RESUMEN
Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies; however, the domains important for protective response are still unknown. In order to further investigate its immunogenic properties, we have characterized the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3 was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity was restored when using chimeric antibodies in which the variable regions of the murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules will enable future investigations of complement-mediated antibody functionality independently of the Fc-mediated differences in complement activation.
Asunto(s)
Adhesinas Bacterianas/inmunología , Anticuerpos Antibacterianos/inmunología , Epítopos/inmunología , Infecciones Meningocócicas/inmunología , Vacunas Meningococicas/inmunología , Neisseria/inmunología , Animales , Medición de Intercambio de Deuterio/métodos , Mapeo Epitopo/métodos , Humanos , RatonesRESUMEN
Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.
Asunto(s)
Adhesinas Bacterianas/inmunología , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Mapeo Epitopo/métodos , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Neisseria meningitidis Serogrupo B/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Multimerización de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , TemperaturaRESUMEN
SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.
Asunto(s)
Anticuerpos Antibacterianos/farmacología , Formación de Anticuerpos , Infecciones por Escherichia coli , Proteínas de Escherichia coli/inmunología , Polisacárido Liasas/antagonistas & inhibidores , Factores de Virulencia/inmunología , Animales , Animales no Consanguíneos , Anticuerpos Antibacterianos/metabolismo , Células Cultivadas , Escherichia coli Enteropatógena/crecimiento & desarrollo , Escherichia coli Enteropatógena/inmunología , Escherichia coli Enteropatógena/metabolismo , Activación Enzimática/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Femenino , Intestinos/microbiología , Ratones , Ratones Endogámicos CBA , Polisacárido Liasas/inmunología , Polisacárido Liasas/metabolismo , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/metabolismoRESUMEN
Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of â¼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Epítopos/inmunología , Vacunas Meningococicas/inmunología , Neisseria meningitidis/inmunología , Neisseria meningitidis/patogenicidad , Factores de Virulencia/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Técnicas de Visualización de Superficie Celular , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Mapeo Epitopo , Epítopos/química , Espectrometría de Masas , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Modelos Moleculares , Péptidos/química , Péptidos/inmunología , Unión Proteica/inmunología , Resonancia por Plasmón de Superficie , Factores de Virulencia/químicaRESUMEN
Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.
Asunto(s)
Antígenos Bacterianos/genética , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/genética , Escherichia coli/genética , Genoma Bacteriano/genética , Meningitis por Escherichia coli/microbiología , Animales , Secuencia de Bases , Biología Computacional , Escherichia coli/inmunología , Finlandia , Islas Genómicas/genética , Humanos , Ratones , Datos de Secuencia Molecular , Vías Secretoras/genética , Análisis de Secuencia de ADNRESUMEN
UNLABELLED: In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site. IMPORTANCE: Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Inmunoglobulina A Secretora/metabolismo , Activación Neutrófila , Factores de Virulencia/metabolismo , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Técnicas de Inactivación de Genes , Humanos , Evasión Inmune , Ratones , Modelos Moleculares , Conformación Proteica , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
UNLABELLED: The increasing antibiotic resistance of pathogenic Escherichia coli species and the absence of a pan-protective vaccine pose major health concerns. We recently identified, by subtractive reverse vaccinology, nine Escherichia coli antigens that protect mice from sepsis. In this study, we characterized one of them, ECOK1_0290, named FdeC (factor adherence E. coli) for its ability to mediate E. coli adhesion to mammalian cells and extracellular matrix. This adhesive propensity was consistent with the X-ray structure of one of the FdeC domains that shows a striking structural homology to Yersinia pseudotuberculosis invasin and enteropathogenic E. coli intimin. Confocal imaging analysis revealed that expression of FdeC on the bacterial surface is triggered by interaction of E. coli with host cells. This phenotype was also observed in bladder tissue sections derived from mice infected with an extraintestinal strain. Indeed, we observed that FdeC contributes to colonization of the bladder and kidney, with the wild-type strain outcompeting the fdeC mutant in cochallenge experiments. Finally, intranasal mucosal immunization with recombinant FdeC significantly reduced kidney colonization in mice challenged transurethrally with uropathogenic E. coli, supporting a role for FdeC in urinary tract infections. IMPORTANCE: Pathogenic Escherichia coli strains are involved in a diverse spectrum of diseases, including intestinal and extraintestinal infections (urinary tract infections and sepsis). The absence of a broadly protective vaccine against all these E. coli strains is a major problem for modern society due to high costs to health care systems. Here, we describe the structural and functional properties of a recently reported protective antigen, named FdeC, and elucidated its putative role during extraintestinal pathogenic E. coli infection by using both in vitro and in vivo infection models. The conservation of FdeC among strains of different E. coli pathotypes highlights its potential as a component of a broadly protective vaccine against extraintestinal and intestinal E. coli infections.