RESUMEN
Capsular polysaccharide produced by Haemophilus influenzae b (Hib) is the main virulent agent and used as the antigen in the vaccine formulation. In this study, an improved process of polysaccharide purification was established based on tangential flow ultrafiltration using detergents (cocamidopropyl betaine and sodium deoxycholate), two selective ethanol precipitations steps, and extensive enzymatic hydrolysis as strategy. The relative purity (RP) related to protein and nucleic acids were 122~263 and 294~480, respectively, and compatible with the specifications established by the World Health Organization for Hib vaccine, RP≥100. These results make this process simple, cheaper, efficient, environmentally friendly, and prone to be scaled up.
Asunto(s)
Cápsulas Bacterianas/aislamiento & purificación , Haemophilus influenzae tipo b/metabolismo , Ultrafiltración/métodos , Cápsulas Bacterianas/biosíntesis , Glucosa/farmacología , Haemophilus influenzae tipo b/efectos de los fármacos , Haemophilus influenzae tipo b/crecimiento & desarrolloRESUMEN
There is ample evidence that Staphylococcus aureus capsular polysaccharide (CP) promotes virulence. Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. This study was conducted to determine the relative prevalence of nonencapsulated S. aureus in patients with chronic and acute osteomyelitis. Only 76/118 (64%) S. aureus isolates from patients with osteomyelitis expressed CP, whereas all 50 isolates from blood cultures of patients with infections other than osteoarticular infections expressed CP (P = 0.0001). A significantly higher prevalence of nonencapsulated S. aureus was found in patients with chronic osteomyelitis (53%) than in those with acute osteomyelitis (21%) (P = 0.0046). S. aureus isolates obtained from multiple specimens from five of six patients with chronic osteomyelitis exhibited phenotypic (expression of CP, alpha-hemolysin, beta-hemolysin, slime, and the small-colony variant phenotype) and/or genotypic (pulsed-field gel electrophoresis and spa typing) differences. Nonencapsulated S. aureus was recovered from at least one specimen from each chronic osteomyelitis patient. Fourteen isolates obtained from two patients with acute osteomyelitis were indistinguishable from each other within each group, and all produced CP5. In conclusion, we demonstrated that nonencapsulated S. aureus is more frequently isolated from patients with chronic osteomyelitis than from those with acute osteomyelitis, suggesting that loss of CP expression may be advantageous to S. aureus during chronic infection. Our findings on multiple S. aureus isolates from individual patients allow us to suggest that selection of nonencapsulated S. aureus is likely to have occurred in the patient during long-term bone infection.
Asunto(s)
Cápsulas Bacterianas/biosíntesis , Variación Genética , Osteomielitis/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Adolescente , Adulto , Anciano , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Staphylococcus aureus/aislamiento & purificación , Estados Unidos , Factores de Virulencia/biosíntesis , Adulto JovenRESUMEN
AIMS: To determine whether the presence and type of exopolysaccharides (EPS), slime-EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. METHODS AND RESULTS: Phage-host interactions between eight EPS-producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (phiYsca, phi3, phi5, phi6, phi8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular-producing strains were more sensitive to phage attacks than the noncapsular-producing strains. Streptococcus thermophilus CRL1190 (capsular-producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular-negative, EPS low-producing mutant of strain CRL1190 was reduced, especially for phiYcsa and phi8. CONCLUSIONS: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Capsular-producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.
Asunto(s)
Cápsulas Bacterianas/biosíntesis , Microbiología de Alimentos , Fagos de Streptococcus/fisiología , Streptococcus thermophilus/virología , Acoplamiento Viral , Argentina , Cápsulas Bacterianas/química , Dermatoglifia del ADN , ADN Bacteriano/genética , Polisacáridos Bacterianos/análisis , Técnica del ADN Polimorfo Amplificado Aleatorio , Streptococcus thermophilus/clasificación , Streptococcus thermophilus/genética , Streptococcus thermophilus/aislamiento & purificaciónRESUMEN
The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.
Asunto(s)
Pared Celular/metabolismo , Cryptococcus neoformans/metabolismo , Polisacáridos Bacterianos/metabolismo , Vesículas Secretoras/metabolismo , Animales , Cápsulas Bacterianas/biosíntesis , Transporte Biológico , Línea Celular , Centrifugación por Gradiente de Densidad , Espacio Extracelular/metabolismo , Glucosilceramidas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Polisacáridos/metabolismo , Esteroles/metabolismoRESUMEN
AIM: An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed-batch cultivation in bioreactor. METHODS AND RESULTS: Streptococcus pneumoniae serotype 23F was cultivated in a 5-l bioreactor with nitrogen-sparging and followed by addition of air in the stationary phase. The amount of CPS released in the supernatant progressively increased under air sparging. The profile of cellular viability and optical density was similar in both cultures. Immunoelectron microscopy showed that the amount of tightly cell-bound CPS was higher in bacteria cultivated under nitrogen than under air. CONCLUSIONS: The stress caused by the addition of air at the stationary phase promoted a large increase of free CPS into the medium, as a consequence of the morphologic change in the capsule. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of air in the stationary phase of the culture would greatly simplify the subsequent downstream process, allowing CPS purification from the supernatant. The direct consequence of this process improvement is the reduction of vaccine production costs.
Asunto(s)
Aire , Cápsulas Bacterianas/biosíntesis , Streptococcus pneumoniae/metabolismo , Cápsulas Bacterianas/ultraestructura , Biomasa , Reactores Biológicos , Recuento de Colonia Microbiana , Medios de Cultivo , Glucosa/metabolismo , Ácido Láctico/biosíntesis , Microscopía Inmunoelectrónica , Estrés Oxidativo , Vacunas Neumococicas/biosíntesis , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/inmunologíaRESUMEN
A simple, specific, sensitive and reproducible ELISA has been developed to quantify the level of CPS (capsular polysaccharide) production in supernatants of Streptococcus pneumoniae cell cultures. CPSs from Strep. pneumoniae have been widely used as vaccine antigens. The quantification method is based on two type-23F serotype-specific polyclonal antibodies: IgG, purified from sera of mice immunized with a pneumococcal type-23F CPS conjugate, used in the coating step, and a serotype-specific rabbit serum as the second antibody. Solutions of purified type-23F CPS were used as standards. The relationship between A(492) and type-23F CPS concentration was linear over the range 1-310 ng/ml (r=0.989), with 1 ng/ml as the lower limit of sensitivity. The specificity of ELISA was assessed because purified type-19F CPS and cell-wall polysaccharide samples were not detected after their evaluation by the ELISA described in the present study. Repeatability and intermediate precision of the assay were good, the coefficients of variation being 3 and 10% respectively. This ELISA allowed selection of an appropriate vaccine strain, for a natural polysaccharide vaccine, among several 23F pneumococcal clinical isolates and constituted a valuable analytical tool for Strep. pneumoniae fermentation and CPS purification follow-up.
Asunto(s)
Cápsulas Bacterianas/química , Ensayo de Inmunoadsorción Enzimática/métodos , Meningitis Neumocócica/diagnóstico , Polisacáridos Bacterianos/análisis , Streptococcus pneumoniae/clasificación , Animales , Cápsulas Bacterianas/biosíntesis , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Ratones , Ratones Endogámicos BALB C , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/aislamiento & purificación , Reproducibilidad de los Resultados , Streptococcus pneumoniae/metabolismoRESUMEN
The glucose 1-phosphate uridylyltransferase (GalU) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of Streptococcus pneumoniae. The pneumococcal GalU protein was overexpressed in Escherichia coli, and purified. GalU showed a pI of 4.23, and catalyzed the reversible formation of UDP-glucose and pyrophosphate from UTP and glucose 1-phosphate with K(m) values of 0.4 mM: for UDP-glucose, 0.26 mM: for pyrophosphate, 0.19 mM: for glucose 1-phosphate, and 0.24 mM: for UTP. GalU has an optimum pH of 8-8.5, and requires Mg(2+) for activity. Neither ADP-glucose nor TDP-glucose is utilized as substrates in vitro. The purification of GalU represents a fundamental step to provide insights on drug design to control the biosynthesis of the main pneumococcal virulence factor.
Asunto(s)
Cápsulas Bacterianas/biosíntesis , Polisacáridos Bacterianos/biosíntesis , Streptococcus pneumoniae/enzimología , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Escherichia coli/genética , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/genética , Streptococcus pneumoniae/genéticaRESUMEN
The hemagglutinin/protease (HA/P) seems to be an attractive locus for the insertion of heterologous tags in live cholera vaccine strains. A deltaCTXphi spontaneous mutant derived from a pathogenic strain of O139 Vibrio cholerae was sequentially manipulated to obtain hapA Colon, two colons celA derivatives which were later improved in their environmental safety by means of a thyA mutation. All the strains here obtained showed similar phenotypes in traits known to be remarkable for live cholera vaccines irrespective of their motility phenotypes, although the hapA mutants had a 10-fold decrease in their colonisation capacity compared with their parental strains in the infant mouse cholera model. However, the subsequent thyA mutation did not affect their colonisation properties in the same model. These preliminary results pave the way for further clinical assays to confirm the possibilities of these vaccine prototypes as safe and effective tools for the prevention of O139 cholera.
Asunto(s)
Vacunas contra el Cólera/inmunología , Antígenos O/inmunología , Vibrio cholerae O139/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/inmunología , Cápsulas Bacterianas/biosíntesis , Cápsulas Bacterianas/inmunología , Celulasa/genética , Cólera/prevención & control , Toxina del Cólera/biosíntesis , Toxina del Cólera/genética , Clostridium/genética , Resistencia a Medicamentos , Farmacorresistencia Bacteriana Múltiple , Genes Sintéticos , Pruebas de Hemaglutinación , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Metaloendopeptidasas/genética , Mutagénesis Insercional , Conejos , Seguridad , Estreptomicina/farmacología , Combinación Trimetoprim y Sulfametoxazol/farmacología , Vibrio cholerae O139/efectos de los fármacos , Vibrio cholerae O139/enzimología , Vibrio cholerae O139/genéticaRESUMEN
We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.