RESUMEN
Background: The multicomponent meningococcal serogroup B vaccine (4CMenB) is an outer membrane vesicle and recombinant protein-based vaccine licensed to protect against serogroup B meningococcal disease. It remains unknown whether this vaccine will prevent carriage or transmission, key aspects in long-term vaccine success and disease eradication. Methods: Using a "humanized" transgenic mouse model of nasal colonization, we took a systematic approach to estimate the potential for carriage prevention against antigenically diverse Neisseria meningitidis strains and to compare this protection to an invasive meningococcal disease challenge model. Results: The 4CMenB vaccine prevented morbidity and mortality after lethal invasive doses of all meningococcal strains tested. Immunization effectively prevented carriage with only 1 of 4 single antigen-matched strains but reduced or prevented nasal colonization by all 4 isolates with multiple cross-reacting antigens. Each immunized mouse had substantial immunoglobulin G targeting the challenge strains, indicating that antibody correlates with protection against sepsis but not nasal carriage. Conclusions: Immunization with the 4CMenB vaccine elicits a robust humoral response that correlates with protection against invasive challenge but not with prevention of asymptomatic colonization. This suggests that widespread use of this vaccine will reduce morbidity and mortality rates in immunized individuals, with the potential to contribute to herd protection against a subset of strains.
Asunto(s)
Portador Sano/inmunología , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/inmunología , Cavidad Nasal/microbiología , Neisseria meningitidis Serogrupo B/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Neisseria meningitidis/metabolismo , Factores de Virulencia/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Elementos Transponibles de ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Prueba de Complementación Genética , Pruebas Genéticas , Lipoproteínas/genética , Proteínas de la Membrana/genética , Mutagénesis Insercional , Neisseria meningitidis/genética , Transporte de Proteínas , Factores de Virulencia/genéticaRESUMEN
Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.
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Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Neisseria meningitidis/metabolismo , Zinc/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Conformación Proteica , Sepsis/microbiologíaRESUMEN
Host recognition of pathogen-associated molecular patterns (PAMPs) initiates an innate immune response that is critical for pathogen elimination and engagement of adaptive immunity. Here we show that mammalian cells can detect and respond to the bacterial-derived monosaccharide heptose-1,7-bisphosphate (HBP). A metabolic intermediate in lipopolysaccharide biosynthesis, HBP is highly conserved in Gram-negative bacteria, yet absent from eukaryotic cells. Detection of HBP within the host cytosol activated the nuclear facto κB pathway in vitro and induced innate and adaptive immune responses in vivo. Moreover, we used a genome-wide RNA interference screen to uncover an innate immune signaling axis, mediated by phosphorylation-dependent oligomerization of the TRAF-interacting protein with forkhead-associated domain (TIFA) that is triggered by HBP. Thus, HBP is a PAMP that activates TIFA-dependent immunity to Gram-negative bacteria.
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Proteínas Adaptadoras Transductoras de Señales/inmunología , Bacterias Gramnegativas/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Fosfatos de Azúcar/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Burkholderia/inmunología , Citosol/química , Citosol/inmunología , Escherichia coli/inmunología , Flagelina/inmunología , Pruebas Genéticas , Bacterias Gramnegativas/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Células Jurkat , FN-kappa B/inmunología , Neisseria gonorrhoeae/inmunología , Neisseria meningitidis/inmunología , Interferencia de ARN , Fosfatos de Azúcar/análisis , Fosfatos de Azúcar/metabolismo , Factor 6 Asociado a Receptor de TNF/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
BACKGROUND: The effect of protein-based meningococcal vaccines on prevention of nasopharyngeal colonization has been difficult to investigate experimentally because a reliable animal colonization model did not exist. METHODS: Human CEACAM1 transgenic mice, which can be colonized by meningococci, were immunized IP with one of two meningococcal native outer membrane vesicle (NOMV) vaccines prepared from mutants with attenuated endotoxin (lpxL1 knockout) and over-expressed sub-family B Factor H-binding proteins (FHbp). Animals were challenged intranasally two weeks after the third dose with wild-type strain H44/76, or were treated IP with anti-NOMV serum before and during the bacterial challenge. RESULTS: The NOMV-1 vaccine, prepared from the serogroup B H44/76 mutant, elicited â¼40-fold higher serum bactericidal antibody titers against the wild-type H44/76 challenge strain than the NOMV-2 vaccine prepared from a heterologous serogroup W mutant strain with different PorA and FHbp amino acid sequence variants. Compared to aluminum hydroxide-immunized control mice, the efficacy for prevention of any H44/76 colonization was 93% (95% confidence interval, 52-99, P<0.0001) for the NOMV-1 vaccine, and 19% (-3-36, P=0.23) for NOMV-2. NOMV-2-vaccinated mice had a 5.6-fold decrease in geometric mean CFU of bacteria per animal in tracheal washes compared to control mice (P=0.007). The efficacy of passive administration of serum from NOMV-1-vaccinated mice to immunologically naïve mice against colonization was 44% (17-61; P=0.002). CONCLUSIONS: Both NOMV vaccines protected against meningococcal colonization but there was greater protection by the NOMV-1 vaccine with antigens matched with the challenge strain. Meningococcal vaccines that target protein antigens have potential to decrease colonization.
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Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Meningococicas/inmunología , Nasofaringe/microbiología , Neisseria meningitidis Serogrupo B/inmunología , Neisseria meningitidis/crecimiento & desarrollo , Animales , Antígenos Bacterianos/genética , Antígenos CD/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Moléculas de Adhesión Celular/genética , Endotoxinas/inmunología , Humanos , Inmunización Pasiva , Inyecciones Intraperitoneales , Infecciones Meningocócicas/prevención & control , Ratones , Ratones Transgénicos , Mutación , Neisseria meningitidis/inmunología , Neisseria meningitidis Serogrupo B/crecimiento & desarrollo , Determinación de Anticuerpos Séricos Bactericidas , VacunaciónRESUMEN
Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Despite several studies showing increased susceptibility to secondary bacterial infections following IAV infection, information on the direct effect of S. pneumoniae on IAV in vitro is unknown. This is an important area of investigation as S. pneumoniae is a common commensal of the human upper respiratory tract, present as an important coinfecting pathogen with IAV infection. A recent study showed that S. pneumoniae enhances human metapneumovirus infection in polarized bronchial epithelial cells in vitro. The aim of the current study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication using a standard immunofluorescence assay (IFA). For this study we used four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus. Our results suggested that pretreatment of MDCK cells with 7.5×10(6) colony-forming units (CFUs) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (0.5 to 4 hr). But, pretreatment of cell lines with 7.5×10(5) and lower CFUs of S. pneumoniae had no detectable effect on either the morphology of cells or on the IAV replication. However, unlike in epithelial cell lines, due to influence of secreted host factors the effect of pneumococci on IAV replication may be different during coinfections in vivo in the human upper respiratory tract, and in vitro with primary human polarized bronchial epithelial cells.
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Células Epiteliales/microbiología , Virus de la Influenza A/fisiología , Streptococcus pneumoniae/fisiología , Replicación Viral , Animales , Calibración , Perros , Técnicas In Vitro , Células de Riñón Canino Madin Darby , Microscopía Fluorescente , Streptococcus pneumoniae/clasificaciónRESUMEN
Streptococcus pneumoniae is an important human pathogen that requires carbohydrates for growth. The significance of carbohydrate acquisition is highlighted by the genome encoding more than 27 predicted carbohydrate transporters. It has long been known that about 60% of pneumococci could utilize the fructooligosaccharide inulin as a carbohydrate source, but the mechanism of utilization was unknown. Here we demonstrate that a predicted sucrose utilization locus is actually a fructooligosaccharide utilization locus and imparts the ability of pneumococci to utilize inulin. Genes in strain TIGR4 predicted to encode an ABC transporter (SP_1796-8) and a ß-fructosidase (SP_1795) are required for utilization of several fructooligosaccharides longer than kestose, which consists of two ß(2-1)-linked fructose molecules with a terminal α(1-2)-linked glucose molecule. Similar to other characterized pneumococcal carbohydrate utilization transporter family 1 transporters, growth is dependent on the gene encoding the ATPase MsmK. While the majority of pneumococcal strains encode SP_1796-8 at this genomic location, 19% encode an alternative transporter. Although strains encoding either transporter can utilize short-chain fructooligosaccharides for growth, only strains encoding SP_1796-8 can utilize inulin. Exchange of genes encoding the SP_1796-8 transporter for those encoding the alternative transporter resulted in a TIGR4 strain that could utilize short-chain fructooligosaccharide but not inulin. These data demonstrate that the transporter encoded at this locus determines the ability of the bacteria to utilize long-chain fructooligosaccharides and explains the variation in inulin utilization between pneumococcal strains.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Oligosacáridos/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Transporte Biológico/genética , Metabolismo de los Hidratos de Carbono/genética , Regulación Bacteriana de la Expresión Génica , Inulina/metabolismo , Oligosacáridos/química , Streptococcus pneumoniae/enzimología , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismoRESUMEN
Streptococcus pneumoniae relies exclusively on carbohydrates as a carbon source and devotes 30% of all transport mechanisms to carbohydrate import. Pneumococci utilize at least 32 carbohydrates in vitro. However, some proposed substrates are not human-derived, so it is unclear where they are encountered in the host niche, and other substrates remain unidentified. The majority of transporter loci are conserved, arguing against redundancy and instead for distinct roles during pathogenesis. Despite this, expression and regulation of carbohydrate transporters in vivo remain ill defined. Recent work has also demonstrated that multiple ABC transporters share an ATPase; whether this evolved for genome minimization or for transporter regulation remains unknown. Continued efforts to understand carbohydrate import may reveal novel vaccine and therapeutic targets and increase our understanding of pneumococcal pathogenesis.