RESUMO
Sepsis, a leading cause of death worldwide, involves exacerbated proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here, we used a phage display library to identify two peptides in Escherichia coli that interact with host innate receptors. One of these peptides, encoded by the wzxE gene of E. coli K-12, was involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen. Peptide-receptor interactions induced CD16-mediated inhibitory immunoreceptor tyrosine-based activating motif signaling, blocking the production of ROS and bacterial killing. This CD16-mediated inhibitory signaling was abrogated in a WzxE(-/-) mutant of E. coli K-12, restoring the production of ROS and bacterial killing. Taken together, the two novel CD16 ligands identified negatively regulate bacterial killing and inflammation. Our findings may contribute toward the development of new immunotherapies for E. coli-mediated infectious diseases and inflammation.
Assuntos
Antígenos de Bactérias/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Proteínas de Membrana Transportadoras/imunologia , Fagocitose , Receptores de IgG/imunologia , Animais , Escherichia coli , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biblioteca de Peptídeos , Fagócitos/imunologia , Sepse/prevenção & controle , Transdução de SinaisRESUMO
We report the oral vaccination of SWISS mice with an Aro attenuated Salmonella enterica var. Typhimurium vaccine strain expressing the 14-kDa Schistosoma mansoni antigen, Sm14. Bacterial adjuvants, including (i) Lactococcus lactis expressing interleukin-12 (IL-12) and (ii) Lactobacillus delbrueckii UFV-H2b20, were also employed in oral immunization assays. Detection assays to specific IgG and IgA anti-Sm14 antibodies were performed to evaluate humoral immune responses in vaccinated mice. An increase in specific IgG titers was observed; however, no IgA production was detected. The protection levels against schistosomiasis (34.9-49.5%) obtained with all experimental formulations in this work were very similar to values reported by previous studies, which used purified recombinant Sm14 for parenteral vaccination of mice. There was a slight reduction in hepatic granulomas of mice vaccinated with Salmonella. Oogram studies showed diminished numbers of S. mansoni eggs in the intestinal wall of vaccinated mice, but individual female worm fecundity did not seem to be affected by our immunization protocol.
Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Proteínas de Membrana Transportadoras/imunologia , Vacinas contra Salmonella/uso terapêutico , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose/imunologia , Animais , Antígenos de Helmintos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Proteínas de Transporte de Ácido Graxo , Feminino , Proteínas de Helminto/biossíntese , Proteínas de Helminto/efeitos dos fármacos , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Camundongos , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Esquistossomose/prevenção & controleRESUMO
The development of a defined anti-schistosomiasis vaccine would contribute to the current control strategy mainly because immunization provides long-lasting immunity to the disease. Sm14, one of the six Schistosoma mansoni antigens selected by WHO as a candidate to compose a subunit vaccine against schistosomiasis, has been associated with resistance to S. mansoni infection in human beings and is able to induce protection in the murine model. To identify human T cell epitopes in Sm14, we used the TEPITOPE algorithm to select peptides that would most likely bind to several HLA-DR molecules. In this study, three Sm14 epitopes were selected and produced as synthetic peptides. Human T cell responses from schistosomiasis patients living in endemic areas in Brazil were determined by proliferation assay and IL-5 and IFN-gamma measurements. Differential peptide recognition and cytokine production in response to Sm14 epitopes were observed in individuals resistant to S. mansoni infection versus susceptible individuals. Sm14(32-48) and Sm14(53-69) peptides were preferentially recognized by peripheral blood mononuclear cells (PBMCs) of S. mansoni-resistant individuals, and Sm14(53-69) induced significant production of IFN-gamma. Additionally, Sm14(32-48) and Sm14(53-69) were "promiscuous" peptides, since they were able to induce cellular immune responses in individuals carrying 10 and 8, respectively, of the 11 HLA-DR molecules expressed in the studied population. Among Sm14 synthetic peptides tested in this study, we identified Sm14(32-48) and Sm14(53-69) as promising candidates to compose an anti-schistosomiasis vaccine, since they seem to be related to resistance to human schistosomiasis.
Assuntos
Proteínas de Transporte/imunologia , Epitopos de Linfócito T/imunologia , Proteínas de Helminto/imunologia , Proteínas de Membrana Transportadoras/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Linfócitos T/imunologia , Animais , Doenças Endêmicas , Mapeamento de Epitopos , Proteínas de Transporte de Ácido Graxo , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/metabolismo , Antígenos HLA-DR/imunologia , Humanos , Peptídeos/imunologia , Schistosoma mansoni/química , Esquistossomose mansoni/patologia , Esquistossomose mansoni/prevenção & controleRESUMO
Paramyosin and Sm14 are two of the six antigens selected by the World Health Organization as candidates to compose a subunit vaccine against schistosomiasis. Both antigens are recognized by individuals naturally resistant to Schistosoma mansoni infection and induced protective immunity in the murine model. Three Sm14 epitopes and eleven paramyosin epitopes were selected by their ability to bind to different HLA-DR molecules using the TEPITOPE computer program, and these peptides were synthetically produced. The cellular recognition of Sm14 and paramyosin epitopes by peripheral blood mononuclear cells of individuals living in endemic area for schistosomiasis was tested by T cell proliferation assay. Among all Sm14 and paramyosin epitopes studied, Sm14-3 was preferentially recognized by individuals naturally resistant to S. mansoni infection while Para-5 was preferentially recognized by individuals resistant to reinfection. These two peptides represent promising antigens to be used in an experimental vaccine against schistosomiasis, since their preferential recognition by resistant individuals suggest their involvement in the induction of protective immunity.
Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Proteínas de Membrana Transportadoras/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Tropomiosina/imunologia , Vacinas/imunologia , Algoritmos , Animais , Epitopos/imunologia , Proteínas de Transporte de Ácido Graxo , Feminino , Antígenos HLA-DR/imunologia , Proteínas de Helminto/administração & dosagem , Humanos , Leucócitos Mononucleares , Masculino , Proteínas de Membrana Transportadoras/administração & dosagem , Esquistossomose mansoni/prevenção & controle , Linfócitos T/imunologia , Tropomiosina/administração & dosagem , Vacinas/administração & dosagemRESUMO
The sucrose binding protein (SBP) has been implicated as an important component of the sucrose uptake system in plants. SBP-mediated sucrose transport displays unique kinetic features and the protein is not similar to other transport proteins. Here, we report the characterization of a member of the SBP family from soybean [Glycine max (L) Merrill] designated S64 or SBP2. Subcellular fractionation and precipitation by GTP-agarose demonstrated that S64/SBP2 is a membrane-associated protein that exhibits GTP binding activity. Purified recombinant S64/SBP2 protein, expressed as a histidine-tagged protein in Escherichia coli, exhibited nucleotide-binding specificity to guanine nucleotides. The GTP binding site was mapped to an imperfect Walker A type-sequence, Ala279-Leu-Ala-Pro-Thr-Lys-Lys-Ser286, by site-directed mutagenesis. Escherichia coli-produced wild-type protein and a truncated version of the protein containing the putative binding-sequence-bound GTP, although not with the same efficiency. In contrast, replacement of Thr283 and Lys284 residues to Leu and Glu residues prevented GTP binding. The site directed mutant failed to bind GTP but retained the ability to undergo oligomerization andto promote growth of the susy7 yeast strain, deficient inutilizing extracellular sucrose, on medium containing sucrose as the sole carbon source. Our results indicate that GTP binding and sucrose transport by SBP are separable and function independently. The implications of our findings with respect to the function and membrane topology of SBP are discussed.