Autotransporter-Mediated Display of Complement Receptor Ligands by Gram-Negative Bacteria Increases Antibody Responses and Limits Disease Severity
Kristen M Holland-TummilloLauren E. ShoudyDonald SteinerSudeep KumarSarah J. RosaPrachi NamjoshiAnju SinghTimothy J. SellatiEdmund J. GosselinKarsten R. O. Hazlett
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The targeting of immunogens/vaccines to specific immune cells is a promising approach for amplifying immune responses in the absence of exogenous adjuvants. However, the targeting approaches reported thus far require novel, labor-intensive reagents for each vaccine and have primarily been shown as proof-of-concept with isolated proteins and/or inactivated bacteria. We have engineered a plasmid-based, complement receptor-targeting platform that is readily applicable to live forms of multiple gram-negative bacteria, including, but not limited to, Escherichia coli, Klebsiella pneumoniae, and Francisella tularensis. Using F. tularensis as a model, we find that targeted bacteria show increased binding and uptake by macrophages, which coincides with increased p38 and p65 phosphorylation. Mice vaccinated with targeted bacteria produce higher titers of specific antibody that recognizes a greater diversity of bacterial antigens. Following challenge with homologous or heterologous isolates, these mice exhibited less weight loss and/or accelerated weight recovery as compared to counterparts vaccinated with non-targeted immunogens. Collectively, these findings provide proof-of-concept for plasmid-based, complement receptor-targeting of live gram-negative bacteria.Keywords:
Heterologous
Gram-Negative Bacteria
At present, development of effective vaccines of new generation is an actual problem, in particular concerning the tularemia causative agent.It determines the need to search antigen determinants with high immunogenic activity.Some authors demonstrate that outer membrane proteins of Francisella tularensis possess immunological activity.This fact gave occasion to isolation and comprehensive study of Francisella tularensis cellular envelopes as a perspective component in vaccine engineering.Toxicity and immunogenic activity of Cell Envelopes (CE) of Francisella tularensis different subspecies is complex studied.It is shown that these preparations in doses 6.3; 19.0; 57.0 and 100 µg fail to possess toxicity.The immunizing dose (95 µg) of CE Francisella tularensis subsp.holarctica 306, Francisella tularensis subsp.mediasiatica А-61 and Francisella tularensis subsp.tularensis B-399 A-Cole protecting white mice against experimental tularemia infection was determined.It is experimentally demonstrated that 83 % animals survive only after using CE Francisella tularensis subsp.mediasiatica А-61.It is shown that F. tularensis CE preparations influence on activation of T and B lympho-cytes of white mice blood cells.New data concerning the possibility of F. tularensis antigen preparation application for increase of experimental animal resistance to F. tularensis are obtained.On the basis of the findings there is need for the further detailed investigation of immunogenic properties of CE F. tularensis subsp.holarctica 306, F. tularensis subsp.
Tularemia
Francisella
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ABSTRACT The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The Δ pdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, Δ iglG and Δ iglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis .
Tularemia
Francisella
Pathogenicity island
Intracellular parasite
Pyroptosis
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ABSTRACT We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis . Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida , respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida , and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica . Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival.
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Francisella
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Immunogenic properties of cell wall (CW) preparations of Francisella tularensis four subspecies are investigated. It is shown that the preparations from F. tularensis urea lysates are not toxic for experimental animals. Besides, CW of F. tularensis А-61 subsp. mediasiatica and F. tularensis B-399 A-Cole subsp. tularensis possess immunogenic activity in experimental tularemia caused by F. tularensis 306 subsp. holarctica.
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Francisella
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Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine.We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose-6-phosphate isomerase (pgi) of primary metabolic pathway to increase glucose-6-phosphate pool inside the host. Disruption was carried out by lambda Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2-deoxy-scyllo-inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Delta pgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine.Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis.This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5- and 4,6-disubtituted route of DOS-containing aminoglycosides.
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Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth.
Proteome
Tularemia
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Attenuated vaccine
Francisella
Pathogenicity island
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Francisella tularensis, the causative agent of tularemia, is a HHS Tier 1 select agent. Tularemia is the most commonly reported human and animal infection caused by a bacterial select agent in the ...
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Francisella
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Zoonotic disease
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Escherichia coli has long been the primary expression system for the synthesis of heterologous proteins, but its utility is limited because many heterologous proteins are either prone to be degraded by the cellular proteases or to accumulate in insoluble form, typically as inclusion bodies. This paper reviews recent advances in the strategies for expression soluble heterologous proteins in Escherichia coli so as to improve the recovery yield of heterologous gene products in a biologically active form.
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Heterologous expression
Inclusion bodies
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The ability of Francisella tularensis to replicate in macrophages has led many investigators to assume that it resides primarily intracellularly in the blood of mammalian hosts. We have found this supposition to be untrue. In almost all cases, the majority of F. tularensis recovered from the blood of infected mice was in plasma rather than leukocytes. This distribution was observed irrespective of size of inoculum, route of inoculation, time after inoculation, or virulence of the infecting strain. Our findings yield new insight into the pathogenesis of tularemia and may have important ramifications in the search for anti- Francisella therapies.
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Francisella
Pathogenesis
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Survey of <i>Francisella tularensis</i> in Wild Animals in Japan in Areas Where Tularemia is Endemic
Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.
Tularemia
Zoonotic disease
Francisella
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