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At least 631 records · Page 35Linked to original sources
PubMed1988

Antigens recognised by the human immune response to infection with Leptospira interrogans serovar hardjo.

Serum samples from patients infected with Leptospira interrogans serovar hardjo were tested by the microscopic agglutination test (MAT), enzyme immunoassay (EIA) and immunoblotting. There was no apparent correlation between MAT titre and EIA optical density (OD) for individual serum samples, but sequential serum samples produced similar profiles in both tests during the course of an infection. Immunoblotting of hardjo sonicate with patients' sera revealed reactions with a number of bands, in the mol. wt (10(3] range 14.4-95. However, all serum samples reacted with the major 28 x 10(3)-mol. wt sub-unit of hardjo lipopolysaccharide (LPS) and most reacted with a (34.5-35) x 10(3)-mol. wt flagella doublet. Examination of sequential serum samples obtained over a period of about 3 months after infection revealed little change in the antigens detected after the second to third week of infection. Absorption of patients' sera with whole viable leptospires revealed that antibodies to several exposed antigens, including LPS, were produced. Sera which reacted with hardjo flagella also reacted with bands of similar mol. wts in preparations from other serovars.

Agglutination Tests↗
PubMed1989

Experimental immunisation of hamsters with lipopolysaccharide antigens of Leptospira interrogans.

Hamsters were immunised with leptospiral lipopolysaccharide (LPS) or the polysaccharide (PS) fraction of LPS from Leptospira interrogans serovar copenhageni and the antibody responses were measured by agglutination tests. Maximum titres were observed approximately 6 weeks after immunisation and protection against lethal challenge with the homologous strain was afforded by immunisation with as little as 2.5 micrograms of LPS or PS. All animals produced IgM agglutinins but a higher proportion of-animals immunised with PS produced IgG agglutinins than did those immunised with LPS. Immunisation of guinea-pigs with autoclaved PS showed that the preparation retained some but not all of its immunogenic activity.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1990

Vaccination of mice with lipopolysaccharide (LPS) and LPS-derived immuno-conjugates from Leptospira interrogans.

Mice were vaccinated with lipopolysaccharide (LPS) from Leptospira interrogans serovar pomona or hardjo, or with the polysaccharide (PS) fraction of the LPS, or with an immunoconjugate of PS and diphtheria toxoid (DT). Maximum agglutinin titres were found 6-10 weeks after vaccination with LPS or PS-DT conjugate; the latter elicited antibody titres at least 10 times higher than those produced in response to LPS. Animals failed to react significantly to PS. Titres elicited by antigens of serovar pomona were higher than those elicited by serovar hardjo.

Animals↗
PubMed1999

Expression in Escherichia coli of flaB, the gene coding for a periplasmic flagellin of Leptospira interrogans serovar pomona.

A periplasmic flagellin gene, flaB, of Leptospira interrogans serovar pomona (strain Pomona) was expressed in Escherichia coli for the production and antigenic characterisation of the protein. The flaB structural gene, which was previously cloned into pUC118, was derived by PCR from the recombinant plasmid and used to generate an expression construct with the trc promoter-driven pProEx HT system. Under the conditions employed, the flaB was expressed as inclusion bodies formed within E. coli, yielding c. 120 mg of the recombinant protein/L of culture. A polyhistidine tag introduced at the amino-acid terminus of the FlaB protein allowed for the purification of the protein by nickel-chelate affinity chromatography. The expressed protein reacted with both mouse and bovine antisera to L. interrogans on Western blots, indicating that it could be of use in the diagnosis of leptospirosis. The recombinant leptospiral flagellin may also be of value in studying its role in the pathogenesis of leptospirosis.

Animals↗
PubMed1976

Growth, survival, antigenic stability, and virulence of Leptospira interrogans serotype canicola.

Leptospira interrogans serotype canicola (strain NADL A-13) grew from inocula as small as two cells in liquid polysorbate 80 medium (P-80 medium, in P-60, P-40 and P-20 media, and in P-80 medium from which polysorbate, NH4C1 or thiamine had been omitted. It grew well initially in vitamin B12-deleted P-80 medium, but only with inocula as large as 26 x 10(4) cells per ml. P-80 medium lacking both polysorbate and NH4Cl supported light growth from small inocula, but the omission of thiamine and vitamin B12 in addition seriously affected the properties of the medium. Where readily detectable growth did not develop in liquid nutrient-deleted medium, viable ortanisms could often be demonstrated indirectly by subculture to semisolid medium, and their occurrence was influenced by the presence of albumin, thiamine, and vitamin B12. Growth on semisolid media was comparable with that in liquid media of similar composition. The absence of polysorbate 80, thiamine, or vitamin B12 prevented the appearance of Dinger's zones of growth from small inocula. Antigenic composition as measured by microscopic agglutination tests with homologous and heterologous antisera was not appreciably affected by repeated subculturing in various complete and incomplete media. Homogenates of infected-hamster-kidney tissue in bovine serum-albumin diluent still contained viable organisms after 60 days' storage at 23-25 degrees C. Organisms derived from this material after 3 and 16 days' storage showed no loss of virulence. Organisms grown in artificial culture showed no loss of virulence after storage in bovine albumin diluent or phosphate buffer for 7 days at 23-25 degrees C. Cultures of the organism survived without loss of virulence for 15 months in 13 semisolid media of differing complexity. Single colonies derived from five different solid media were grown in semisolid forms of the parent media and stored at 23-25 degrees C for 10 months without loss of virulence.

Animals↗
PubMed1976

Immunising potency of Leptospira interrogans serotype canicola after heat inactivation at different temperatures.

The immunogenicity of Leptospira interrogans serotype canicola suspensions inactivated by various degrees of heat exposure was examined in hamsters. No differences between leptospires killed at 50 degrees C and at 98 degrees C were shown. After exposure to 121 degrees C, suspensions retained their ability to protect against lethal infections but lost their ability to prevent leptospiruria. Tests with vaccines inactivated at or below 98 degrees C showed that the doses required for complete protection varied with the interval between vaccination and challenge. Larger doses were required to prevent the development of leptospiruria than to prevent death.

Animals↗
PubMed1995

IS1500, an IS3-like element from Leptospira interrogans.

Copies of an insertion-sequence (IS)-like element were isolated from two closely related serovars of Leptospira interrogans sensu stricto. Nucleotide sequence analysis of the 1236 bp element showed a characteristic IS structure with terminal imperfect inverted repeats (IRs) flanking a 1159 bp central region. This element was designated IS1500. Four open reading frames (orfA-orfD) were found in the central 'unique' region of IS1500. Similarities were detected between ORFA and ORFB and the putative transposases from members of the IS3 family of transposable elements. IS1500 or IS1500-like sequences were also detected in all other pathogenic leptospiral serovars, but not in the saprophytic species L. biflexa. Differences in IS1500 copy numbers in members of the same species suggest that this element can transpose. Physical mapping of IS1500 insertions in L. interrogans serovars icterohaemorrhagiae and pomona showed insertions were only on the large chromosomal replicon. The location of some IS1500 insertions coincides with regions of the genome that have undergone large rearrangements.

Amino Acid Sequence↗
PubMed2003

Leptospira interrogans serovar Valbuzzi: a cause of severe pulmonary haemorrhages in the Andaman Islands.

Outbreaks of leptospirosis that present with predominant pulmonary signs and symptoms have been occurring in the Andaman Islands since the late 1980s. Before this, pulmonary haemorrhage had not been observed as a common complication of leptospirosis in India. During an outbreak on North Andaman in 1997, four leptospire isolates were obtained from blood of a fatal case and three other patients who recovered. These isolates were characterized using serological and molecular techniques. Cross-agglutination absorption tests and microscopic agglutination tests using mAbs were used for serological characterization. Genetic typing was done using DNA sequencing of PCR products. Serologically, the isolates were closely related to strain Valbuzzi serovar Valbuzzi of serogroup Grippotyphosa. The sequences of PCR products from these isolates were compared with those of 45 strains belonging to seven species. The isolates showed 97.5-100 % sequence similarity to reference strains belonging to Leptospira interrogans, indicating that the isolates belong to L. interrogans. Serogroups Icterohaemorrhagiae and Australis have been incriminated as the cause of pulmonary haemorrhage in China, Korea and Australia. The four isolates characterized in the present study were obtained from patients with similar symptoms. However, they belonged to serovar Valbuzzi of serogroup Grippotyphosa, indicating that serogroups other than Icterohaemorrhagiae and Australis can also cause pulmonary haemorrhage.

Adolescent↗
PubMed2003

Identification of a novel antigen of pathogenic Leptospira spp. that reacted with convalescent mice sera.

The virulence of leptospires isolated from human patients against C3H/HeJ mice was investigated. Infection with clinical isolates from patients with severe leptospirosis was lethal to C3H/HeJ mice, suggesting that C3H/HeJ mice are suitable as an acute lethal model of severe leptospirosis. Using this model, a novel antigen of pathogenic Leptospira spp. (named LAg42), which reacted with convalescent mice sera, was identified. LAg42 is a 42 kDa inner-membrane protein and its immunogenic region is located in the C-terminal region. The gene for LAg42 is conserved among pathogenic leptospires but not among non-pathogenic leptospires, which suggests its involvement in virulence.

Animals↗
PubMed2006

An improved multiple-locus variable number of tandem repeats analysis for Leptospira interrogans serovar Australis: a comparison with fluorescent amplified fragment length polymorphism analysis and its use to redefine the molecular epidemiology of this serovar in Queensland, Australia.

In this study, an improved multiple-locus variable number of tandem repeats analysis (MLVA) method based upon a previously published method is described. Improvements to the method included redesigned primers and PCR conditions, combined with pooled capillary electrophoresis using multicolored dyes. Allele sizes were converted into an allele string, and each unique allele string was assigned a numerical MLVA type (MVT). The improved MLVA method was then applied to 96 previously characterized Leptospira interrogans serovar Australis isolates from human and animal sources. The improved MLVA was found to have between six and 13 alleles at each locus, compared with three to eight in the original. The mean Hunter-Gaston diversity index (HGDI) for the improved MLVA method was 0.654, compared with 0.599 in the original; this increase in diversity was largely due to changes in the analysis of the variable number of tandem repeat (VNTR) data. When the improved MLVA method was compared with the fluorescent amplified fragment length polymorphism (FAFLP) method, there was a high level of concordance between the profiles; however, the MLVA method produced an additional four unique profiles amongst the subset of 30 isolates tested. Given that the improved MLVA method was found to be superior to the original MLVA method, it was subsequently used to redefine the molecular epidemiology of L. interrogans serovar Australis in Queensland, Australia. Using cluster analysis, the authors were able to demonstrate clonal links amongst rodent isolates, rodent and human isolates, and rodent and canine isolates. These results highlight the role of rodents in the disease, and also the potential role of MLVA in defining the molecular epidemiology of L. interrogans.

Animals↗
PubMed2003

Construction and complementation of the first auxotrophic mutant in the spirochaete Leptospira meyeri.

In bacteria, the first reaction of the tryptophan biosynthetic pathway involves the conversion of chorismate and glutamine to anthranilate by the action of anthranilate synthase, which is composed of the alpha (trpE gene product) and beta (trpG gene product) subunits. In this study, the tryptophan biosynthetic gene trpE of the spirochaete Leptospira meyeri was interrupted by a kanamycin-resistance cassette by homologous recombination. The trpE double cross-over mutant was not able to grow on solid or in liquid EMJH medium. In contrast, the trpE mutant showed a wild-type phenotype when tryptophan or anthranilate was added to the media, therefore showing that disruption of the L. meyeri trpE gene resulted in tryptophan auxotrophy. The authors have also characterized a second selectable marker that allows the construction of a spectinomycin-resistant L. meyeri-E. coli shuttle vector and the functional complementation of the L. meyeri trpE mutant.

Anthranilate Synthase↗
PubMed2002

Cloning, high-level expression, purification and crystallization of peptide deformylase from Leptospira interrogans.

A new peptide deformylase (PDF; EC 3.5.1.27) gene from Leptospira interrogans was identified and cloned into expression plasmid pET22b(+) and was highly expressed in Escherichia coli BL21(DE3). With DEAE-Sepharose anion-exchange chromatography followed by Superdex G-75 size-exclusion chromatography, 60 mg of PDF from L. interrogans was purified from 1 l of cell culture. Crystallization screening of the purified enzyme resulted in two crystal forms, from one of which a 3 A resolution X-ray diffraction data set has been collected.

Amidohydrolases↗
PubMed2003

Co-crystallization of Leptospira interrogans peptide deformylase with a potent inhibitor and molecular-replacement schemes with eight subunits in an asymmetric unit.

Translation initiation in eubacteria involves a formylmethionine at the N-terminus of newly synthesized polypeptides. This N-formyl group is removed by peptide deformylase (PDF) during the post-translation process. Such a formylation/deformylation cycle is essential for the cell survival of eubacteria, but is not utilized in eukaryotic cytosolic protein biosynthesis. In view of the absence of deformylase activity in mammalian cells, this is an attractive target for the design of novel antibiotic drugs. Co-crystallization of peptide deformylase from Leptospira interrogans (LiPDF) with its natural inhibitor actinonin produced diffraction-quality crystals that belong to space group P2(1), with unit-cell parameters a = 87.5, b = 119.1, c = 95.8 A, beta = 111.6 degrees. The 3.1 A resolution data set collected in-house was used to obtain phases by molecular replacement. Three schemes for the correction of the preliminary solutions were proposed and proved successful in determining the structure of LiPDF with eight subunits in the asymmetric unit.

Amidohydrolases↗
PubMed2004

Investigation of zinc-containing peptide deformylase from Leptospira interrogans by X-ray absorption near-edge spectroscopy.

Peptide deformylase (PDF, EC 3.5.1.27) is essential for the normal growth of eubacterium but not for mammalians. Recently, PDF has been studied as a target for new antibiotics. Its activity is strongly dependent on the bound metal ion. The crystallographic studies did not show any significant structural difference upon various bound metal ions. In this paper, X-ray absorption spectroscopy was employed to determine the local structure around the zinc ion of PDF from Leptospira interrogans in dry powder. XANES (X-ray absorption near-edge structure) calculations were performed and the local geometry of the active center was reconstructed successfully. By comparing with the crystal structure of an enzyme-product complex, the results from calculations show that a water molecule has moved towards the zinc ion and lies in the distance range to coordinate with the zinc ion weakly.

Amidohydrolases↗
PubMed2006

Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans.

Ferredoxin-NADP+ reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 angstroms resolution at a synchrotron source.

Crystallization↗
PubMed2006

Purification, crystallization and preliminary crystallographic studies on 2-dehydro-3-deoxygalactarate aldolase from Leptospira interrogans.

2-Dehydro-3-deoxygalactarate (DDG) aldolase is a member of the class II aldolase family and plays an important role in the pyruvate-metabolism pathway, catalyzing the reversible aldol cleavage of DDG to pyruvate and tartronic semialdehyde. As it is a potential novel antibiotic target, it is necessary to elucidate the catalytic mechanism of DDG aldolase. To determine the crystal structure, crystals of DDG aldolase from Leptospira interrogans were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 A resolution using a Cu K alpha rotating-anode X-ray source. The crystal belonged to space group C2, with unit-cell parameters a = 293.5, b = 125.6, c = 87.6 A, beta = 100.9 degrees. The V(M) is calculated to be 2.4 A3 Da(-1), assuming there to be 12 protein molecules in the asymmetric unit.

Aldehyde-Lyases↗
PubMed1978

Nature of antigenic determinant of serovar-specific antigen of Leptospira interrogans serovar hebdomadis.

The nondialyzable delipidized serovar-specific main antigen (NDTM antigen) of Leptospira interrogans serovar hebdomadis strain Hebdomadis (a variant which can grow in a synthetic medium) showed a strong inhibition of the complement fixation between the serovar-specific main (TM) antigen of this strain and the homologous antiserum. The inhibitory effect of the NDTM antigen was completely lost by treating the antigen with proteolytic enzymes, and the fractions of TM antigen containing amino sugar, neutral sugars, and lipids did not show any inhibition of complement fixation, indicating that the antigenic determinant of this strain is related to proteins. NDTM antigen contained more hydrophilic amino acids than hydrophobic amino acids, whereas TM antigen contained more hydrophobic amino acids than hydrophilic amino acids. The amino acid compositions of NDTM antigens of hebdomadis strain Hebdomadis (variant) and kremastos strain Kyoto, which belonged to the same serogroup, were considerably similar. Difference was found in the amounts of methionine, arginine, lysine and glutamine acid.

Amino Acids↗
PubMed1984

Sugar synthesis in Leptospira. I. Presence of glucosephosphate isomerase.

The presence of glucosephosphate isomerase, one of the key enzymes in carbohydrate metabolism, was confirmed for the first time in the cell-free extract of Leptospira biflexa. The glucosephosphate isomerase of L. biflexa was heat-labile and its optimum pH was about 8.5. The enzyme showed an optimal temperature of about 45 C but was more stable at 30 C. Km value of the enzyme was 5.6 X 10(-3)M. The activity of the enzyme was inhibited by the inhibitor, 6-phosphogluconate. From this study, the presence of a metabolic pathway, the phosphogluconate pathway, other than non-oxidative pentose phosphate pathway presented by Baseman and Cox was suggested.

Citric Acid Cycle↗