RESUMO
We developed a modified flagellar type III secretion apparatus to secrete heterologous polypeptides into the growth medium of Escherichia coli. The secretion was facilitated by fusing the 173-bp untranslated DNA fragment upstream of the gene fliC (encoding flagellin) as well as a transcriptional terminator from fliC, into the gene encoding the polypeptide of interest. The polypeptides secreted into the growth medium at concentrations ranging from 1 to 15 mg/l were from Campylobacter jejuni (262 residues in length), Streptococcus pneumoniae (434 residues), Staphylococcus aureus (115 residues), and N-terminal FliC hybrid proteins, for example, the eukaryotic green fluorescent protein (238 residues). The expressed proteins represented >50% of total secreted protein. Previously reported protein yields from extracellular secretion of foreign proteins in E. coli have been low, approximately 100 microg/l. The strengths of our method are the concentration and purity of the secreted proteins and its versatility with regard to the proteins' length and origin.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/crescimento & desenvolvimento , Flagelos/fisiologia , Flagelina/metabolismo , Peptídeos/metabolismo , Proteínas de Bactérias/genética , Campylobacter jejuni/química , Meios de Cultura/química , DNA Bacteriano , DNA Recombinante , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelos/metabolismo , Flagelina/química , Flagelina/genética , Deleção de Genes , Genes Bacterianos , Proteínas de Fluorescência Verde/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/química , Streptococcus pneumoniae/químicaRESUMO
Immunisation efforts save millions of lives every year, but vaccines hold the potential to deliver even greater health benefits for mankind. Vaccine research and development is highly complex, and it requires concerted public funding efforts to support. In this paper we discuss EU funding priorities and the resulting recent advancements in European vaccine research, and we lay out the EU strategy for aiding promising vaccine candidates to successfully reach the market.
Assuntos
Controle de Doenças Transmissíveis/métodos , União Europeia , Imunização , Vacinas , Vacinas contra a AIDS/economia , Pesquisa Biomédica/economia , Controle de Doenças Transmissíveis/economia , Doenças Transmissíveis , Surtos de Doenças , Descoberta de Drogas/métodos , Humanos , Malária/prevenção & controle , Tuberculose/prevenção & controle , Vacinação , Vacinas/economiaRESUMO
The European Commission (EC) supports a large number of research activities in tuberculosis through the EU Framework Programmes for Research and Development (FP). By utilizing a variety of funding instruments, the EC has established a mixed portfolio of research projects, ranging from small discovery projects to large multidisciplinary consortia with sufficient critical mass to undertake translational and clinical research. The European investments in TB research have generated promising results with new vaccine candidates, drug leads, diagnostic markers and basic research results starting to emerge. In the light of a rapidly changing global research environment it has therefore become timely to review and update the priorities for TB research. To facilitate this process, a high-level conference on "Challenges for the future: research on HIV/AIDS, Malaria and Tuberculosis" was convened in Brussels on November 2008. This review gives an overview of the present portfolio of EC funded TB research, and summarises the conclusions from the conference on future perspectives for TB research in Europe and beyond.
Assuntos
Antituberculosos , Pesquisa Biomédica , União Europeia , Apoio à Pesquisa como Assunto , Tuberculose/prevenção & controle , Antituberculosos/economia , Pesquisa Biomédica/economia , Pesquisa Biomédica/tendências , Congressos como Assunto , Humanos , Apoio à Pesquisa como Assunto/economia , Apoio à Pesquisa como Assunto/tendências , Tuberculose/tratamento farmacológico , Tuberculose/economiaAssuntos
Proteínas de Bactérias , Ativadores de Plasminogênio/metabolismo , Virulência/fisiologia , Yersinia pestis/patogenicidade , Proteínas da Membrana Bacteriana Externa/metabolismo , Linhagem Celular , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Humanos , Peptídeo Hidrolases , Porinas/metabolismoRESUMO
Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.
Assuntos
Adesinas de Escherichia coli/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Diarreia/veterinária , Enterócitos/metabolismo , Vacinas contra Escherichia coli/metabolismo , Escherichia coli/imunologia , Doenças dos Suínos/prevenção & controle , Adesinas de Escherichia coli/metabolismo , Animais , Diarreia/metabolismo , Diarreia/microbiologia , Diarreia/prevenção & controle , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Plantas Geneticamente Modificadas , Sus scrofa , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologia , Nicotiana/metabolismo , Transformação GenéticaRESUMO
The O-antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express beta-barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O-antigen repeats on wild-type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O-antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6-Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla-mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O-antigen prevented PgtE-mediated bacterial adhesion to basement membrane. Substitution of Arg-138 and Arg-171 of the motif for protein binding to lipid A 4'-phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O-antigen sterically prevents recognition of large-molecular-weight substrates. Loss of O-antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.