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1.
Mol Cell Proteomics ; 14(8): 2138-49, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26018414

RESUMEN

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.


Asunto(s)
Proteínas Bacterianas/metabolismo , Lipoproteínas/metabolismo , Microdominios de Membrana/metabolismo , Streptococcus pyogenes/metabolismo , Medios de Cultivo , Células HEK293 , Humanos , Microdominios de Membrana/efectos de los fármacos , Peso Molecular , Mutación/genética , Penicilinas/farmacología , Programas Informáticos , Streptococcus pyogenes/efectos de los fármacos , Receptor Toll-Like 2/metabolismo
2.
Mol Cell Proteomics ; 11(6): M111.015693, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22286755

RESUMEN

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.


Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/administración & dosificación , Infecciones Estreptocócicas/prevención & control , Streptococcus pyogenes/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Análisis por Conglomerados , Femenino , Citometría de Flujo , Hemólisis , Humanos , Ratones , Faringitis/sangre , Faringitis/inmunología , Faringitis/microbiología , Análisis por Matrices de Proteínas , Proteoma/inmunología , Proteoma/metabolismo , Ovinos , Infecciones Estreptocócicas/sangre , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/metabolismo , Vacunación
3.
J Biol Chem ; 285(10): 7517-24, 2010 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-20048164

RESUMEN

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading frame spr0075 of the strain R6 genome. This locus encodes for an approximately 120-kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and six repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, because fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C-terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, whereas sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggest that, by directly interacting with fibronectin, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Células Epiteliales/microbiología , Fibronectinas/metabolismo , Plasminógeno/metabolismo , Streptococcus pneumoniae/metabolismo , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Células Epiteliales/citología , Humanos , Ratones , Microesferas , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Infecciones Neumocócicas/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
4.
Vaccine ; 28(20): 3609-16, 2010 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-20079873

RESUMEN

Pili of gram-positive bacteria are key virulence factors and their subunits are considered excellent vaccine candidates. Streptococcus suis is an emerging zoonotic agent that can cause epidemics of life-threatening infections in humans, but the functional role or immunoprotective potential of its pilus components have not been studied yet. Using a selective proteomics approach, we have identified a surface protein of serotype 2 S. suis showing features of an ancillary pilus subunit, as evidenced by bioinformatics analysis, immunoblot and immunoelectron microscopy. Immunization with recombinant fragments of this protein, designated herein as PAPI-2b, markedly protected mice from systemic S. suis infection.


Asunto(s)
Proteínas Fimbrias/inmunología , Fimbrias Bacterianas/inmunología , Proteómica , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/inmunología , Streptococcus suis/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Biología Computacional , Femenino , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus suis/inmunología
5.
J Proteomics ; 73(12): 2365-9, 2010 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-20656083

RESUMEN

Streptococcus suis serotype 2 is a major Gram-positive swine pathogen, causing also zoonoses. We describe here the immunoprotective activity in an in vivo animal model of a serotype-2 cell wall protein, designated Sat, which was identified by a previously validated proteomics approach consisting of the protease digestion of live bacteria and the selective recovery of exposed domains, followed by LC/MS/MS analysis. Increased survival rate (80%) and decreased bacterial burden were observed in mice immunized with a recombinant Sat fragment, suggesting that this protein is a potential vaccine candidate against serotype-2 infection.


Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas de la Membrana/inmunología , Infecciones Estreptocócicas/veterinaria , Vacunas Estreptocócicas/uso terapéutico , Enfermedades de los Porcinos/prevención & control , Animales , Proteínas Bacterianas/uso terapéutico , Proteínas de la Membrana/uso terapéutico , Ratones , Proteómica , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/inmunología , Streptococcus suis/inmunología , Porcinos
6.
Peptides ; 30(10): 1936-9, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19631246

RESUMEN

Brucella melitensis and Brucella abortus are responsible for brucellosis in bovine and ovine species and for Malta fever in humans. The lipopolysaccharide (LPS) of Brucella is an important virulence factor and can elicit protective antibodies. Because of their potential importance in vaccine design and in serological diagnosis, we developed peptides mimicking the antigenic properties of distinctive antigenic determinants of Brucella LPS. These peptides were selected from several phage display random peptide libraries for their ability to bind monoclonal antibodies directed against the A- or C-type epitopes of Brucella LPS. Plasmids encoding for two of the isolated peptides induced, after DNA immunization, LPS-specific antibody responses. Although these responses were only moderate in extent, these data further suggest the feasibility of using peptide mimics of carbohydrate epitopes as immunogens, a property which may be useful in the design of novel anti-Brucella vaccines.


Asunto(s)
Antígenos Bacterianos/inmunología , Brucella , Epítopos/inmunología , Lipopolisacáridos/inmunología , Imitación Molecular , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Brucella/química , Brucella/inmunología , Vacuna contra la Brucelosis/genética , Vacuna contra la Brucelosis/inmunología , Bovinos , Epítopos/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/genética , Péptidos/inmunología , Ovinos
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