RESUMEN
The lack of knowledge about pathogenicity mechanisms of Streptococcus (S.) suis is, at least partially, attributed to limited methods for its genetic manipulation. Here, we established a Cre-lox based recombination system for markerless gene deletions in S. suis serotype 2 with high selective pressure and without undesired side effects.
Asunto(s)
Recombinación Homóloga , Integrasas/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Streptococcus suis/genética , Streptococcus suis/metabolismo , Orden Génico , Sitios GenéticosRESUMEN
Suilysin is a pore-forming cholesterol-dependent cytolysin secreted by Streptococcus suis (S. suis), an important swine and zoonotic pathogen. The role of suilysin in S. suis host-cell interaction is still unclear. We found a higher adherence and invasion rate of an unencapsulated sly-positive strain in comparison to its isogenic sly-negative mutant. Electron microscopy revealed that formation of membrane ruffles accompanying invasion of the sly-positive strain was abolished in the sly-negative mutant. Inhibition experiments showed that the actin cytoskeleton was involved in suilysin-mediated effects. Point-mutation of the domain putatively responsible for macropore-formation resulted in abolished hemolytic and cytolysin activity, but had no effect on S. suis host cell association. Concluding, our results indicate that subcytolytic suilysin promotes S. suis association with epithelial cells.
Asunto(s)
Proteínas Hemolisinas/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Infecciones Estreptocócicas/microbiología , Streptococcus suis/efectos de los fármacos , Streptococcus suis/fisiología , Enfermedades de los Porcinos/microbiología , Animales , Línea Celular , Células Epiteliales/microbiología , Proteínas Hemolisinas/toxicidad , Humanos , Mutación , Streptococcus suis/genética , Streptococcus suis/ultraestructura , PorcinosRESUMEN
Bartonella (B.) henselae is the zoonotic agent of cat scratch disease. B. henselae has been associated with therapy-resistant Lyme disease in humans suggesting that B. henselae and Borrelia burgdorferi sensu lato might be transmitted concurrently by ticks. In the present study we found that 16 (6.9%) of 230 Ixodes ricinus collected from humans harboured DNA of Bartonella spp. Fifteen positive ticks were infected with B. henselae and one tick with B. clarridgeiae. Twenty-five percent of the 16 Bartonella positive ticks were co-infected with Borrelia burgdorferi sensu lato. Our data show that B. henselae is present in Ixodes ricinus and that ticks may serve as source of infection for humans.
Asunto(s)
Bartonella henselae/aislamiento & purificación , Grupo Borrelia Burgdorferi/aislamiento & purificación , Ixodes/microbiología , Animales , Bartonella henselae/genética , Grupo Borrelia Burgdorferi/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , AlemaniaRESUMEN
Streptococcus suis is one of the most important pathogens in pigs and is also an emerging zoonotic agent. After crossing the epithelial barrier, S. suis causes bacteraemia, resulting in meningitis, endocarditis and bronchopneumonia. Since the host environment seems to be an important regulatory component for virulence, we related expression of virulence determinants of S. suis to glucose availability during growth and to the sugar metabolism regulator catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, representing arcABC operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao and epf, differed significantly between exponential and early stationary growth of a highly virulent serotype 2 strain. Deletion of ccpA altered the expression of the surface-associated virulence factors arcB, sao and eno, as well as the two currently proven virulence factors in pigs, ofs and cps2A, in early exponential growth. Global expression analysis using a cDNA expression array revealed 259 differentially expressed genes in early exponential growth, of which 141 were more highly expressed in the CcpA mutant strain 10ΔccpA and 118 were expressed to a lower extent. Interestingly, among the latter genes, 18 could be related to capsule and cell wall synthesis. Correspondingly, electron microscopy characterization of strain 10ΔccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with enhanced binding to porcine plasma proteins and a reduced resistance to killing by porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on the capsule synthesis and virulence properties of S. suis.
Asunto(s)
Cápsulas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucosa/metabolismo , Proteínas Represoras/metabolismo , Streptococcus suis/patogenicidad , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , ADN Complementario/genética , ADN Complementario/metabolismo , Perfilación de la Expresión Génica , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Represoras/genética , Streptococcus suis/genética , Streptococcus suis/crecimiento & desarrollo , Streptococcus suis/metabolismo , Porcinos , Virulencia , Factores de Virulencia/genéticaRESUMEN
Bartonella species are Gram-negative, fastidious bacteria. Bartonella henselae is found in cats and transmitted to humans via cat scratches or bites causing cat-scratch disease, characterized by clinical symptoms with varying severity. The prevalence of bartonellosis among humans in Germany appears to be high, and severe clinical cases have been described. However, epidemiological data of B. henselae in cats are rare. In this study we determined the detection rates of Bartonella ssp. in cats by culture and real-time PCR. Furthermore, B. henselae isolates were genetically characterized by highly discriminatory amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Bartonella spp. were isolated by culture from 11 (2.2%) of 507 blood samples. Out of 169 blood samples additionally analyzed by PCR, 28 (16.6%) were found positive for Bartonella spp., illustrating the advantage of PCR in Bartonella spp. detection. PCR-REA identified B. henselae in 27 cats and Bartonella clarridgeiae in one cat. B. henselae isolates from different geographical regions in Germany were genetically characterized by AFLP and MLST. Both methods confirmed genetic diversity of B. henselae on the strain level. MLST identified 11 new sequence types, all of them assigned to three clonal complexes as determined by eBURST. AFLP typing revealed genetic relation among the B. henselae isolates from the same geographical region. Combining AFLP typing and MLST/eBURST analyses revealed that B. henselae of the same AFLP subcluster belonged to the same clonal complex. Altogether these results indicate that B. henselae may evolve clonally.
Asunto(s)
Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Infecciones por Bartonella/veterinaria , Bartonella henselae/genética , Variación Genética , Tipificación de Secuencias Multilocus , Animales , Técnicas de Tipificación Bacteriana , Infecciones por Bartonella/epidemiología , Infecciones por Bartonella/microbiología , Bartonella henselae/clasificación , Bartonella henselae/aislamiento & purificación , Secuencia de Bases , Gatos , ADN Bacteriano/genética , Ensayo de Inmunoadsorción Enzimática , Geografía , Alemania/epidemiología , Filogenia , Reacción en Cadena de la Polimerasa/veterinaria , Prohibitinas , Especificidad de la EspecieRESUMEN
OBJECTIVE: The transcription factor CCAAT/enhancer- binding protein (C/EBP) beta is involved in inflammatory responses in immune cells, including myelomonocytic cells. In this study, signal transduction pathways regulating C/EBPbeta expression were investigated. METHODS: The expression of C/EBPbeta mRNA in cells treated with various activators and inhibitors of PKA and PKC was analyzed by Northern blot hybridization. C/EBPbeta promoter activity was investigated by transient transfection assays with C/EBPbeta promoter CAT constructs. RESULTS: Phorbol 12-myristate 13-acetate (PMA), forskolin and 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cAMP and cGMP phosphodiesterases, but not cGMP, when added to chicken myelomonocytic HD11 cells, markedly stimulated the C/EBPbeta mRNA expression. However, transfection experiments using HD11 cells showed that CAT constructs controlled by the 5' flanking sequence from -704 to +24 of chicken C/EBPbeta gene were activated by PMA, but not by forskolin. In contrast to forskolin, IBMX was able to activate the C/EBPbeta promoter CAT constructs. Further transient transfection experiments using other cell lines demonstrated that the chicken C/EBPbeta promoter was responsive to forskolin in mouse fibroblasts NIH3T3, but not in human hepatoma HepG2 cells. Increase in C/EBPbeta mRNA stability in HD11 cells was induced by forskolin and PMA. CONCLUSION: The results indicate that the C/EBPbeta gene is regulated transcriptionally as well as post-transcriptionally in response to forskolin and PMA, and the forskolin responsiveness of the C/EBPbeta promoter seems to depend on cellular cAMP turnover.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Monocitos/citología , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , 1-Metil-3-Isobutilxantina/metabolismo , Animales , Secuencia de Bases , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular , Pollos , Colforsina/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Inhibidores de Fosfodiesterasa/metabolismo , Estabilidad del ARN , Alineación de Secuencia , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/metabolismoRESUMEN
We compared immunogenicity in pigs of whole cell lysate proteins (WCP) with murein-associated proteins (MAP) obtained from a virulent serotype 2 strain of Streptococcus (S.) suis grown at 32 or 42 degrees C. Protein fractions were tested for their ability to induce antibodies in 3-week-old piglets by enzyme-linked immunosorbent assay and Western blot analysis. We found a significant increase in the antibody levels in all sera irrespective of the preparation used for immunization. However, alpha-WCP sera showed higher reactivities than alpha-MAP sera, and piglets immunized with 32 degrees C preparations (alpha-32 sera) showed higher responses than those immunized with 42 degrees C preparations (alpha-42 sera). Western blot analysis revealed that alpha-WCP sera in part reacted with different proteins when compared with alpha-MAP sera. Furthermore, some proteins were only detected by alpha-32 but not by alpha-42 sera. In conclusion, the results demonstrate the immunogenicity of cell wall MAP of S. suis, and highlight the importance of considering growth conditions in the preparation of subunit vaccines.
Asunto(s)
Peptidoglicano/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus suis/inmunología , Enfermedades de los Porcinos/inmunología , Animales , Anticuerpos Antibacterianos/análisis , Western Blotting/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Calor , Serotipificación , Infecciones Estreptocócicas/inmunología , Streptococcus suis/clasificación , Estrés Fisiológico , Porcinos , Enfermedades de los Porcinos/sangreRESUMEN
Streptococcus suis is a porcine and human pathogen causing invasive diseases, such as meningitis or septicaemia. Host cell interactions of S. suis have been studied mainly with serotype 2 strains, but multiple capsular serotypes as well as non-typeable strains exist with diverse virulence features. At present, S. suis is considered an extracellular pathogen. However, whether or not it can also invade host cells is a matter of controversial discussions. We have assessed adherence and invasion of S. suis for HEp-2 epithelial cells by comparing 10 serotype 2 strains and four non-typeable (NT) strains. Only the NT strains and a non-encapsulated serotype 2 mutant strain, but none of the serotype 2 strains, adhered strongly and were invasive. Invasion seemed to be affected by environmental signals, as suggested from comparison of strains grown in different media. Further phenotypic and genotypic characterization revealed a high diversity among the different strains. Electron microscopic analysis of invasion of selected invasive NT strains indicated different uptake mechanisms. One strain induced large invaginations comparable to those seen in 'caveolae' mediated uptake, whereas invasion of the other strains was accompanied by formation of filipodia-like membrane protrusions. Invasion of all strains, however, was similarly susceptible to hypertonic sucrose, which inhibits receptor-mediated endocytosis. Irrespective of the uptake pathway, streptococci resided in acidified phago-lysosome like vacuoles. All strains, except one, survived intracellularly as well as extracellular acidic conditions. Survival seemed to be associated with the AdiS protein, an environmentally regulated arginine deiminase of S. suis. Concluding, invasion and survival of NT strains of S. suis in epithelial cells revealed novel evidence that S. suis exhibits a broad variety of virulence-associated features depending on genetic variation and regulation.
Asunto(s)
Cápsulas Bacterianas/fisiología , Células Epiteliales/microbiología , Streptococcus suis/patogenicidad , Ácidos/farmacología , Antibacterianos/farmacología , Adhesión Bacteriana , Cápsulas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Citoplasma/microbiología , Endocitosis , Células Epiteliales/ultraestructura , Variación Genética , Genotipo , Humanos , Hidrolasas/metabolismo , Microscopía Electrónica , Mutación , Fenotipo , Serotipificación , Streptococcus suis/crecimiento & desarrollo , Streptococcus suis/ultraestructura , Sacarosa/metabolismo , Vacuolas/microbiología , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Streptococcus suis (Sc. suis) can cause very different clinical entities. In contrast to Sc. suis-associated pneumonia, the induction of meningitis, septicemia, and polyarthritis by certain Sc. suis strains requires the expression of virulence factors that contribute to the invasiveness of the pathogen. In the presented study, we examined the occurrence of known virulence-associated factors in Sc. suis isolates from samples sent to the Institute of Microbiology, School of Veterinary Medicine Hannover, in order to evaluate their significance as potential virulence factors in different disease complexes in Northern Germany. The results show that (i) MRP + EF + serotype 2 and MRP* EF-serotype 9 strains are statistically significant associated with the disease complex meningitis/septicemia/arthritis and, thus, have to be considered invasive strains, (ii) serotyping alone is not sufficient for identification of virulent strains, (iii) there is a remarkable heterogeneity among pneumonia-associated Sc. suis strains and (iv) activity of haemolysin or suilysin appears to be not appropriate as virulence marker. Finally, it has to be noted that at present only half of the Sc. suis isolates from pigs with meningitis/septicemia/poyarthritis can be characterised by the detection of virulence-associated factors. Thus, the identification and characterisation of additional, serotype independent virulence factors of Sc. suis is a very important issue in future studies.
Asunto(s)
Infecciones Estreptocócicas/veterinaria , Streptococcus suis/patogenicidad , Enfermedades de los Porcinos/microbiología , Animales , Alemania/epidemiología , Proteínas Hemolisinas/metabolismo , Prevalencia , Infecciones Estreptocócicas/diagnóstico , Infecciones Estreptocócicas/epidemiología , Streptococcus suis/clasificación , Porcinos , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/epidemiología , VirulenciaRESUMEN
Due to the hard environmental and climatic situation in late winter 1999, a herd of about 200 free-ranging, semi-domesticated reindeer was gathered in a paddock in northern Norway for emergency feeding. About the same number of reindeer was not corralled but supplementary fed on their winter pastures. The fodder was of relatively good quality but very dusty and fed in a very dry environment. Six weeks later, an outbreak of eye-infection was diagnosed in one third of the corralled reindeer; mild symptoms were observed in most of them, but 11 animals showed severe signs of disease. No signs of disease were found in the non-corralled animals. Ten reindeer died through emaciation, the eleventh was sacrificed. Histopathological diagnosis of two severely affected eyes revealed a severe purulent kerato-conjunctivitis with bacteria and plant particles embedded in purulent exudates on the cornea and conjunctiva. In one eye from the two most affected animals Actinomyces pyogenes, coagulase-negative Staphylococci and Escherichia coli and in the other one Staphylococcus aureus and Escherichia coli were found. The bacteria encountered in this study are not considered the primary cause of disease. They seem rather to be opportunistic infectious agents of eyes that have been irritated mechanically through exposure to dusty fodder in a dry environment. The stress through unfamiliar corralling of the reindeer, that followed an insufficient fodder supply, could be considered as an additional infection supporting factor. This case-report emphasises on the importance of different factors involved in favouring outbreaks of disease in reindeer, under intensified husbandry conditions. Even though crowding and emergency feeding may be, at certain circumstances, the only means of survival for reindeer, a negative impact of implied crowding diseases on their productivity, must be considered, as well.
Asunto(s)
Crianza de Animales Domésticos/métodos , Polvo , Queratoconjuntivitis/veterinaria , Reno , Alimentación Animal/microbiología , Animales , Brotes de Enfermedades/veterinaria , Ingestión de Alimentos , Queratoconjuntivitis/epidemiología , Queratoconjuntivitis/etiología , Queratoconjuntivitis/microbiología , Noruega/epidemiología , Densidad de Población , Factores de Riesgo , Estaciones del Año , Estrés Fisiológico/complicaciones , Estrés Fisiológico/etiología , Estrés Fisiológico/veterinariaRESUMEN
Samples of faeces were taken from 166 healthy domesticated reindeer (Rangifer tarandus tarandus) from three flocks in different reindeer husbandry districts in northern Norway and examined bacteriologically for the presence of Clostridium perfringens. The organism was isolated from 98 (59 per cent) of the reindeer. The isolates were classified into C perfringens toxin types by PCR analysis specific for the genes encoding the four major toxins (alpha, beta, epsilon and tau) and were subclassified by the detection of the genes encoding C perfringens beta2-toxin and enterotoxin. All the isolates belonged to C perfringens toxin type A. In addition, 15 of the 98 isolates were PCR-positive for the beta2-toxin gene, and two of the isolates had the the gene encoding for enterotoxin.
Asunto(s)
Toxinas Bacterianas/genética , Clostridium perfringens/aislamiento & purificación , Reno/microbiología , Animales , Clostridium perfringens/clasificación , Clostridium perfringens/patogenicidad , Noruega , Reacción en Cadena de la PolimerasaAsunto(s)
Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Infecciones por Clostridium/veterinaria , Clostridium perfringens/genética , Enfermedades de los Perros/epidemiología , Animales , Infecciones por Clostridium/epidemiología , Clostridium perfringens/clasificación , Clostridium perfringens/aislamiento & purificación , Cartilla de ADN , Diarrea/epidemiología , Diarrea/microbiología , Diarrea/veterinaria , Enfermedades de los Perros/microbiología , Perros , Ensayo de Inmunoadsorción Enzimática/veterinaria , Alemania/epidemiología , Reacción en Cadena de la Polimerasa/veterinaria , PrevalenciaRESUMEN
The phagosomes containing viable pathogenic mycobacteria, such as Mycobacterium (M.) tuberculosis and Mycobacterium avium ssp. avium (M. avium), are known to be limited in their ability to both acidify and fuse with late (but not early) endocytic organelles. Here, we analysed the pH and fusogenicity of phagosomes containing M. avium ssp. paratuberculosis (M. ptb), the causative agent of paratuberculosis in ruminants. Using the murine J774 macrophage cell line, we compared viable and heat-killed M. ptb and, in addition, viable or dead M. avium, as well as two non-pathogenic mycobacteria, Mycobacterium smegmatis and Mycobacterium gordonae. Electron microscopic analysis revealed that M. ptb persisted intracellularly in phagosomes for up to 15 days. The phagosomes containing live M. ptb and M. avium were significantly reduced in their ability to acquire some markers for the endocytic pathway, such as internalized calcein, BSA-gold or the membrane protein Lamp 2. However, they were almost completely accessible to 70 kDa fluorescein isothiocyanate (FITC)-dextran and Lamp 1. Overall, the phagosomes containing dead pathogenic mycobacteria behaved similarly to the ones containing live non-pathogenic mycobacteria in all experiments. Using FITC-dextran in a novel fluorescence-activated cell sorting (FACS)-based method, we could also show that the bulk of endocytic compartments, including phagosomes, were only very mildly acidified to approximately pH 6.3 over at least 72 h in J774 cells infected with live M. ptb and M. avium. In contrast, J774 cells treated with heat-killed M. ptb or BSA-coated latex beads showed substantial acidification of the phagosome/endocytic compartments to a pH value of approximately 5.2. After infection with M. smegmatis and M. gordonae, acidification was initially (1-5 h after infection) inhibited, but increased after longer infection to levels similar to those with dead mycobacteria.
Asunto(s)
Concentración de Iones de Hidrógeno , Macrófagos/microbiología , Mycobacterium avium subsp. paratuberculosis/patogenicidad , Mycobacterium avium/patogenicidad , Fagosomas/microbiología , Animales , Membranas Intracelulares , Fusión de Membrana , Ratones , Mycobacterium/patogenicidad , Especificidad de la EspecieRESUMEN
The lysozyme gene is activated in myelomonocytic HD11 cells in response to LPS. In this study, we described the involvement of LPS-activated signal transduction pathways in activation of the lysozyme gene. Pre-treatment of HD 11 cells with H-89, H-7, TMB-8, or KN-93 resulted in inhibition of the LPS-enhanced lysozyme expression, suggesting that PKA, PKC, and Ca2+-dependent protein kinases participate in the LPS activation. CaMKII seems to be required for the processing of lysozyme transcripts. TPA and calcium ionophore A23187, when separately added to HD11 cells, stimulated the lysozyme expression effectively, and forskolin was ineffective. It is interesting that simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a potentiated increase in lysozyme mRNA expression, indicating a synergistic cooperation of PKA and Ca2+. This synergistic effect of PKA and Ca2+ was observed on the expression of a stably integrated CAT construct, controlled by the lysozyme promoter and the -6.1-kb enhancer containing binding sites for C/EBP and NF-kappaB/Rel. Therefore, we discussed the role of C/EBPbeta(NF-M), CREB, and NF-kappaB/Rel as possible targets for phosphorylation mediated by PKA, PKC, and Ca2+.
Asunto(s)
Calcio/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Muramidasa/biosíntesis , Proteína Quinasa C/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Calcimicina/farmacología , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Línea Celular , Pollos , Colforsina/farmacología , AMP Cíclico/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Sinergismo Farmacológico , Elementos de Facilitación Genéticos , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Ionóforos/farmacología , Ligasas/metabolismo , Monocitos/enzimología , Muramidasa/genética , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas , Proteína Quinasa C/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Empalme del ARN/efectos de los fármacos , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Transactivadores/metabolismoRESUMEN
We evaluated the genetic diversity of Streptococcus suis isolates of different serotypes by macrorestriction analysis and elucidated possible relationships between the genetic background, expression of potential virulence traits, and source of isolation. Virulence traits included expression of serotype-specific polysaccharides, muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin activity, and adherence to epithelial cells. Macrorestriction analysis of streptococcal DNA digested with restriction enzymes SmaI and ApaI allowed differentiation of single isolates that could be assigned to four major clusters, named A1, A2, B1, and B2. Comparison of the genotypic and phenotypic features of the isolates with their source of isolation showed that (i) the S. suis population examined, which originated mainly from German pigs, exhibited a genetic diversity and phenotypic patterns comparable to those found for isolates from other European countries; (ii) certain phenotypic features, such as the presence of capsular antigens of serotypes 2, 1, and 9, expression of MRP and EF, and hemolysin activity (and in particular, combinations of these features), were strongly associated with the clinical background of meningitis and septicemia; and (iii) isolates from pigs with meningitis and septicemia showed a significantly higher degree of genetic homogeneity compared to that for isolates from pigs with pneumonia and healthy pigs. Since the former isolates are considered highly virulent, this supports the theory of a clonal relationship among highly virulent strains.
Asunto(s)
Antígenos Bacterianos , Infecciones Estreptocócicas/veterinaria , Streptococcus suis/clasificación , Streptococcus suis/patogenicidad , Enfermedades de los Porcinos/microbiología , Porcinos/microbiología , Animales , Adhesión Bacteriana , Proteínas Bacterianas/genética , Electroforesis en Gel de Campo Pulsado , Células Epiteliales/microbiología , Genotipo , Proteínas Hemolisinas/análisis , Meningitis Bacterianas/microbiología , Meningitis Bacterianas/veterinaria , Fenotipo , Filogenia , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/veterinaria , Mapeo Restrictivo , Sepsis/microbiología , Sepsis/veterinaria , Serotipificación , Infecciones Estreptocócicas/microbiología , Streptococcus suis/genética , Streptococcus suis/aislamiento & purificación , VirulenciaRESUMEN
We have characterized antigens from Actinobacillus (A.) pleuropneumoniae grown under iron restriction with respect to their immunogenic and protective potential. Antigens were the cell-free culture supernatants (CFS) obtained after treatment of A. pleuropneumoniae broth cultures with sodium deoxycholate. Using the iron-repressible transferrin-binding lipoprotein TbpB and the constitutively expressed outer membrane lipoprotein OmlA as markers, we have shown that the detergent extraction enriched the CFS with lipoproteins from the outer membrane (OM). Extractions with 0.05% of sodium deoxycholate increased the lipoprotein contents in the CFS, but did not affect the integrity of the OM. This was demonstrated by the absence of the iron-repressible integral OM transferrin-binding protein TbpA. Furthermore, the absence of periplasmic and cytoplasmic proteins in CFS after extraction was determined in immunoblot analyses with anti-bacterial alkaline phosphatase and anti-Hsp60 antisera, demonstrating that there was no rupture of the OMs or the plasma membranes due to the extraction procedure. Antigen preparations from A. pleuropneumoniae serotype 2 and 9 grown under iron restrictive conditions were combined, emulsified, and tested for their ability to confer protection in pigs. Pigs immunized with CFS from sodium deoxycholate extracted cultures developed a strong antibody response and, upon challenge with A. pleuropneumoniae serotype 2, the immunized pigs showed no or only mild clinical signs of disease and had a significantly lower degree of lung damage than the control pigs.
Asunto(s)
Actinobacillus pleuropneumoniae/inmunología , Vacunas Bacterianas/aislamiento & purificación , Infecciones por Actinobacillus/inmunología , Infecciones por Actinobacillus/prevención & control , Infecciones por Actinobacillus/veterinaria , Actinobacillus pleuropneumoniae/crecimiento & desarrollo , Actinobacillus pleuropneumoniae/metabolismo , Animales , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/farmacología , Ácido Desoxicólico , Detergentes , Hierro/metabolismo , Masculino , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/prevención & control , Neumonía Bacteriana/veterinaria , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & control , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/aislamiento & purificación , Vacunas de Subunidad/farmacologíaRESUMEN
Mycobacterium avium subspecies paratuberculosis is the etiologic agent of paratuberculosis (Johnes disease), a chronic enteritis in ruminants, which is one of the most widespread bacterial diseases of domestic animals, causing enormous economic losses worldwide. Though the disease was first described more than a century ago, the biology of the infecting organism and the mechanisms of its interactions with the host still remain a mystery. In this review, recent advances made on pathogenesis of paratuberculosis are summarized and future challenges are discussed.
Asunto(s)
Mycobacterium avium subsp. paratuberculosis , Paratuberculosis/microbiología , Rumiantes/microbiología , Animales , Mycobacterium avium subsp. paratuberculosis/inmunología , Mycobacterium avium subsp. paratuberculosis/patogenicidad , Mycobacterium avium subsp. paratuberculosis/fisiología , Paratuberculosis/inmunología , Paratuberculosis/patologíaRESUMEN
Lysozyme is increasingly expressed in macrophages in inflammatory response to bacterial LPS. In this study, we investigated the mechanisms that control expression of the lysozyme gene in myelomonocytic HD11 cells activated by LPS. Nuclear run-on transcription assays showed that LPS caused a 15-fold increase in the transcription rate of the lysozyme gene. However, Northern analyses with lysozyme cDNA and intron sequences revealed that the LPS-induced increase in nuclear lysozyme transcripts greatly exceeded the increase in transcription rate. Furthermore, nuclear lysozyme transcripts in untreated cells with a t(1/2) of <10 min were more unstable than those accumulated in LPS-activated cells. We suggested, therefore, that the increased lysozyme expression following LPS treatment was largely due to a nuclear stabilization of the primary transcript. Interestingly, the increase in stability of the lysozyme primary transcript was accompanied by changes in nuclear processing including an increase in poly(A) tail length, which gradually shortened after entering the cytoplasm. The long lysozyme poly(A) tail, however, did not result in any increase in polysomal recruitment for translation or in stability of the cytoplasmic lysozyme mRNA.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Muramidasa/genética , ARN Mensajero/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Humanos , Muramidasa/biosíntesis , Precursores del ARN/metabolismo , Empalme del ARN , Transcripción Genética/efectos de los fármacosRESUMEN
C/EBPbeta has been shown to mediate the lipopolysaccharide (LPS)-induced expression of the lysozyme gene through enhanced binding to the -6.1-kb lysozyme enhancer. In this study, we describe the LPS regulation of the C/EBPbeta gene in myelomonocytic HD11 cells. Northern analysis showed that the steady state level of C/EBPbeta mRNA increased in response to LPS. The half life of C/EBPbeta mRNA of about 1 h in HD11 cells was not affected by exposure to LPS. Nuclear run-on transcription experiments with isolated nuclei revealed that the rate of C/EBPbeta gene transcription was enhanced by LPS, demonstrating that the C/EBPbeta gene in HD11 cells was regulated at the transcriptional level in response to LPS. Furthermore, the LPS-induced binding activity of C/EBPbeta to the -6.1-kb lysozyme enhancer was dependent not only on protein synthesis, but also on transcription. Thus, these results suggested that the LPS-induced binding activity to the -6.1-kb lysozyme enhancer in HD11 cells was regulated by an enhanced transcription-dependent de novo synthesis of C/EBPbeta.
Asunto(s)
Proteínas de Unión al ADN/genética , Muramidasa/genética , Proteínas Nucleares/genética , Transcripción Genética/efectos de los fármacos , Animales , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular Transformada , Pollos , Proteínas de Unión al ADN/biosíntesis , Dactinomicina/análogos & derivados , Dactinomicina/farmacología , Semivida , Lipopolisacáridos/farmacología , Muramidasa/metabolismo , Proteínas Nucleares/biosíntesis , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismoRESUMEN
Macrophages respond to lipopolysaccharide (LPS) with the activation of various genes, including the lysozyme gene. Here, we show that the level of lysozyme mRNA increases following treatment of chicken myelomonocytic HD11 cells with LPS. By transient and stable transfection of the chloramphenicol acetyltransferase (CAT) gene controlled by regulatory elements of the lysozyme gene, we identified a subfragment of the -6.1 kilobase (kb) lysozyme enhancer that mediates the LPS-induced lysozyme expression. This subfragment contains two elements (D and E), each of which matches the highly degenerate consensus sequence of binding sites for C/EBP-like transcription factors. Furthermore, we found protein complexes to interact with elements D and E whose binding activity to elements D and E is LPS-inducible in myelomonocytic HD11 cells. Immunomobility shift assays show that NF-M, a myeloid-specific C/EBP beta-related transcription factor is an essential component of these protein complexes. Mutations of the C/EBP binding sites within D and E cause a reduction of basal activity and abolish LPS responsiveness of the -6.1 kb lysozyme enhancer. These results show that the -6.1 kb lysozyme enhancer, in addition to its role in cell type-specific expression, can mediate, by interacting with NF-M, LPS-induced expression of the lysozyme gene in chicken myelomonocytic cells.