RESUMO
Serum samples obtained from patients hospitalized in Barbados with severe leptospirosis were tested by the microscopic agglutination test (MAT), enzyme immunoassay (EIA) and immunoblotting with leptospires that had been isolated from these patients. While serum samples taken a few days after onset of symptoms often showed no apparent correlation between MAT and EIA, later sequential serum samples produced similar profiles in both tests during the course of infection. Immunoblotting sonicate from Leptospira interrogans serovars arborea, copenhageni and bim with patients' sera, revealed reactions with a number of bands that corresponded with outer envelope components. These components included lipopolysaccharide (LPS), flagella and other outer membrane proteins, in addition to a low-molecular-weight (MW) carbohydrate cross-reactive with members of the Leptospiraceae. IgM antibodies elicited in the first to second week after infection reacted mainly with LPS and the low-MW cross-reactive carbohydrate. Comparative analysis of isolates of the same serovar by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting showed that while two serovar arborea isolates were identical, serovar bim isolates differed significantly from each other. This difference was also observed in comparative MAT testing.
Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Leptospira interrogans/imunologia , Doença de Weil/imunologia , Testes de Aglutinação , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/imunologia , Barbados , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Leptospira interrogans/análise , Lipopolissacarídeos/análiseRESUMO
Whole-cell and detergent-soluble proteins, enriched for outer membrane antigens, of the Leptospira interrogans serovars present in commercially available pentavalent vaccines (hardjo, pomona, icterohaemorrhagiae, grippotyphosa, and canicola) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). Protein and antigenic profiles of these serovars, representing several serogroups, were compared with similar profiles of the most common North American bovine pathogen, serovar hardjo type hardjo bovis. The reference strain of serovar balcanica and a hardjoprajitno bovine field isolate (serovar hardjo) were also assayed. Coomassie blue-stained gels revealed extensive protein similarities, and Western blots demonstrated antigenic relatedness throughout the low- and high-molecular-weight regions. Possible quantitative differences in protein expression among the strains were noted, as were similarities in the protein profiles of type hardjo bovis and the balcanica reference strain. A cocktail composed of these homologous antigens may serve as an efficacious subunit vaccine for leptospirosis.
Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Leptospira interrogans/imunologia , Animais , Vacinas Bacterianas/imunologia , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Leptospira interrogans/análise , Coelhos , SorotipagemRESUMO
Outer membrane and secreted proteins were isolated from Leptospira interrogans serovar pomona and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblot and radioimmunoprecipitation techniques. The L. interrogans outer membranes were extracted with Triton X-114 and contained several proteins. The major cellular protein with a molecular mass of 31 kDa was associated exclusively with the L. interrogans outer membrane. Using a whole cell immunoprecipitation method, five hydrophobic, Triton X-114 extractable proteins (22, 26, 31, 36 and 42 kDa) were exposed on the surface of L. interrogans. The 31 kDa protein was heat labile and was a potent antigen in animals experimentally infected with L. interrogans serovar pomona. Several proteins were secreted by L. interrogans including a 60 kDa protein tentatively identified as the L. interrogans hemolysin.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Bactérias/metabolismo , Leptospira interrogans/análise , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/imunologia , Proteínas Hemolisinas/metabolismo , Temperatura Alta , Leptospira interrogans/crescimento & desenvolvimento , Leptospira interrogans/metabolismo , Proteínas de Membrana/isolamento & purificação , Peso Molecular , SuínosRESUMO
Surface components of virulent and attenuated Leptospira interrogans serovar grippotyphosa were compared by using Triton X-114 solubilization and phase partitioning, immunoprecipitation of intact organisms, and freeze-fracture electron microscopy. Removal of the leptospiral outer membrane by using 0.1% Triton X-114 was demonstrated by whole-mount electron microscopy and by essentially complete solubilization of a lipopolysaccharidelike substance (LLS) from the outer membrane. Triton X-114 (0.1%) did not solubilize subsurface proteins, such as endoflagellar filaments or penicillin-binding proteins, which are markers for the periplasmic space and inner membrane, respectively. Triton X-114 solubilized material from both the virulent and attenuated strains, which partitioned into the hydrophobic, detergent phase, contained LLS and major proteins of 41 and 44 kDa, which were also immunoprecipitable from intact organisms. The virulent strain contained greater amounts of an LLS component with an apparent molecular mass of 30 kDa (R(f) = 0.57), whereas the attenuated strain contained larger amounts of an LLS component with an apparent molecular mass of 20 kDa (R(f) = 0.74). Differences in protein components between virulent and attenuated organisms were also detected; whereas the 41- and 44-kDa proteins were immunoprecipitated in equal amounts from both the virulent and attenuated strains, a 33-kDa protein was immunoprecipitated in significantly greater amounts from the attenuated strain. Quantitation of outer membrane particle density by freeze-fracture electron microscopy showed that both strains had a low transmembrane outer membrane protein content compared with that of typical gram-negative bacteria. The virulent and attenuated strains had 443 and 990 particles (P less than 0.000001) per micron, respectively, in the concave outer membrane fracture face. These findings suggest that in vitro cultivation of L. interrogans is accompanied by quantitative and qualitative changes in both LLS and outer membrane-associated proteins.
Assuntos
Antígenos de Superfície/análise , Proteínas da Membrana Bacteriana Externa/análise , Leptospira interrogans/patogenicidade , Polissacarídeos Bacterianos/análise , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Cricetinae , Eletroforese em Gel de Poliacrilamida , Técnica de Fratura por Congelamento , Leptospira interrogans/análise , Leptospira interrogans/imunologia , Leptospira interrogans/ultraestrutura , Peso Molecular , Octoxinol , Polietilenoglicóis , Polissacarídeos Bacterianos/imunologia , Testes de Precipitina , Coelhos , Inoculações Seriadas , Solubilidade , VirulênciaRESUMO
Fatty acid profiles of six leptospira strains representative of genera, species, and serogroups within the family Leptospiraceae were determined by gas liquid chromatography (GLC) of fatty acid methyl ester (FAME) derivatives. The influence of methodological and biological variables on FAME profiles of the same strain was tested. FAME profiles were sharply affected by the fatty acid composition of the culture medium but not by the growth phase. Twenty-four FAME peaks were selected on the basis of their presence in repeated gas chromatographic runs of single strains. Inter-strain divergences of FAME profiles were quantified by linear regression analysis (LR). Step-wise divergences in FAME profiles were observed between strains at serogroup, species, and genus levels.
Assuntos
Ácidos Graxos/análise , Leptospira/análise , Spirochaetaceae/análise , Cromatografia Gasosa , Ésteres , Leptospira/classificação , Leptospira interrogans/análise , Leptospira interrogans/classificação , Leptospira interrogans serovar canicola/análise , Leptospira interrogans serovar canicola/classificação , Análise de Regressão , Spirochaetaceae/classificaçãoRESUMO
We applied SDS-PAGE, 2D-PAGE and Western blot to analyse the outer envelopes protein and LPS of five strains of leptospires. The work would lay foundations for taxonomy, the development of vaccination regimens and the elucidation of pathogenic mechanisms. The outer envelope proteins of leptospires were analyzed by SDS-PAGE and silver staining. We found that the protein profiles of the pathogenic leptospires were basically identical. A comparison of the protein profiles of the pathogenic L. with those exhibited by two nonpathogenic L. indicated that there was no obvious relationship between these organisms and any of the L. interrogans strains examined. The quantity of 21.5 kd protein of strain 017 was greater than that of strain 601 and 156. Approximately 200, 225, 238 distinct polypeptides were detected in the strain 017, 601 and 156 in 2D-PAGE by silver staining respectively. The profiles of 2D-PAGe showed obvious differences in pI. The pI of strain 017, 601 and 156 were mainly 6.68-7.4, 6.55-6.9, 5.85-7.1 respectively. The 21.5 kd protein of strain 017 was made up of six polypeptides. Our immunoblots revealed that McAb (LB1) reacted with a 41 kd antigen, which was common to the three virulent leptospires tested. SDS-PAGE profiles of silver stained outer envelope LPS of pathogenic L. differed greatly from those of the nonpathogenic L. There was a distinct differences between strain Patoc I and 3055. Our studies showed that each of the five strains of leptospires possessed characteristic outer envelope LPS, which may be used to identify the genus, species and serovars of a strain of L.
Assuntos
Proteínas de Bactérias/análise , Leptospira/análise , Lipopolissacarídeos/análise , Western Blotting , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Leptospira/classificação , Leptospira interrogans/análiseRESUMO
Lipopolysaccharides (LPSs) from Leptospira interrogans serovar hardjo (reference strain hardjoprajitno and strain hardjobovis) were prepared by the hot phenol-water procedure. High yields of LPSs were found in the phenol phase. Gel electrophoresis of the phenol phase LPSs showed similar patterns for all strains in contrast to the different patterns found in the water phase LPSs. Sugar composition was also similar among all strains with rhamnose as the predominant sugar. Mannosamine was detected by high performance thin layer and gas-liquid chromatography. 2-Keto-3-deoxyoctonic acid (KDO) was comparable with authentic KDO by paper chromatography. Periodate oxidation at near neutral pH with or without prior hydrolysis showed that most of the KDO was substituted. The fatty acid composition of strain hardjobovis LPS was slightly different from that of the reference strain hardjoprajitno. Myristic and 3-hydroxymyristic acid were not detected in any of the LPS preparations. In conjunction with genetic and other data, the two strains are sufficiently different to be regarded as members of two separate species sharing common antigens. There is sufficient evidence to rename the hardjoprajitno strain type L. interrogans hardjo-p, and the hardjobovis strain type L. borgpeterseni hardjo-b.
Assuntos
Leptospira interrogans/análise , Lipopolissacarídeos/isolamento & purificação , Carboidratos/análise , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/análise , Imuno-Histoquímica , Leptospira interrogans/classificação , Fenol , Fenóis , Dodecilsulfato de Sódio , Especificidade da EspécieRESUMO
Acredtita-se que as lesöes teciduais na leptospirose possam decorrer da açäo direta das leptospiras, de toxinas sintetizadas ou liberadas durante sua lise. O presente estudo visou a extraçäo química da glicolipoproteína (GLP) da aleptospira, a produçäo de anti-soro anti-GLP e a avaliaçäo de sua distribuiçäo em cortes de fígado e rim de cobaias inoculadas e sacrificadas em estudo sequencial diário até o 6§ dia de infecçäo, correspondente ao pico da doença. Procurou-se também correlacionar a expressäo tecidual da GLP com o grau de lesöes locais, em busca de novos subsídios para a compreensäo da patogenia da leptospiros. A GLP foi detectada em fígado e rim de 2 dentre 6 cobaias no 5§ dia e em todas as 6 no 6§ dia de infecçäo, sob a forma de grânulos no citoplasma de macrófagos, livres no interstício ou acolados à membrana de células endoteliais e parenquimatosas, especialmente nas regiöes mais lesadas. A cronologia do aparecimento da GLP e sua distribuiçäo sugerem tratar-se de produto de lise de leptospiras fagocitadas por macrófagos e que esta substância, conquanto näo comprovada como iniciadora das lesöes, asocia-se a seu agravamento nas etapas mais avançadas da leptospirose
Assuntos
Animais , Cobaias , Doença de Weil/etiologia , Glicoproteínas/toxicidade , Leptospira interrogans/análise , Proteínas de Bactérias/toxicidade , Fígado/análise , Glicoproteínas/análise , Rim/análise , Proteínas de Bactérias/análiseRESUMO
Tissue damage in leptospirosis has been ascribed to direct effect of the microorganisms and/or their virulence, including products synthetized by leptospires or released during their lysis. This study aimed at chemical extraction of the glycolipoprotein (GLP) from virulent leptospires, production of a rabbit anti-GLP and analysis of its distribution in liver and kidney of inoculated guinea-pigs, sacrificed sequentially from the 1st to 6th day of infection, covering the whole, spectrum of acute leptospirosis. The comparison of GLP expression to local injuries aimed at new pathogenetic data. GLP was detected in liver and kidney in 2 out of 6 guinea-pigs on the 5th day and in all 6 animals on the 6th day of infection. Granular forms were seen in the cytoplasm of macrophages, free in interstitium or adhered to endothelial and parenchymal cell membranes, especially in the most damaged sites. These findings lead us to the hypothesis of GLP as a toxic factor resulting from leptospiral lysis by macrophages. Although it was not proved as a promoter of initial lesions, it seems to be related to the enhancement of tissue damage late in the course of the disease.
Assuntos
Proteínas de Bactérias/toxicidade , Glicoproteínas/toxicidade , Leptospira interrogans/análise , Doença de Weil/etiologia , Animais , Proteínas de Bactérias/análise , Glicoproteínas/análise , Cobaias , Rim/análise , Fígado/análiseRESUMO
A modified method, differential centrifugation followed by sucrose density centrifugation, was used to purify axial filaments from three strains of Leptospires. Ultrastructure of the axial filaments was studied and profiles of the axial filaments were characterized and compared. The results have shown that all the three strains of Leptospires, i.e., L. interrogsans serovar Lai strain 017, L. biflexa serovar patoc strain Patoc I and L. illini strain 3055, have two axial filaments in one cell. The axial filament is 20 nm in diameter. It is the first observation that the end which inserts the cytoplasms cylinder is wider in diameter than the free one. An insertion pore structure is observed. The new method yields 1.5mg axial filaments from 12 g leptospires cells. SDS-PAGE was first employed in the analysis of axial filaments of leptospires. The results have also shown that there are 6 proteins in the axial filaments of strain 017, MW 26,000-50,000 while 7 proteins in the axial filaments of strain Patoc I and strain 3055. MW 29,000-80,000 and 28,500-80,000 respectively. Interestingly, all the axial filaments of the three strains have a common protein band of MW 31,500. The possibility of using axial filament proteins as a new criterion for typing and a serodiagnosis antigen is discussed.
Assuntos
Proteínas do Citoesqueleto/isolamento & purificação , Leptospira/análise , Animais , Eletroforese em Gel de Poliacrilamida/métodos , Leptospira/ultraestrutura , Leptospira interrogans/análise , Leptospira interrogans/ultraestruturaRESUMO
Purified preparations of lipopolysaccharides (LPS) extracted from two different strains of Leptospira interrogans have been electrophoretically analyzed in order to determine their location at the level of outer envelope (OE). Evidence has been collected for the presence of some LPS fractions in the OE, suggesting that a part of this molecule is embedded in the membrane structure. The serological specificity of the LPS has been in addition tested by means of monoclonal antiserovar antibodies (Moabs); the results indicated that the LPS structure is endowed of the immunodeterminants of the serovar. The remarkable relevance of this finding for the Leptospira taxonomy is discussed.
Assuntos
Leptospira interrogans/análise , Lipopolissacarídeos/análise , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Membrana Celular/análise , Eletroforese em Gel de Poliacrilamida , Epitopos , Leptospira interrogans/classificação , Leptospira interrogans/ultraestrutura , Lipopolissacarídeos/imunologia , Testes de PrecipitinaRESUMO
Leptospira interrogans, serovar copenhageni was extracted by the phenol-water method and the resulting preparation examined for chemical composition and endotoxic activity. Chemical analysis revealed that a number of sugars were present, however, the amount of lipid content was very low. Further, the preparation was devoid of characteristic endotoxic properties, like lethal toxicity, pyrogenicity and the property to induce the local Shwartzman reaction. The extract, however, was active in the limulus lysate gelation test and in the induction of monocyte activation. It is concluded that the leptospira preparation is devoid of endotoxin properties, both from the chemical and from the biological point of view.
Assuntos
Leptospira interrogans/análise , Lipopolissacarídeos/análise , Animais , Carboidratos/análise , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/análise , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Fenol , Fenóis , Fenômeno de Shwartzman , ÁguaRESUMO
Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.
Assuntos
DNA Bacteriano/análise , Leptospira interrogans/classificação , Animais , Enzimas de Restrição do DNA , Leptospira interrogans/análise , Leptospira interrogans/genética , Ratos , SorotipagemRESUMO
Strain Ictero No. I, the first isolate of Leptospira, isolated by Inada and Ido in 1914, was found to be sufficiently qualified to be the type strain of Leptospira interrogans rather than strain RGA. In an agglutinin absorption test, anti-Ictero No. I serum was not absorbed completely with strain RGA, and 25% of the homologous titer remained unabsorbed, while anti-RGA serum was completely absorbed with strain Ictero No. I. Thus, strain Ictero No. I was not serologically identical with strain RGA, and the two strains were considered to be different serovars. A protein band with a molecular weight of approximately 33,000 daltons was detected in strain Ictero No. I but not in strain RGA by SDS-PAGE. By Western blotting, this protein band was detectable with anti-Ictero No. I serum but not with anti-RGA serum. The presence of the 33K protein in strain Ictero No. I, but not in strain RGA, was confirmed by radioimmunoprecipitation using [125I]-labeled antigens, indicating that the protein antigen was surface-exposed. Only 8 of the 89 enzymes activities were different between strains Ictero No. I and RGA (line Sapporo). From the above results, we propose that strain Ictero No. I should be designated as the type strain of L. interrogans instead of strain RGA.
Assuntos
Leptospira interrogans/classificação , Testes de Aglutinação , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Reações Cruzadas , Enzimas/isolamento & purificação , Leptospira interrogans/análise , Leptospira interrogans/imunologia , Especificidade da EspécieRESUMO
The aqueous layer was isolated from Leptospira interrogans serovar canicola strain Moulton by the hot phenol-water method. After ultracentrifugation, the precipitate was designated as lipopolysaccharide-like substance (LLS) fraction and the chemical composition was compared with that of bacterial LPS. The LLS fraction consists of 35.2% carbohydrate, 3.8% amino sugar, 36.4% lipid, 15.2% protein, and 0.3% phosphorus. Neutral sugars were detected as rhamnose, arabinose, xylose, 4-O-methylmannose, mannose, galactose, and a small amount of erythrose, fucose and glucose by gas-liquid chromatography (GLC), but 2-keto-3-deoxyoctonic acid was not detected in the LLS by thiobarbituric acid test and high voltage paper electrophoresis. Fatty acids detected by GLC were decanoic acid (C10: 0), dodecanoic acid (C12: 0), dodecenoic acid (C12: 1), tridecenoic acid (C13: 1), tetradecanoic acid (C14: 0), hexadecanoic acid (C16: 0), hexadecenoic acid (C16: 1), and octadecenoic acid (C18: 1). With SDS-polyacrylamide gel electrophoresis, bacterial LPS showed many orderly bands, while the banding pattern of the leptospiral LLS was very simple. These findings demonstrate that the physicochemical properties and chemical composition of LLS fraction from Leptospira are different from those of LPS extracted from gram-negative bacteria such as Enterobacteriaceae, and suggesting that Leptospira has no typical LPS.
Assuntos
Leptospira interrogans/análise , Lipopolissacarídeos/análise , Amino Açúcares/análise , Proteínas de Bactérias/análise , Carboidratos/análise , Cromatografia Gasosa , Eletroforese em Papel , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/análise , Lipídeos/análise , Fósforo/análise , Açúcares Ácidos/análiseRESUMO
Growth of leptospiras in the presence of diphenylamine (DPA) caused a decrease in the content of leptospiral lipopolysaccharide (LPS). In association with this decrease of LPS, leptospiras became susceptible to anti-leptospiral action of normal rabbit serum (NRS), leptospiricidal action of antibody and complement, and killing by phagocytes. DPA-treated leptospiras were eliminated rapidly from the blood of infected mice and could not grow in the animals.
Assuntos
Compostos de Anilina/farmacologia , Atividade Bactericida do Sangue , Difenilamina/farmacologia , Leptospira interrogans/efeitos dos fármacos , Lipopolissacarídeos/análise , Fagocitose , Animais , Leptospira interrogans/análise , Leptospira interrogans/imunologia , Camundongos , Doença de Weil/imunologiaRESUMO
Lipopolysaccharide (LPS) from Leptospira interrogans serovar copenhageni was prepared from the aqueous phase of a phenol/water extract. Electron microscopic examination of negatively stained LPS showed a mixture of ribbon-like, round and ring structures. Carbohydrate analysis of the preparations revealed pentoses, hexoses, heptoses, hexosamines, and a 2-keto-3-deoxyonic acid which was chromatographically different from authentic 2-keto-3-deoxyoctonic acid (KDO). The major fatty acids of the LPS were hydroxylauric, palmitic and oleic acids. Although the leptospiral LPS preparations did not contain KDO or hydroxymyristic acid, they were otherwise morphologically and chemically similar to the LPS of other Gram-negative bacteria.