RESUMEN
Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.4- and 2.4/2.4-linked, and a branched 2.4[2.8]2.4-linked Kdo tetrasaccharide occur in Chlamydiaceae. While the 2.8/2.4-linked trisaccharide contains a family-specific epitope, the branched Kdo oligosaccharide occurs only in Chlamydophila psittaci and antibodies against it will be useful in human and veterinarian diagnostics. To overcome the generation of cross-reactive antibodies that bind with high affinity to a dominant epitope formed by 2.4/2.4-linked Kdo, we immunized mice with a synthetic 2.4[2.8]-linked branched Kdo trisaccharide and used phage display of scFv to isolate recombinant antibody fragments (NH2240-31 and SAG506-01) that recognize the branched Kdo oligosaccharide with a K(D) of less than 10 nM. Importantly, although these antibodies used germline genes coding for an inherited Kdo recognition site, they were able clearly to distinguish between 2.4[2.8]2.4- and 2.4/2.4-linked Kdo. Sequence determination, binding data, and X-ray structural analysis revealed the basis for the improved discrimination between similar Kdo ligands and indicated that the alteration of a stacking interaction from a phenylalanine residue in the center of the combining site to a tyrosine residue facing away from the center favors recognition of branched 2.4[2.8]2.4-linked Kdo residues. Immunofluorescence tests of infected cell monolayers using this antibody show specific staining of C. psittaci elementary bodies that allow it to be distinguished from other pathogenic chlamydiae.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Carbohidratos/inmunología , Chlamydophila psittaci/inmunología , Lipopolisacáridos/inmunología , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos Antiidiotipos/genética , Carbohidratos/genética , Chlamydiaceae/genética , Chlamydiaceae/inmunología , Chlamydophila psittaci/química , Chlamydophila psittaci/genética , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Lipopolisacáridos/análisis , Lipopolisacáridos/química , Ratones , Oligosacáridos/genética , Oligosacáridos/inmunología , Proteínas Recombinantes/inmunología , Trisacáridos/genética , Trisacáridos/inmunología , Rayos XRESUMEN
Traditionally, the amount of infective chlamydiae in a given sample is determined by inoculating dilution series into cell cultures and physically counting chlamydial inclusions. This approach is time consuming, tedious, and error prone, mainly when dealing with high titers. Therefore, this paper describes a largely automated technique that was developed to standardize the determination of chlamydial load in vitro. Cells are fixed at 36 h post-inoculation and bacteria visualized using standard immunological detection methods. Consequently, for 81 microscopic fields, an image is recorded at the interpolated focal plane. These images are then automatically processed using an ImageJ plugin and the obtained results are imported into Excel to determine the number of inclusion forming units per mL in the sample. The main advantage of this technique is that no or minimal sample dilution is required, thus minimizing dilution errors. In addition, this technique was employed during the early, middle and late growth stages of the chlamydial developmental cycle and results correlated well (P < 0.01) with 16S rRNA values from previous experiments, thereby proving its suitability to follow chlamydial growth in vitro. The method described is highly suitable for high throughput titration of cell culture inoculated samples and assessment of possible antichlamydial effects of novel compounds throughout the chlamydial growth cycle.
Asunto(s)
Técnicas Bacteriológicas/métodos , Chlamydophila psittaci/crecimiento & desarrollo , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Animales , Línea Celular , Pollos , Chlamydophila psittaci/química , Viabilidad MicrobianaRESUMEN
To explore the molecular basis of antigen recognition by germline antibodies, we have determined to high resolution the structures of the near-germline monoclonal antibody S25-2 in complex with seven distinct carbohydrate antigens based on the bacterial sugar 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (Kdo). In contrast to previous findings, the inherited germline Kdo monosaccharide binding site is not restricted to this bacterial sugar but is able to accommodate an array of substitutions and chemical modifications of Kdo, including naturally occurring antigens containing the related monosaccharide d-glycero-alpha-d-talo-oct-2-ulosonic acid as well as nonterminal Kdo residues. However, we show by surface plasmon resonance and ELISA how antibody S25-2 specificity is so dependent on the context in which the antigen is presented that a free disaccharide displays strong binding while the same lipid-A-bound disaccharide does not bind. These structures provide insight into how inherited germline genes code for immunoglobulins of limited flexibility that are capable of binding a range of epitopes from which affinity-matured antibodies are generated.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Productos Biológicos/química , Productos Biológicos/inmunología , Epítopos/química , Epítopos/inmunología , Animales , Antígenos/química , Antígenos/metabolismo , Chlamydophila psittaci/química , Chlamydophila psittaci/inmunología , Ensayo de Inmunoadsorción Enzimática , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Ratones , Modelos Moleculares , Estructura Molecular , Unión Proteica , Estructura Terciaria de Proteína , Azúcares Ácidos/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
The lipopolysaccharide (LPS) of Chlamydia trachomatis serotype E was isolated from tissue culture-grown elementary bodies and analyzed structurally by mass spectrometry and 1H, 13C and 31P nuclear magnetic resonance. The LPS is composed of the same pentasaccharide bisphosphate alphaKdo-(2-8)-alphaKdo-(2-4)-alphaKdo-(2-6)-betaGlcN-4P-(1-6)-alphaGlcN-1P (Kdo is 3-deoxy-alpha-d-manno-oct-2-ulosonic acid) as reported for C. trachomatis serotype L2[Rund, S., Lindner, B., Brade, H. and Holst, O. (1999) J. Biol. Chem. 274, 16819-16824]. The glucosamine disaccharide backbone is substituted with a complex mixture of fatty acids with ester or amide linkage whereby no ester-linked hydroxy fatty acids were found. The LPS was purified carefully (with contaminations by protein or nucleic acids below 0.3%) and tested for its ability to induce proinflammatory cytokines in several readout systems in comparison to LPS from C. trachomatis serotype L2 and Chlamydophila psittaci strain 6BC as well as enterobacterial smooth and rough LPS and synthetic hexaacyl lipid A. The chlamydial LPS were at least 10 times less active than typical endotoxins; specificity of the activities was confirmed by inhibition with the LPS antagonist, B1233, or with monoclonal antibodies against chlamydial LPS. Like other LPS, the chlamydial LPS used toll-like receptor TLR4 for signalling, but unlike other LPS activation was strictly CD14-dependent.
Asunto(s)
Chlamydia trachomatis/química , Chlamydophila psittaci/química , Proteínas de Drosophila , Lipopolisacáridos/química , Lipopolisacáridos/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Células CHO/metabolismo , Infecciones por Chlamydia , Chlamydia trachomatis/clasificación , Chlamydophila psittaci/clasificación , Cricetinae , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/metabolismo , Citometría de Flujo , Humanos , Interleucina-8/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Receptores de Lipopolisacáridos/inmunología , Receptores de Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/metabolismo , FN-kappa B/metabolismo , Resonancia Magnética Nuclear Biomolecular , Receptores de Superficie Celular/metabolismo , Serotipificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Receptor Toll-Like 4 , Receptores Toll-Like , TransfecciónRESUMEN
Lipopolysaccharides (LPSs) of Chlamydophila psittaci 6BC and Chlamydophila pneumoniae Kajaani 6 contain 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), GlcN, organic bound phosphate, and fatty acids in the molar ratios of approximately 3:2:2.2:4.8 and approximately 2.9:2:2.1:4.9, respectively. The LPSs were immunoreactive with a monoclonal antibody against a family-specific epitope of chlamydial LPS. This finding, together with methylation analyses of both LPSs and MALDI-TOF MS experiments on de-O-, and de-O,N-acylated LPSs, indicate the presence of a Kdo trisaccharide proximal to lipid A having a structure alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo, which appears to be the main component of the core region in the native chlamydial LPSs. In the de-O-acylated LPSs from Chl. psittaci 6BC and Chl. pneumoniae Kajaani 6, two major molecular species are present that differ in distribution of amide-bound hydroxy fatty acids over both GlcN. It appears that either two (R)-3-hydroxy-18-methylicosanoic acids or one (R)-3-hydroxy-18-methylicosanoic acid and one (R)-3-hydroxyicosanoic acid are attached to the GlcN residues. In contrast, the de-O-acylated LPS of Chl. psittaci PK 5082 contains one major molecular species that has two (R)-3-hydroxyicosanoic acid residues attached to two GlcN residues.
Asunto(s)
Chlamydophila pneumoniae/química , Chlamydophila psittaci/química , Lipopolisacáridos/química , Western Blotting , Secuencia de Carbohidratos , Ácidos Grasos/análisis , Lipopolisacáridos/análisis , Datos de Secuencia Molecular , Estructura Molecular , Organofosfatos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Azúcares Ácidos/análisisRESUMEN
Eight strains of Chlamydia psittaci were isolated in Japan from the nasal and conjunctival swabs of six household cats using the L929 cell line of mouse fibroblast origin. The isolates were identified as C. psittaci on the basis of the formation of characteristic inclusion bodies in the cell culture detected by Giemsa stain and immunofluorescence. Comparison of nucleotide sequences of the ompA gene amplified from the three isolates with the published sequence of feline FEPN strain of C. psittaci showed almost 100% homology.
Asunto(s)
Enfermedades de los Gatos/microbiología , Infecciones por Chlamydia/veterinaria , Chlamydophila psittaci/aislamiento & purificación , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/biosíntesis , Gatos , Células Cultivadas , Infecciones por Chlamydia/microbiología , Chlamydophila psittaci/química , Chlamydophila psittaci/genética , Conjuntivitis/microbiología , Conjuntivitis/veterinaria , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Cuerpos de Inclusión/microbiología , Ratones , Reacción en Cadena de la Polimerasa/veterinaria , Rinitis/microbiología , Rinitis/veterinariaRESUMEN
The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/análisis , Chlamydophila psittaci/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Disulfuros/química , Peso MolecularRESUMEN
The oligosaccharides alpha-Kdop-(2-->8)-alpha-Kdop-(2-->6)-beta-D- GlcpNAc-(1-->OAll) 4, alpha-Kdop-(2-->4)-alpha- Kdop-(2-->4)-alpha-Kdop-(2-->6)-beta-D-GlcpNAc-(1-->OAll+ ++) 10, and the branched Kdo tetrasaccharide alpha- Kdop-(2-->4)-[alpha-Kdop-(2-->8)]-alpha-Kdop-(2-->4)-a lpha-Kdop-(2-->OAll) 21 have been prepared using en bloc transfer of Kdo oligosaccharide bromide donors to protected mono- or disaccharide acceptors. Radical addition of cysteamine to the anomeric allyl glycosides afforded good yields of the corresponding 3-(2-aminoethylthio)propyl glycosides 5, 11 and 22. The spacer ligands were activated with thiophosgene and reacted with bovine serum albumin to give the neoglycoconjugates 6, 12 and 23 which were used to prepare solid-phase antigens in enzyme immuno-assays for the characterization of monoclonal antibodies against chlamydial LPS. The data showed that the (2-->8)-linked Kdo disaccharide and the (2-->8)-(2-->4)-linked Kdo trisaccharide portion of the neoglycoconjugate 23 were not available for binding of antibodies which recognize these structures as di- and trisaccharide, respectively.
Asunto(s)
Chlamydophila psittaci/inmunología , Epítopos/inmunología , Glicoproteínas/síntesis química , Lipopolisacáridos/inmunología , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Bovinos , Chlamydophila psittaci/química , Glicoproteínas/química , Glicoproteínas/inmunología , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/inmunologíaRESUMEN
The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.
Asunto(s)
Chlamydophila psittaci/química , Lipopolisacáridos/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Línea Celular , Cloroformo/química , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Hidrazinas/química , Hidróxidos/farmacología , Lípido A/química , Lípido A/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Ratones , Datos de Secuencia Molecular , Fenol/química , Compuestos de Potasio/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Azúcares Ácidos/química , Factores de Tiempo , Agua/químicaAsunto(s)
Lipopolisacáridos/metabolismo , Oligosacáridos/aislamiento & purificación , Fosfatos/aislamiento & purificación , Acilación , Conformación de Carbohidratos , Secuencia de Carbohidratos , Chlamydophila psittaci/química , Chlamydophila psittaci/genética , Cromatografía por Intercambio Iónico , Lipopolisacáridos/química , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Oligosacáridos/química , Fosfatos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transferasas/química , Transferasas/genética , Transferasas/metabolismoRESUMEN
The EUO gene of chlamydia is highly expressed early in the developmental cycle, relative to other genes, but continues to be expressed throughout the active growth phases. The precise function of EUO protein is not known, but it binds to DNA in vitro. In this study, we developed a selection and amplification scheme for identifying chlamydial genomic fragments to which EUO preferentially binds in vitro. The scheme involved mixing recombinant EUO with a Chlamydia psittaci genomic library in a pBluescript plasmid vector in vitro, trapping EUO-bound plasmid clones on filters, and amplifying the clones in Escherichia coli. After nine rounds of enrichment, the EUO binding sites of the three most highly enriched clones were identified by DNase I footprint analysis. All three clones had multiple binding sites of various sizes with no clear distinguishing feature other than they were AT-rich and were usually not located in putative promoter regions. We used limited site-specific mutagenesis to characterize the strongest binding site of the most-highly-enriched clone, which represented about 50% of the population after nine rounds. This mutagenesis identified a core binding site of 15 nucleotides (nt) whose sequence was used to find related sequences within each of the strong binding sites in the other two clones. Using the frequency of bases at specific positions within this group of sequences as a guide, we carried out trial-and-error searching with many related sequences, eliminating those which identified nonfootprinted sites. This process led us to the consensus 15-nt sequence AHGAAAWVTYTWDAY, which, when allowing two mismatches, picked out all of the strong binding sites and no nonfootprinting sites within the three enriched clones. This sequence may be useful for predicting additional possible EUO binding sites in the chlamydial genome.
Asunto(s)
Chlamydophila psittaci/química , ADN/metabolismo , Endopeptidasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Desoxirribonucleasa I/farmacología , Datos de Secuencia Molecular , Mutación , Proteínas Recombinantes/metabolismo , Transcripción GenéticaRESUMEN
A monoclonal antibody (mAb) S45-18 was generated against a synthetic neoglycoconjugate containing the trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) which represents a structure of the lipopolysaccharide (LPS) from Chlamydophila psittaci 6BC. The antibody was characterized by binding and inhibition assays in ELISA using: (i) the immunizing antigen and chemically synthesized derivatives thereof; (ii) chlamydial elementary bodies (EB); and (iii) LPS of Chl. psittaci 6BC and Chlamydia trachomatis L2. The specificity was determined in comparison to that of mAb S25-23 recognizing the alphaKdo(2-->8)alphaKdo(2-->4)alphaKdo trisaccharide which represents an epitope shared by all species of the family. MAb S45-18 bound to an epitope of the structure alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo, with lower reactivity with the (2-->8)-(2-->4)-linked analog. Using chlamydial EB or LPS, mAb S45-18 bound preferentially to LPS and EB of Chl. psittaci. Therefore, Chl. psittaci LPS contains, in addition to the known genus-specific epitope, a species-specific epitope.
Asunto(s)
Anticuerpos Monoclonales , Chlamydophila psittaci/inmunología , Lipopolisacáridos/inmunología , Trisacáridos/química , Trisacáridos/inmunología , Animales , Anticuerpos Antibacterianos , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Secuencia de Carbohidratos , Chlamydophila psittaci/química , Epítopos/química , Glicoconjugados/química , Glicoconjugados/inmunología , Lipopolisacáridos/química , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Especificidad de la EspecieRESUMEN
A lipopolysaccharide (LPS) of Chlamydia psittaci PK 5082 strain associated with enzootic abortion in ewes was isolated from embryonated hen eggs-grown elementary bodies (EBs) by a phenol/water procedure. Compositional analyses revealed the presence of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), glucosamine (GlcN), phosphorus, and fatty acids in a molar ratio of 2.6:2.0:2.4:4.4. GlcN was the only amino sugar detected. Methylation analysis of the LPS confirmed the presence of a Kdo trisaccharide proximal to lipid A having the structure Kdo-(2-->8)-Kdo-(2-->4)-Kdo, which appears to be a highly conserved region in native chlamydial LPSs. The complex fatty acid composition revealed the presence of ten different straight or branched (iso and anteiso) nonhydroxy fatty acids and thirteen 3-hydroxy fatty acids. The major nonhydroxy fatty acid was icosanoic acid and the most prominent 3-hydroxy fatty acid was 3-hydroxyicosanoic acid followed by 3-hydroxy-18-methylicosanoic acid. The 3-hydroxy fatty acids represented more than two thirds of the total fatty acid content and most of them were bound in amide linkages. In contrast, most nonhydroxy fatty acids were ester-linked. It appears that LPSs from various chlamydial species differ in fatty acid composition and distribution.
Asunto(s)
Chlamydophila psittaci/química , Lipopolisacáridos/química , Animales , Secuencia de Carbohidratos , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/análisis , Cromatografía de Gases y Espectrometría de Masas , Datos de Secuencia Molecular , Azúcares Ácidos/análisisRESUMEN
The putative outer membrane location of the OMP90 (formerly POMP) family from the ovine abortion strain of Chlamydia psittaci was investigated by immunoelectron microscopy. Using a non-embedding technique, antigens were shown to be localised on the outer membrane surface of both elementary and reticulate bodies, the infectious and non-infectious forms of Chlamydiae respectively. Antibodies affinity-purified against the expressed amino- and carboxy-terminal halves of one of the family members. OMP90A, demonstrated that the amino half is surface-exposed while the carboxyl half is most probably localised internally. Surface localisation on elementary bodies indicates the importance of these proteins as protective antigen candidates.
Asunto(s)
Antígenos Bacterianos/análisis , Proteínas de la Membrana Bacteriana Externa/análisis , Chlamydophila psittaci/química , Aborto Veterinario/microbiología , Animales , Anticuerpos Monoclonales , Electroforesis en Gel de Poliacrilamida , Femenino , Immunoblotting , Inmunohistoquímica , Microscopía Inmunoelectrónica , Embarazo , Psitacosis/microbiología , Psitacosis/veterinaria , Ovinos , Enfermedades de las Ovejas/microbiologíaRESUMEN
The EUO gene (for early upstream open reading frame) of Chlamydia psittaci was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with C. psittaci 6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp -200 to +67 promoter region of the cysteine-rich envelope protein (crp) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects crp DNA from DNase I from approximately bp -60 to -9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the crp operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the crp operon with high affinity in vitro (Kd of about 15 nM), it is not known whether the protein binds the crp DNA in vivo.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chlamydophila psittaci/metabolismo , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Chlamydophila psittaci/química , Datos de Secuencia Molecular , OperónRESUMEN
The kdsA gene encoding 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo-8-P) synthase of Chlamydia psittaci 6BC was cloned by complementing the temperature-sensitive kdsA mutant Salmonella enterica serovar Typhimurium AG701i50. The sequence analysis of a recombinant DNA fragment revealed an open reading frame of 807 nucleotides which codes for a polypeptide of 269 amino acids with a high degree of similarity to known KdsA proteins. In addition, alignments of Kdo-8-P synthases with bacterial and fungal 3-deoxy-D-arabino-2-heptulosonate-7-phosphate (Dha-7-P) synthases suggested that both classes of enzymes are structurally related and may belong to a family of 2-keto-3-deoxy-aldonic acid synthases. The chlamydial protein was overexpressed and functionally characterized in vitro to synthesize Kdo-8-P from D-arabinose 5-phosphate and phosphoenolpyruvate. A chlamydial DNA region upstream of the gene exhibiting similarities to the consensus sequence of sigma 70 promoters of Escherichia coli was responsible for the heterologous expression of kdsA.
Asunto(s)
Aldehído-Liasas/genética , Chlamydophila psittaci/enzimología , Chlamydophila psittaci/genética , Genes Bacterianos/fisiología , Aldehído-Liasas/biosíntesis , Aldehído-Liasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Chlamydophila psittaci/química , Clonación Molecular , Datos de Secuencia Molecular , Azúcares Ácidos/metabolismoRESUMEN
Four major clusters, designated A, B, C and D, were distinguished in Western Blots by a monoclonal antibody specific for the "antigen family at 90 kDa" after two-dimensional electrophoretic analysis on immobilized pH gradient of chlamydial elementary bodies of abortifacient C. psittaci. Clusters B, C, and D were closely related with molecular mass (kDa) pI values of 91.5/5.2-5.4, 90/5.0-5.2 and 90.5/5.6-5.8, respectively. Cluster A was larger, with molecular mass/pI of 104.7/5.1-5.3. Evidence for the antigenic relationship between cluster A and clusters B, C and D was further supported by immunological cross-reaction with affinity-purified antibodies from serum of ewes with chlamydial-induced abortion. The experimental values obtained for size and pI of the four clusters correlated well with the calculated values from known sequences coded by multiple chlamydial genes. Direct evidence for the correspondence between the immunoreactive clusters B, C and D and the retrieved genes was provided by antibody binding experiments to recombinant polypeptides representing fragments of the deduced proteins. The 4-member antigen family at 90/104 kDa is the first example of proteins coded by multiple genes within the genus Chlamydia.
Asunto(s)
Proteínas Bacterianas/análisis , Chlamydophila psittaci/química , Electroforesis en Gel Bidimensional/métodos , Aborto Veterinario/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/inmunología , Western Blotting , Chlamydophila psittaci/inmunología , Femenino , Punto Isoeléctrico , Datos de Secuencia Molecular , Peso Molecular , Embarazo , Ovinos/microbiología , Enfermedades de las Ovejas/microbiologíaRESUMEN
Analysis by two-dimensional gel electrophoresis of the N-laurylsarkosinate(Sarkosyl)-insoluble envelope complexes of L-[35]S-cysteine-labeled elementary bodies of Chlamydia pneumoniae strain IOL-207, Chlamydia trachomatis serovar LGV2, D, and F, and Chlamydia psittaci strain 6BC showed differences in the molecular charges of chlamydial outer membrane proteins. The apparent isoelectric point (pI) of the major outer membrane protein of C. pneumoniae strain IOL-207 was 6.4, whereas the pI of the major outer membrane protein of the C. trachomatis and C. psittaci strains differed little from one another, ranging from 5.3 to 5.5. The 60-kDa cysteine-rich protein of C. pneumoniae was the only 60-kDa chlamydial protein with a pI value (5.9) more acidic than that of the corresponding major outer membrane protein. As a general rule, the charges of both the 60-kDa and the low-molecular-mass (12-15 kDa) cysteine-rich proteins were widely variable, depending on the strain. However, in each individual strain, the variation of the charge of the 60-kDa protein had a compensatory change in the low-molecular-mass cysteine-rich protein.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Chlamydia trachomatis/química , Chlamydophila pneumoniae/química , Chlamydophila psittaci/química , Electroforesis en Gel de Poliacrilamida , Punto Isoeléctrico , Peso MolecularRESUMEN
A chlamydial agent was recovered from the placental cotyledons of an aborting cow from a 100-cow dairy herd in Cumbria. Immunoblotting analysis of purified elementary bodies of the isolate revealed a reactivity pattern typical of serotype I Chlamydia psittaci strains. Nucleotide sequencing of the major outer membrane protein (MOMP) gene further confirmed the isolate, BA1, as a serotype I strain. The sequence was identical to that of the type strain of ovine enzootic abortion, B577. In both the antigenic and MOMP sequencing analyses BA1 was distinguishable from serotype II C. pecorum strains. A sequential series of sera obtained from the aborting cow, from which BA1 was recovered, was analysed by immunoblotting against the homologous isolate, and demonstrated reactivity to major chlamydial antigens over a 110-day period. Close contact between ruminant species on the farm suggested that the C. psittaci strain may have been transmitted to cattle from infected sheep.