RESUMEN
Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein of Neisseria meningitidis and a component of the Bexsero vaccine. NHBA is characterized by the presence of a highly conserved Arg-rich region involved in binding to heparin and heparan sulphate proteoglycans present on the surface of host epithelial cells, suggesting a possible role of NHBA during N. meningitidis colonization. NHBA has been shown to be cleaved by the meningococcal protease NalP and by human lactoferrin (hLF), a host protease presents in different body fluids (saliva, breast milk and serum). Cleavage occurs upstream or downstream the Arg-rich region. Since the human nasopharynx is the only known reservoir of infection, we further investigated the susceptibility of NHBA to human proteases present in the saliva to assess whether proteolytic cleavage could happen during the initial steps of colonization. Here we show that human saliva proteolytically cleaves NHBA, and identified human kallikrein 1 (hK1), a serine protease, as responsible for this cleavage. Kallikrein-related peptidases (KLKs) have a distinct domain structure and exist as a family of 15 genes which are differentially expressed in many tissues and in the central nervous system. They are present in plasma, lymph, urine, saliva, pancreatic juices, and other body fluids where they catalyze the proteolysis of several human proteins. Here we report the characterization of NHBA cleavage by the tissue kallikrein, expressed in saliva and the identification of the cleavage site on NHBA both, as recombinant protein or as native protein, when expressed on live bacteria. Overall, these findings provide new insights on NHBA as target of host proteases, highlights thepotential role of NHBA in the Neisseria meningitidis nasopharyngeal colonization, and of kallikrein as a defensive agent against meningococcal infection.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Infecciones Meningocócicas/microbiología , Proteolisis , Saliva/química , Calicreínas de Tejido/metabolismo , Secuencia de Aminoácidos , Humanos , Neisseria meningitidis/fisiología , ProteómicaRESUMEN
During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.
Asunto(s)
Interacciones Huésped-Patógeno , Neisseria meningitidis/fisiología , Análisis por Matrices de Proteínas/métodos , Staphylococcus aureus/fisiología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Sitios de Unión , Células CHO , Complemento C1q/metabolismo , Cricetulus , Humanos , Unión Proteica , Proteínas Recombinantes/metabolismo , Receptores Depuradores de Clase E/metabolismoRESUMEN
Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.
Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/inmunología , Proteínas Hemolisinas/química , Proteínas Hemolisinas/inmunología , Imitación Molecular , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus , Proteína ADAM10/metabolismo , Animales , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Línea Celular , Citotoxinas , Epítopos/inmunología , Escherichia coli/genética , Proteínas Hemolisinas/administración & dosificación , Proteínas Hemolisinas/genética , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Modelos Moleculares , Ingeniería de Proteínas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , VacunaciónRESUMEN
Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/química , Receptor Toll-Like 7/química , Absceso/patología , Inmunidad Adaptativa , Animales , Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Antígenos/inmunología , Humanos , Ratones , Modelos Animales , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus , Células TH1/inmunologíaRESUMEN
Neisseria meningitidis adhesin A (NadA) is a meningococcus surface protein thought to assist in the adhesion of the bacterium to host cells. We have previously shown that NadA also promotes bacterial internalization in a heterologous expression system. Here we have used the soluble recombinant NadA (rNadA) lacking the membrane anchor region to characterize its internalization route in Chang epithelial cells. Added to the culture medium, rNadA internalizes through a PI3K-dependent endocytosis process not mediated by the canonical clathrin or caveolin scaffolds, but instead follows an ARF6-regulated recycling pathway previously described for MHC-I. The intracellular pool of rNadA reaches a steady state level within one hour of incubation and colocalizes in endocytic vesicles with MHC-I and with the extracellularly labeled chaperone Hsp90. Treatment with membrane permeated and impermeable Hsp90 inhibitors 17-AAG and FITC-GA respectively, lead to intracellular accumulation of rNadA, strongly suggesting that the extracellular secreted pool of the chaperone is involved in rNadA intracellular trafficking. A significant number of intracellular vesicles containing rNadA recruit Rab11, a small GTPase associated to recycling endosomes, but do not contain transferrin receptor (TfR). Interestingly, cell treatment with Hsp90 inhibitors, including the membrane-impermeable FITC-GA, abolished Rab11-rNadA colocalization but do not interfere with Rab11-TfR colocalization. Collectively, these results are consistent with a model whereby rNadA internalizes into human epithelial cells hijacking the recycling endosome pathway and recycle back to the surface of the cell via an ARF6-dependent, Rab11 associated and Hsp90-regulated mechanism. The present study addresses for the first time a meningoccoccal adhesin mechanism of endocytosis and suggests a possible entry pathway engaged by N. meningitidis in primary infection of human epithelial cells.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Adhesinas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Factor 6 de Ribosilación del ADP , Línea Celular , Humanos , Espacio Intracelular , Neisseria meningitidis/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Unión Proteica , Transporte de Proteínas , Proteolisis , Proteínas Recombinantes , TemperaturaRESUMEN
Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. We provide evidence that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes.
Asunto(s)
Axones/fisiología , Proteínas de la Membrana/química , Factores de Crecimiento Nervioso/química , Receptores de Superficie Celular/química , Proteínas Supresoras de Tumor/química , Animales , Movimiento Celular , Receptor DCC , Fibronectinas/química , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/ultraestructura , Receptores de Netrina , Netrina-1 , Neuronas/fisiología , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/ultraestructura , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/ultraestructuraRESUMEN
Group A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of the spy0269 gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interact in vitro with the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cells in vitro and that Lactococcus lactis expressing Spy0269 on its cell surface could adhere to mammalian cells in vitro and to mice nasal mucosa in vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (Streptococcus pyogenes Adhesion and Division protein).
Asunto(s)
Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/inmunología , Streptococcus pyogenes/metabolismo , Antígenos Bacterianos , Proteínas Bacterianas/genética , Línea Celular , Clonación Molecular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/microbiología , Eliminación de Gen , Humanos , Lactococcus lactis/metabolismo , Unión Proteica , Streptococcus pyogenes/citología , Streptococcus pyogenes/genéticaRESUMEN
In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.
Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas/química , Proteoma/química , Staphylococcus aureus/metabolismo , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Minería de Datos , Ratones , Ratones Endogámicos , Proteoma/genética , Proteoma/metabolismo , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/inmunologíaRESUMEN
Staphylococcus aureus is a human pathogen causing globally significant morbidity and mortality. The development of antibiotic resistance in S. aureus highlights the need for a preventive vaccine. In the present paper we explore the structure and function of FhuD2 (ferric-hydroxamate uptake D2), a staphylococcal surface lipoprotein mediating iron uptake during invasive infection, recently described as a promising vaccine candidate. Differential scanning fluorimetry and calorimetry studies revealed that FhuD2 is stabilized by hydroxamate siderophores. The FhuD2-ferrichrome interaction was of nanomolar affinity in surface plasmon resonance experiments and fully iron(III)-dependent. We determined the X-ray crystallographic structure of ligand-bound FhuD2 at 1.9 Å (1 Å=0.1 nm) resolution, revealing the bilobate fold of class III SBPs (solute-binding proteins). The ligand, ferrichrome, occupies a cleft between the FhuD2 N- and C-terminal lobes. Many FhuD2-siderophore interactions enable the specific recognition of ferrichrome. Biochemical data suggest that FhuD2 does not undergo significant conformational changes upon siderophore binding, supporting the hypothesis that the ligand-bound complex is essential for receptor engagement and uptake. Finally, immunizations with FhuD2 alone or FhuD2 formulated with hydroxamate siderophores were equally protective in a murine staphylococcal infection model, confirming the suitability and efficacy of apo-FhuD2 as a protective antigen, and suggesting that other class III SBPs might also be exploited as vaccine candidates.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Proteínas de Unión Periplasmáticas/química , Staphylococcus aureus/metabolismo , Factores de Virulencia/química , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Compuestos Férricos/metabolismo , Ferricromo/metabolismo , Genes Bacterianos , Humanos , Ácidos Hidroxámicos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Proteínas de Transporte de Membrana/metabolismo , Ratones , Modelos Moleculares , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/inmunología , Proteínas de Unión Periplasmáticas/metabolismo , Estabilidad Proteica , Sideróforos/metabolismo , Vacunas Estafilocócicas/química , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidad , Electricidad Estática , Transferrina/metabolismo , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
The factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis that confers to the bacterium the ability to resist killing by human serum. The determination of its three-dimensional structure revealed that the carboxyl terminus of the protein folds into an eight-stranded ß barrel. The structural similarity of this part of the protein to lipocalins provided the rationale for exploring the ability of fHbp to bind siderophores. We found that fHbp was able to bind in vitro siderophores belonging to the cathecolate family and mapped the interaction site by nuclear magnetic resonance. Our results indicated that the enterobactin binding site was distinct from the site involved in binding to human factor H and stimulates new hypotheses about possible multiple activities of fHbp.
Asunto(s)
Proteínas Bacterianas/metabolismo , Factor H de Complemento/metabolismo , Neisseria meningitidis/metabolismo , Sideróforos/metabolismo , Electroforesis en Gel de Poliacrilamida , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and ß- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and ß- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Arginina/metabolismo , Toxinas Biológicas/metabolismo , ADP Ribosa Transferasas/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Línea Celular , Toxina del Cólera/metabolismo , Humanos , Datos de Secuencia Molecular , NAD+ Nucleosidasa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , alfa-Defensinas/químicaRESUMEN
Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.
Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Fimbrias Bacterianas/metabolismo , Streptococcus agalactiae/citología , Streptococcus agalactiae/enzimología , Secuencia de Aminoácidos , Aminoaciltransferasas/química , Animales , Proteínas Bacterianas/química , Biocatálisis , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Cisteína Endopeptidasas/química , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Hidrolasas/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Péptidos/química , Péptidos/metabolismo , Peptidil Transferasas/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fracciones Subcelulares/metabolismo , Especificidad por SustratoRESUMEN
The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.
Asunto(s)
Antígenos Bacterianos/inmunología , Diseño de Fármacos , Inmunidad/inmunología , Neisseria meningitidis/inmunología , Animales , Antibacterianos/farmacología , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Cristalografía por Rayos X , Humanos , Inmunidad/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/inmunología , Mutación/genética , Neisseria meningitidis/efectos de los fármacos , Ingeniería de Proteínas , Estructura Secundaria de ProteínaRESUMEN
Vaccine research and development are experiencing a renaissance of interest from the global scientific community. There are four major reasons for this: (1) the lack of efficacious treatment for many devastating infections; (2) the emergence of multidrug resistant bacteria; (3) the need for improving the safety of the more traditional licensed vaccines; and finally, (4) the great promise for innovative vaccine design and research with convergence of omics sciences, such as genomics, proteomics, immunomics, and vaccinology. Our first project based on omics was initiated in 2000 and was termed reverse vaccinology. At that time, antigen identification was mainly based on bioinformatic analysis of a singular genome. Since then, omics-guided approaches have been applied to its full potential in several proof-of-concept studies in the industry, with the first reverse vaccinology-derived vaccine now in late stage clinical trials and several vaccines developed by omics in preclinical studies. In the meantime, vaccine discovery and development has been further improved with the support of proteomics, functional genomics, comparative genomics, structural biology, and most recently vaccinomics. We illustrate in this review how omics biotechnologies and integrative biology are expected to accelerate the identification of vaccine candidates against difficult pathogens for which traditional vaccine development has thus far been failing, and how research will provide safer vaccines and improved formulations for immunocompromised patients in the near future. Finally, we present a discussion to situate omics-guided rational vaccine design in the broader context of global public health and how it can benefit citizens in both developed and developing countries.
Asunto(s)
Salud Global , Vacunas , Adyuvantes Inmunológicos/uso terapéutico , Animales , Antígenos/genética , Antígenos/metabolismo , Investigación Biomédica/tendencias , Industria Farmacéutica/legislación & jurisprudencia , Perfilación de la Expresión Génica , Genómica , Humanos , Modelos Biológicos , Modelos Moleculares , Proteómica , Vacunas/inmunologíaRESUMEN
SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. In this study, we investigated the biochemical features and maturation process of SpyCEP, starting from a recombinant form of the protease expressed and purified from Escherichia coli. We show that active recombinant SpyCEP differs from other bacterial proteases in that it is constituted by 2 noncovalently linked fragments derived from autocatalytic processing, an N-terminal fragment of 210 aa bearing one of the 3 catalytic triad residues, and a 1369-residue C-terminal polypeptide containing the remaining 2 catalytic amino acids. The same type of organization is present in the enzyme obtained from S. pyogenes. Furthermore, N-terminal SpyCEP is not involved in the folding of the mature enzyme. The 2 protease fragments were separately expressed in E. coli as soluble polypeptides that, when combined, reconstituted a fully active enzyme complex. Therefore, SpyCEP appears to possess a completely new structural architecture that has not been described so far for other microbial proteases.
Asunto(s)
Fragmentos de Péptidos/química , Péptido Hidrolasas/química , Clonación Molecular , Humanos , Proteínas Recombinantes , Streptococcus pyogenesRESUMEN
Microbial pathogen entry and survival in the host is mediated by a network of molecular interactions between the two partners, which has been the subject of many research efforts. A complex picture is emerging in which host-pathogen crosstalk involves a high number of proteins, often with redundant functions. In the present study, we investigated the potential of protein microarrays to simultaneously scan interactions between surface proteins from two main human streptococcal pathogens, Streptococcus pyogenes and Streptococcus agalactiae, and three human ligands, fibronectin, fibrinogen, and C4 binding protein, known to play an important role in streptococcal pathogenesis. By using this technology, we confirmed interactions described in the literature and detected a novel set of streptococcal proteins with binding capacities for the human ligands. The observations were validated by Western blot and ELISA techniques. Three of the newly identified proteins were isoforms of a group B streptococcus-secreted component named Fib and displayed differential binding capacities for fibronectin, fibrinogen, and C4BP. The protein regions involved in the interaction with each ligand were identified by constructing fragments of one of the Fib variants. The approach proved valuable for the acquisition of novel insights into the complex network of protein-protein interactions occurring during microbial infection.
Asunto(s)
Proteínas Bacterianas/análisis , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Interacciones Huésped-Patógeno , Streptococcus/química , Proteínas Bacterianas/metabolismo , Proteína de Unión al Complemento C4b , Humanos , Análisis por Matrices de Proteínas/métodos , Unión Proteica , Streptococcus agalactiae/química , Streptococcus pyogenes/químicaRESUMEN
Group B Streptococcus (GBS) is a multiserotype bacterial pathogen representing a major cause of life-threatening infections in newborns. To develop a broadly protective vaccine, we analyzed the genome sequences of eight GBS isolates and cloned and tested 312 surface proteins as vaccines. Four proteins elicited protection in mice, and their combination proved highly protective against a large panel of strains, including all circulating serotypes. Protection also correlated with antigen accessibility on the bacterial surface and with the induction of opsonophagocytic antibodies. Multigenome analysis and screening described here represent a powerful strategy for identifying potential vaccine candidates against highly variable pathogens.
Asunto(s)
Antígenos Bacterianos/inmunología , Genoma Bacteriano , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/inmunología , Streptococcus agalactiae/genética , Streptococcus agalactiae/inmunología , Animales , Animales Recién Nacidos , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/genética , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Proteínas Bacterianas/inmunología , Biología Computacional , Femenino , Humanos , Inmunidad Materno-Adquirida , Ratones , Neutrófilos/inmunología , Proteínas Opsoninas , Fagocitosis , Serotipificación , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/clasificación , VacunaciónRESUMEN
The semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. Their distinctive feature is a conserved 500 amino acid semaphorin domain, a ligand-receptor interaction module also present in plexins and scatter-factor receptors. We report the crystal structure of a secreted 65 kDa form of Semaphorin-3A (Sema3A), containing the full semaphorin domain. Unexpectedly, the semaphorin fold is a variation of the beta propeller topology. Analysis of the Sema3A structure and structure-based mutagenesis data identify the neuropilin binding site and suggest a potential plexin interaction site. Based on the structure, we present a model for the initiation of semaphorin signaling and discuss potential similarities with the signaling mechanisms of other beta propeller cell surface receptors, such as integrins and the LDL receptor.