RESUMO
BACKGROUND: Group B Streptococcus (GBS) is a major cause of invasive disease especially in neonates. In GBS three structurally distinct pilus polymers have been identified as important virulence factors and promising vaccine candidates. The vast majority of Group B Streptococci belonging to the hypervirulent serotype III ST-17 lineage bear pilus types 1 and 2b. The purpose of this study was to investigate the relative contribution of these two pilus types to the pathogenesis of a ST-17 strain. RESULTS: We performed in vivo and in vitro analysis of isogenic knockout mutants derived from the GBS COH1 ST-17 strain deprived of either pilus type 1 or 2b. We compared the two pilus mutants with the wild type strain in a mouse model of invasive disease, in vitro survival in macrophages, and adherence/invasion assays using human brain endothelial and lung epithelial cell lines. Significantly less of the pilus 2b mutant was recovered from the blood, lungs and brain tissue of infected mice compared to the wild-type and pilus 1 mutant strains. Further, while the pilus 2b mutant survived similarly in murine macrophages, it exhibited a lower capacity to adhere and invade human brain epithelial and lung endothelial cell lines. CONCLUSIONS: The data suggest an important role of pilus 2b in mediating GBS infection and host cell interaction of strains belonging to the hypervirulent GBS ST-17 lineage.
Assuntos
Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Meningites Bacterianas/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/patogenicidade , Animais , Aderência Bacteriana , Encéfalo/citologia , Encéfalo/microbiologia , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/microbiologia , Humanos , Pulmão/citologia , Pulmão/microbiologia , Macrófagos/citologia , Macrófagos/microbiologia , Camundongos , Mutação , Streptococcus agalactiae/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Gallbladders from patients affected by both malignant pleural mesothelioma (MPM) and important gallbladder disorders were analyzed to verify the presence of asbestos fibres. Histological thin sections were analyzed by optical microscope and variable pressure scanning electron microscopy coupled with energy dispersive spectroscopy, allowing morphological and chemical characterization of each inorganic phase observed. Fibres of chrysotile and crocidolite, minerals regulated as asbestos, were identified. By immunohistochemical analysis, connective tissue was recognized as the incorporation site. These findings confirm that asbestos fibres can reach the gallbladders of patients with MPM, for whom the development of respiratory diseases confirms asbestos exposure.
Assuntos
Amianto/análise , Doenças da Vesícula Biliar/patologia , Vesícula Biliar/patologia , Neoplasias Pulmonares/complicações , Mesotelioma/complicações , Microscopia Eletrônica de Varredura , Neoplasias Pleurais/complicações , Idoso , Feminino , Histocitoquímica , Humanos , Imuno-Histoquímica , Masculino , Mesotelioma Maligno , Espectrometria por Raios XRESUMO
Immunization of nonpregnant adults could help prevent invasive group B Streptococcus (GBS) infections, but adult immune responses have not been investigated. We defined capsular polysaccharide (CPS) and pilus island (PI) surface antigen distribution and expression and immune responses to GBS infection in nonpregnant adults. Prospective surveillance from 7 hospitals in Houston, Texas, USA, identified 102 adults with GBS bacteremia; 43% had skin/soft tissue infection, 16% bacteremia without focus, and 12% osteomyelitis. CPS-specific IgG was determined by ELISA and pilus-specific IgG by multiplex immunoassay. CPS types were Ia (24.5%), Ib (12.7%), II (9.8%), III (16.7%), IV (13.7%), and V (12.7%); 9.8% were nontypeable by serologic methods. Pili, expressed by 89%, were most often PI-2a. CPS and pilus-specific IgG increased during convalescence among patients with strains expressing CPS or PI. All GBS expressed CPS or PI; 79% expressed both. Increased antibodies to CPS and PI during recovery suggests that GBS bacteremia in adults is potentially vaccine preventable.
Assuntos
Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos/imunologia , Bacteriemia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Polissacarídeos Bacterianos/imunologia , Sorotipagem , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/classificação , Streptococcus agalactiae/genética , Streptococcus agalactiae/isolamento & purificaçãoRESUMO
Neonatal invasive disease caused by group B Streptococcus (GBS) represents a significant public health care concern globally. However, data related to disease burden, serotype distribution, and molecular epidemiology in China and other Asian countries are very few and specifically relative to confined regions. The aim of this study was to investigate the genetic characteristics of GBS isolates recovered from neonates with invasive disease during 2013-2014 at Guangzhou and Changsha hospitals in southern mainland China. We assessed the capsular polysaccharide type, pilus islands (PIs) distribution and hvgA gene presence in a panel of 26 neonatal clinical isolates, of which 8 were recovered from Early Onset Disease and 18 from Late Onset Disease (LOD). Among 26 isolates examined, five serotypes were identified. Type III was the most represented (15 cases), particularly among LOD strains (n = 11), followed by types Ib (n = 5), V (n = 3), Ia (n = 2) and II (n = 1). We performed whole-genome sequencing analysis and antimicrobial susceptibility testing on the 14 serotype III isolates belonging to the hypervirulent Clonal Complex 17 (serotype III-CC17). The presence of PI-2b alone was associated with 13 out of 14 serotype III-CC17 strains. Genome analysis led us to identify two multi-drug resistance gene clusters harbored in two new versions of integrative and conjugative elements (ICEs), carrying five or eight antibiotic resistance genes, respectively. These ICEs replaced the 16 kb-locus that normally contains the PI-1 operon. All isolates harboring the identified ICEs showed multiple resistances to aminoglycoside, macrolide, and tetracycline antibiotic classes. In conclusion, we report the first whole-genome sequence analysis of 14 GBS serotype III-CC17 strains isolated in China, representing the most prevalent lineage causing neonatal invasive disease. The acquisition of newly identified ICEs conferring multiple antibiotic resistance could in part explain the spread of this specific clone among Chinese neonatal isolates and underlines the need for a constant epidemiological surveillance.
RESUMO
BACKGROUND: Group B Streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. A vaccine targeting pregnant women could protect infants through placentally transferred antibodies. The association between GBS maternal antibody concentrations and the risk of neonatal infection has been investigated in US and African populations. Here we studied naturally acquired immunoglobulin G (IgG) responses to GBS capsular polysaccharides (CPS) and pilus proteins in European pregnant women. METHODS: Maternal sera were prospectively collected in 8 EU countries from 473 GBS non-colonized and 984 colonized pregnant women who delivered healthy neonates and from 153 mothers of infants with GBS disease. GBS strains from these colonized women and infected infants were obtained in parallel and their capsular and pilus types were identified by serological and molecular methods. Maternal serum concentrations of IgG anti- Ia, -Ib, -III and -V polysaccharides and anti-BP-1, -AP1-2a and -BP-2b pilus proteins were determined by enzyme-linked immunosorbent assay. Antibody functional activity was quantified by Opsonophagocytic Killing Assay. RESULTS: Antibody levels against CPS and pilus proteins were significantly higher in GBS colonized women delivering healthy babies than in mothers of neonates with GBS disease or non-colonized women. Moreover, maternal anti-capsular IgG concentrations showed a significant correlation with functional titers measured by Opsonophagocytic Killing Assay. CONCLUSIONS: Maternal anti-capsular IgG concentrations above 1 µg/mL mediated GBS killing in vitro and were predicted to respectively reduce by 81% (95% confidence interval, 40%-100%) and 78% (45%-100%) the risk of GBS Ia and III early-onset disease in Europe.
Assuntos
Anticorpos Antibacterianos/sangue , Fímbrias Bacterianas/imunologia , Imunidade Materno-Adquirida , Polissacarídeos Bacterianos/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/imunologia , Europa (Continente)/epidemiologia , Feminino , Humanos , Imunoglobulina G/sangue , Gravidez , Estudos Prospectivos , Infecções Estreptocócicas/epidemiologiaRESUMO
Streptococcus agalactiae (Group B Streptococcus, GBS) causes life-threatening infections in newborns and adults with chronic medical conditions. Serotype IV strains are emerging both among carriers and as cause of invasive disease and recent studies revealed two main Sequence Types (STs), ST-452 and ST-459 assigned to Clonal Complexes CC23 and CC1, respectively. Whole genome sequencing of 70 type IV GBS and subsequent phylogenetic analysis elucidated the localization of type IV isolates in a SNP-based phylogenetic tree and suggested that ST-452 could have originated through genetic recombination. SNPs density analysis of the core genome confirmed that the founder strain of this lineage originated from a single large horizontal gene transfer event between CC23 and the hypervirulent CC17. Indeed, ST-452 genomes are composed by two parts that are nearly identical to corresponding regions in ST-24 (CC23) and ST-291 (CC17). Chromosome mapping of the major GBS virulence factors showed that ST-452 strains have an intermediate yet unique profile among CC23 and CC17 strains. We described unreported large recombination events, involving the cps IV operon and resulting in the expansion of serotype IV to CC23. This work sheds further light on the evolution of GBS providing new insights on the recent emergence of serotype IV.
Assuntos
Genoma Bacteriano/genética , Genômica/métodos , Streptococcus agalactiae/genética , Sequenciamento Completo do Genoma/métodos , Adulto , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Recém-Nascido , Filogenia , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Sorotipagem , Especificidade da Espécie , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/classificação , Streptococcus agalactiae/patogenicidade , Virulência/genéticaRESUMO
OBJECTIVES: To estimate the incidence, case fatality ratio and serotypes associated with early-onset (EOD) and late-onset (LOD) invasive GBS disease in infants in southern mainland China. METHODS: During the six-month study period, infants aged ≤ 90 days with culture-confirmed GBS disease born in the study hospitals or elsewhere, but presenting to a study hospital, were enrolled. GBS-positive cultures were genotyped, serotyped and sequence typed. The incidence rate was calculated for infants born in the study hospitals, and case fatality ratio and causative serotypes identified for all enrolled GBS cases. RESULTS: Ten cases were enrolled: 2 EOD cases born in the study hospitals and 8 LOD cases born elsewhere. Incidence rate was 0.28 (95% confidence interval: 0.08-1.03, n = 2/7061 successfully followed-up consenting subjects); no cases resulted in fatality. In the 8 GBS isolates available for typing, 4 serotypes (Ia, Ib, III and V) and 5 multi-locus sequence types (1, 10, 12, 17 and 23) were identified. CONCLUSIONS: This is the first study specifically investigating the incidence of GBS invasive disease in infants in southern mainland China. Incidence and case fatality were low but further research is needed in larger, more diverse cohorts to estimate disease burden for the broader Chinese population.
RESUMO
Group B Streptococcus (GBS) is cause of neonatal invasive diseases as well as of severe infections in the elderly and immune-compromised patients. Despite significant advances in the prevention and treatment of neonatal disease, sepsis and meningitis caused by GBS still represent a significant public health care concern globally and additional prevention and therapeutic strategies against infection are highly desirable. The introduction of national recommended guidelines in several countries to screen pregnant women for GBS carriage and the use of antibiotics during delivery significantly reduced disease occurring within the first hours of life (early-onset disease), but it has had no effect on the late-onset diseases occurring after the first week and is not feasible in most countries. Availability of an effective vaccine against GBS would provide an effective means of controlling GBS disease. This review provides an overview of the burden of invasive disease caused by GBS in infants and adults, and highlights the strategies for the development of an effective vaccine against GBS infections.
RESUMO
Group B Streptococcus (GBS) expresses 3 structurally distinct pilus types (1, 2a, and 2b) identified as important virulence factors and vaccine targets. These pili are heterotrimeric polymers, covalently assembled on the cell wall by sortase (Srt) enzymes. We investigated the pilus-2b biogenesis mechanism by using a multidisciplinary approach integrating genetic, biochemical, and structural studies to dissect the role of the 2 pilus-2b-associated Srts. We show that only 1 sortase (SrtC1-2b) is responsible for pilus protein polymerization, whereas the second one (Srt2-2b) does not act as a pilin polymerase, but similarly to the housekeeping class A Srt (SrtA), it is involved in cell-wall pilus anchoring by targeting the minor ancillary subunit. Based on its function and sequence features, Srt2-2b does not belong to class C Srts (SrtCs), nor is it a canonical member of any other known family of Srts. We also report the crystal structure of SrtC1-2b at 1.9 Å resolution. The overall fold resembles the typical structure of SrtCs except for the N-terminal lid region that appears in an open conformation displaced from the active site. Our findings reveal that GBS pilus type 2b biogenesis differs significantly from the current model of pilus assembly in gram-positive pathogens.
Assuntos
Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Fímbrias Bacterianas/enzimologia , Streptococcus agalactiae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Fímbrias Bacterianas/genética , Estrutura Terciária de Proteína , Streptococcus agalactiae/genéticaRESUMO
Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.
Assuntos
Proteínas de Bactérias/química , Fímbrias Bacterianas/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Streptococcus agalactiae/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Imunização , Camundongos , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Infecções Estreptocócicas/prevenção & controle , Streptococcus agalactiae/genética , Streptococcus agalactiae/imunologiaRESUMO
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.
Assuntos
Adjuvantes Imunológicos/farmacologia , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/química , Receptor 7 Toll-Like/química , Abscesso/patologia , Imunidade Adaptativa , Animais , Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Antígenos/imunologia , Humanos , Camundongos , Modelos Animais , Infecções Estafilocócicas/imunologia , Staphylococcus aureus , Células Th1/imunologiaRESUMO
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.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Proteoma/química , Staphylococcus aureus/metabolismo , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Mineração de Dados , Camundongos , Camundongos Endogâmicos , Proteoma/genética , Proteoma/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/imunologiaRESUMO
The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response.
Assuntos
Proteínas de Bactérias/metabolismo , Streptococcus agalactiae/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Mapeamento de Epitopos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fagocitose , Homologia de Sequência de Aminoácidos , Streptococcus agalactiae/imunologiaRESUMO
Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date, and their mechanisms of transpeptidation and regulation need to be further investigated. The available 3-dimensional structures of these enzymes reveal a typical sortase fold, except for the presence of a unique feature represented by an N-terminal highly flexible loop known as the "lid." This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an autoinhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work, we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Fímbrias Bacterianas/enzimologia , Streptococcus agalactiae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Western Blotting , Domínio Catalítico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fluorometria , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Filogenia , Polimerização , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteólise , Streptococcus agalactiae/genéticaRESUMO
We evaluated three different PCR-based capsular gene typing methods applied to 312 human and bovine Streptococcus agalactiae (group B Streptococcus [GBS]) isolates and compared the results to serotyping results obtained by latex agglutination. Among 281 human isolates 27% could not be typed by latex agglutination. All 312 isolates except 5 could be typed by the three PCR methods combined. Two of these methods were multiplex assays. Among the isolates that were typeable by both latex agglutination and capsular gene typing, 94% showed agreement between the two methods. However, each of the PCR methods showed limitations. One of the methods did not include all 10 recognized serotypes, one misidentified eight isolates of serotypes Ib and IV as serotype Ia, and one did not distinguish between serotypes VII and IX. For five isolates that showed aberrant patterns in the capsular gene typing, long-range PCR targeting the cps operon disclosed large insertions or deletions affecting the cps gene cluster. A sensitive flow cytometric assay based on serotype-specific antibodies applied to 76 selected isolates that were nontypeable by latex agglutination revealed that approximately one-half of these did express capsular polysaccharide. A procedure for convenient and reliable capsular gene typing to be included in epidemiological and surveillance studies of S. agalactiae is proposed.
Assuntos
Cápsulas Bacterianas/genética , Streptococcus agalactiae/classificação , Streptococcus agalactiae/genética , Animais , Cápsulas Bacterianas/metabolismo , Bovinos , Feminino , Citometria de Fluxo , Loci Gênicos , Humanos , Testes de Fixação do Látex , Tipagem Molecular , Gravidez , Sorotipagem , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/metabolismo , Fatores de Virulência/genéticaRESUMO
Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded ß-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Streptococcus/enzimologia , Catálise , Domínio Catalítico , Cristalografia por Raios X/métodos , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de Proteína , Streptococcus/química , Especificidade por SubstratoRESUMO
Group B Streptococcus pili are covalently linked structures assembled via a sortase-catalyzed transpeptidation mechanism involving specific residues and motifs. A sequence element containing a conserved glutamic acid, called the E-box, has been described to be involved in pilus formation. Although it is known that the glutamic acid is involved in stabilizing the internal isopeptide bonds, its role in pilus assembly still needs to be investigated. Using site-specific mutagenesis and complementation studies of knockout strains, we found that the E-box glutamic residue of the backbone and the major ancillary proteins is essential for pilus protein polymerization. NMR analysis revealed that the mutation of this residue seriously affected the folding of the protein. By contrast, the mutation of the lysine involved in the same isopeptide bond did not engender a structural destabilization, and the native fold was preserved. Moreover, molecular dynamics simulations on the E-box-containing domain of the backbone protein showed that the E-box glutamic acid is necessary to maintain the appropriate dryness of the domain core and that its mutation favors an unfolded state. The data provide the first direct evidence that the E-box has an additional and key role in maintaining the correct protein fold independently of isopeptide bond formation.
Assuntos
Fímbrias Bacterianas/fisiologia , Ácido Glutâmico/fisiologia , Streptococcus agalactiae/fisiologia , Western Blotting , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
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.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Fímbrias Bacterianas/metabolismo , Streptococcus agalactiae/citologia , Streptococcus agalactiae/enzimologia , Sequência de Aminoácidos , Aminoaciltransferases/química , Animais , Proteínas de Bactérias/química , Biocatálise , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Cisteína Endopeptidases/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Hidrolases/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Peptidil Transferases/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/metabolismo , Especificidade por SubstratoRESUMO
Structural vaccinology is an emerging strategy for the rational design of vaccine candidates. We successfully applied structural vaccinology to design a fully synthetic protein with multivalent protection activity. In Group B Streptococcus, cell-surface pili have aroused great interest because of their direct roles in virulence and importance as protective antigens. The backbone subunit of type 2a pilus (BP-2a) is present in six immunogenically different but structurally similar variants. We determined the 3D structure of one of the variants, and experimentally demonstrated that protective antibodies specifically recognize one of the four domains that comprise the protein. We therefore constructed a synthetic protein constituted by the protective domain of each one of the six variants and showed that the chimeric protein protects mice against the challenge with all of the type 2a pilus-carrying strains. This work demonstrates the power of structural vaccinology and will facilitate the development of an optimized, broadly protective pilus-based vaccine against Group B Streptococcus by combining the uniquely generated chimeric protein with protective pilin subunits from two other previously identified pilus types. In addition, this work describes a template procedure that can be followed to develop vaccines against other bacterial pathogens.
Assuntos
Vacinas Bacterianas/síntese química , Proteínas de Fímbrias/química , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/síntese química , Infecções Estreptocócicas/prevenção & controle , Streptococcus agalactiae/imunologia , Animais , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/uso terapêutico , Cristalografia por Raios X , Feminino , Proteínas de Fímbrias/imunologia , Fímbrias Bacterianas/química , Fímbrias Bacterianas/imunologia , Camundongos , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Infecções Estreptocócicas/imunologiaRESUMO
In group B Streptococcus (GBS), 3 structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular-weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies, and in vivo mutagenesis, we performed a broad characterization of GBS sortase C1 of pilus island 2a. The high-resolution X-ray structure of the enzyme revealed that the active site, into the ß-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the "lid." We show that the catalytic triad and the predicted N- and C-terminal transmembrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies, we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild-type protein.