,000 Km(r) clones were screened using J774A.1 macrophages for reduction in cytopathogenicity based on retention of the cell monolayer. A total of 441 candidates with significant host cell retention compared to the parent were identified following screening in a high-throughput format. Retesting at a defined multiplicity of infection followed by in vitro growth analyses resulted in identification of approximately 70 candidates representing 26 unique loci involved in macrophage replication and/or cytotoxicity. Mutants carrying insertions in seven hypothetical genes were screened in a mouse model of infection, and all strains tested appeared to be attenuated, which validated the initial in vitro results obtained with cultured macrophages. Complementation and reverse transcription-PCR experiments suggested that the expression of genes adjacent to the HimarFT insertion may be affected depending on the orientation of the constitutive groEL promoter region used to ensure transcription of the selective marker in the transposon. A hypothetical gene, FTL_0706, postulated to be important for lipopolysaccharide biosynthesis, was confirmed to be a gene involved in O-antigen expression in F. tularensis LVS and Schu S4. These and other studies demonstrate that therapeutic targets, vaccine candidates, or virulence-related genes may be discovered utilizing classical genetic approaches in Francisella.
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PMID:Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages. 1768 43

Francisella tularensis is a gram-negative intracellular bacterium and the causative agent of the zoonotic disease tularemia. F. tularensis is a category A select agent and thus a potential agent of bioterrorism. Whereas an F. tularensis live, attenuated vaccine strain (LVS) is the basis of an investigational vaccine, this vaccine is not licensed for human use because of efficacy and safety concerns. In the present study, we immunized mice with isolated native outer membrane proteins (OMPs), ethanol-inactivated LVS (iLVS), or purified LVS lipopolysaccharide (LPS) and assessed the ability of each vaccine preparation to protect mice against pulmonary challenge with the virulent type A F. tularensis strain SchuS4. Antibody isotyping indicated that both Th1 and Th2 antibody responses were generated in mice after immunization with OMPs or iLVS, whereas LPS immunization resulted in only immunoglobulin A production. In survival studies, OMP immunization provided the greatest level of protection (50% survival at 20 days after infection with SchuS4), and there were associated 3-log reductions in the spleen and liver bacterial burdens (compared to nonvaccinated mice). Cytokine quantitation for the sera of SchuS4-challenged mice indicated that OMP and iLVS immunizations induced high levels of tumor necrosis factor alpha and interleukin-2 (IL-2) production, whereas only OMP immunization induced high levels of IL-10 production. By comparison, high levels of proinflammatory cytokines, including RANTES, granulocyte colony-stimulating factor, IL-6, IL-1alpha, IL-12p40, and KC, in nonvaccinated mice indicated that these cytokines may facilitate disease progression. Taken together, the results of this study demonstrate the potential utility of an OMP subunit (acellular) vaccine for protecting mammals against type A F. tularensis.
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PMID:Native outer membrane proteins protect mice against pulmonary challenge with virulent type A Francisella tularensis. 1850 5

We have developed and evaluated a novel and simplified whole-blood lymphocyte stimulation assay that focuses on the measurement of gamma interferon after 24 h of stimulation with whole-cell tularemia antigen and a tularemia enzyme-linked immunosorbent assay (ELISA) based on highly purified lipopolysaccharide antigen. Comparison of the kinetics of the two assays and those of the traditional tube agglutination test shows that the cellular immune response can be detected earlier by the lymphocyte stimulation assay. This test already shows a high proportion of positive results during the first week after the onset of the disease, may be applicable in everyday laboratory practice, and has the potential of changing routine diagnostics for tularemia. The new ELISA has a high sensitivity and becomes positive to a high degree during the second week of disease.
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PMID:Kinetics of the immune response associated with tularemia: comparison of an enzyme-linked immunosorbent assay, a tube agglutination test, and a novel whole-blood lymphocyte stimulation test. 1856 68

Francisella tularensis causes severe pneumonia that can be fatal if it is left untreated. Due to its potential use as a biological weapon, research is being conducted to develop an effective vaccine and to select and study adjuvant molecules able to generate a better and long-lasting protective effect. PorB, a porin from Neisseria meningitidis, is a well-established Toll-like receptor 2 ligand and has been shown to be a promising vaccine adjuvant candidate due to its ability to enhance the T-cell costimulatory activity of antigen-presenting cells both in vitro and in vivo. BALB/c mice were immunized with lipopolysaccharide (LPS) isolated from the F. tularensis subsp. holarctica live vaccine strain (LVS), with or without PorB from N. meningitidis, and the antibody levels induced during the vaccination regimen and the level of protection against intranasal challenge with LVS were determined. Antigen administered alone induced a specific F. tularensis LPS immunoglobulin M (IgM) response that was not maintained over the weeks and that conferred protection to only 25% of the mice. In contrast, F. tularensis LPS given in combination with neisserial PorB induced consistent levels of specific IgM throughout the immunization and increased the proportion of surviving mice to 70%. Postchallenge cytokine analysis showed that interleukin-6 (IL-6), monocyte chemoattractant protein 1, and gamma interferon were markers of mortality and that IL-1beta was a correlate of survival, independent of the presence of PorB as an adjuvant. These data indicate that neisserial PorB might be an optimal candidate adjuvant for improving the protective effect of F. tularensis LPS and other subunit vaccines against tularemia, but there is still a need to test its efficacy against virulent type A and type B F. tularensis strains.
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PMID:Neisseria meningitidis PorB, a Toll-like receptor 2 ligand, improves the capacity of Francisella tularensis lipopolysaccharide to protect mice against experimental tularemia. 1861 68

Francisella tularensis is the causative agent of tularemia and a potential agent of biowarfare. As an easily transmissible infectious agent, rapid detection and treatment are necessary to provide a positive clinical outcome. As an agent of biowarfare, there is an additional need to prevent infection. We made monoclonal antibodies to the F. tularensis subsp. holarctica live vaccine strain (F. tularensis LVS) by infecting mice with a sublethal dose of bacteria and, following recovery, by boosting the mice with sonicated organisms. The response to the initial and primary infection was restricted to immunoglobulin M antibody directed solely against lipopolysaccharide (LPS). After boosting with sonicated organisms, the specificity repertoire broadened against protein antigens, including DnaK, LpnA, FopA, bacterioferritin, the 50S ribosomal protein L7/L12, and metabolic enzymes. These monoclonal antibodies detect F. tularensis LVS by routine immunoassays, including enzyme-linked immunosorbent assay, Western blot analysis, and immunofluorescence. The ability of the antibodies to protect mice from intradermal infection, both prophylactically and therapeutically, was examined. An antibody to LPS which provides complete protection from infection with F. tularensis LVS and partial protection from infection with F. tularensis subsp. tularensis strain SchuS4 was identified. There was no bacteremia and reduced organ burden within the first 24 h when mice were protected from F. tularensis LVS infection with the anti-LPS antibody. No antibody that provided complete protection when administered therapeutically was identified; however, passive transfer of antibodies against LPS, FopA, and LpnA resulted in 40 to 50% survival of mice infected with F. tularensis LVS.
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PMID:Francisella tularensis infection-derived monoclonal antibodies provide detection, protection, and therapy. 1917 92

Francisella tularensis (Ft), a gram-negative intracellular bacterium, is the etiologic agent of tularemia. Infection of mice with <10 Ft Live Vaccine Strain (Ft LVS) organisms i.p. causes a lethal infection that resembles human tularemia. Here, we show that immunization with as little as 0.1 ng Ft LVS lipopolysaccharide (Ft-LPS), but not Ft lipid A, generates a rapid antibody response that protects wild-type (WT) mice against lethal Ft LVS challenge. Protection is not induced in Ft-LPS-immunized B cell-deficient mice (muMT or JhD), male xid mice, or Ig transgenic mice that produce a single IgH (not reactive with Ft-LPS). Focusing on the cellular mechanisms that underlie this protective response, we show that Ft-LPS specifically stimulates proliferation of B-1a lymphocytes that bind fluorochrome-labeled Ft-LPS and the differentiation of these cells to plasma cells that secrete antibodies specific for Ft-LPS. This exclusively B-1a antibody response is equivalent in WT, T-deficient (TCRalphabeta(-/-), TCRgammadelta(-/-)), and Toll-like receptor 4 (TLR4)-deficient (TLR4(-/-)) mice and thus is not dependent on T cells or typical inflammatory processes. Serum antibody levels peak approximately 5 days after Ft-LPS immunization and persist at low levels for months. Thus, immunization with Ft-LPS activates a rare population of antigen-specific B-1a cells to produce a persistent T-independent antibody response that provides long-term protection against lethal Ft LVS infection. These data support the possibility of creating effective, minimally invasive vaccines that can provide effective protection against pathogen invasion.
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PMID:Antigen-specific B-1a antibodies induced by Francisella tularensis LPS provide long-term protection against F. tularensis LVS challenge. 1925 56

Francisella tularensis, the causative agent of tularemia, interacts with host cells of innate immunity in an atypical manner. For most Gram-negative bacteria, the release of lipopolysaccharide (LPS) from their outer membranes stimulates an inflammatory response. When LPS from the attenuated live vaccine strain (LVS) or the highly virulent Schu S4 strain of F. tularensis was incubated with human umbilical vein endothelial cells, neither species of LPS induced expression of the adhesion molecule E-selectin or secretion of the chemokine CCL2. Moreover, a high concentration (10 microg/ml) of LVS or Schu S4 LPS was required to stimulate production of CCL2 by human monocyte-derived macrophages (huMDM). A screen for alternative proinflammatory factors of F. tularensis LVS identified the heat shock protein GroEL as a potential candidate. Recombinant LVS GroEL at a concentration of 10 microg/ml elicited secretion of CXCL8 and CCL2 by huMDM through a TLR4-dependent mechanism. When 1 microg of LVS GroEL/ml was added to an equivalent amount of LVS LPS, the two components synergistically activated the huMDM to produce CXCL8. Schu S4 GroEL was less stimulatory than LVS GroEL and showed a lesser degree of synergy when combined with Schu S4 LPS. These findings suggest that the intrinsically low proinflammatory activity of F. tularensis LPS may be increased in the infected human host through interactions with other components of the bacterium.
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PMID:GroEL and lipopolysaccharide from Francisella tularensis live vaccine strain synergistically activate human macrophages. 2012 21

Tularemia is a highly contagious infectious zoonosis caused by the bacterial agent Francisella tularensis. Serology is still considered to be a cornerstone in tularemia diagnosis due to the low sensitivity of bacterial culture and the lack of standardization in PCR methodology for the direct identification of the pathogen. We developed a novel immunochromatographic test (ICT) to efficiently detect F. tularensis-specific antibodies in sera from humans and other mammalian species (nonhuman primate, pig, and rabbit). This new tool requires none or minimal laboratory equipment, and the results are obtained within 15 min. When compared to the method of microagglutination, which was shown to be more specific than the enzyme-linked immunosorbent assay, the ICT had a sensitivity of 98.3% (58 positive sera were tested) and a specificity of 96.5% (58 negative sera were tested) on human sera. On animal sera, the overall sensitivity was 100% (22 positive sera were tested) and specificity was also 100% (70 negative sera were tested). This rapid test preferentially detects IgG antibodies that may occur early in the course of human tularemia, but further evaluation with human sera is important to prove that the ICT can be a valuable field test to support a presumptive diagnosis of tularemia. The ICT can also be a useful tool to monitor successful vaccination with subunit vaccines or live vaccine strains containing lipopolysaccharide (e.g., LVS) and to detect seropositive individuals or animals in outbreak situations or in the context of epidemiologic surveillance programs in areas of endemicity as recently recommended by the World Health Organization.
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PMID:Evaluation of an immunochromatographic test for rapid and reliable serodiagnosis of human tularemia and detection of Francisella tularensis-specific antibodies in sera from different mammalian species. 2022 Jan 65

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (<10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.
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PMID:Method for the isolation of Francisella tularensis outer membranes. 2061 13

The lipopolysaccharide (LPS) of Francisella tularensis (Ft), the Gram negative bacterium that causes tularemia, has been shown to be a main protective antigen in mice and humans; we have previously demonstrated that murine anti-Ft LPS IgG2a monoclonal antibodies (MAbs) can protect mice against otherwise lethal intranasal infection with the Ft live vaccine strain (LVS). Here we show that four IgG2a anti-LPS MAbs are specific for the O-polysaccharide (O-antigen [OAg]) of Ft LPS. But whereas three of the MAbs bind to immunodominant repeating internal epitopes, one binds to a unique terminal epitope of Ft OAg. This was deduced from its even binding to both long and short chains of the LPS ladder in Western blots, its rapid decrease in ELISA binding to decreasing solid-phase LPS concentrations, its inability to compete for LPS binding with a representative of the other three MAbs, and its inability to immunoprecipitate OAg despite its superior agglutination titer. Biacore analysis showed the end-binding MAb to have higher bivalent avidity for Ft OAg than the internal-binding MAbs and provided an immunogenicity explanation for the predominance of internal-binding anti-Ft OAg MAbs. These findings demonstrate that non-overlapping epitopes can be targeted by antibodies to Ft OAg, which may inform the design of vaccines and immunotherapies against tularemia.
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PMID:Characterization of monoclonal antibodies to terminal and internal O-antigen epitopes of Francisella tularensis lipopolysaccharide. 2146 82


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