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PubMed2004

Direct chemical synthesis of the beta-mannans: linear and block syntheses of the alternating beta-(1-->3)-beta-(1-->4)-mannan common to Rhodotorula glutinis, Rhodotorula mucilaginosa, and Leptospira biflexa.

Two stereocontrolled syntheses of a methyl glycoside of an alternating beta-(1-->4)-beta-(1-->3)-mannohexaose, representative of the mannan from Rhodotorula glutinis, Rhodotorula mucilaginosa, and Leptospira biflexa, are described. Both syntheses employ a combination of 4,6-O-benzylidene- and 4,6-O-p-methoxybenzylidene acetal-protected donors to achieve stereocontrolled formation of the beta-mannoside linkage. The first synthesis is a linear one and proceeds with a high degree of stereocontrol throughout and an overall yield of 1.9%. The second synthesis, a block synthesis, makes use of the coupling of two trisaccharides, resulting in a shorter sequence and an overall yield of 4.4%, despite the poor selectivity in the key step.

Benzylidene Compounds↗
PubMed1979

A sero-epidemiological study of Leptospira interrogans serovar Balcanica in four brush-tailed possum populations in Victoria, Australia.

The microscopic agglutination (MA) test was utilised to study the prevalence of antibodies to Leptospira interrogans serovar hardjo in 4 populations of brush-tailed possums (Trichosurus vulpecula). The overall antibody prevalence varied from 14% to 66%; however, the age distribution of MA test titres was remarkably similar in all 4 populations. Antibody prevalence was similar in both males and females and demonstrable antibodies were limited to sexually mature animals. The greater prevalence of high titres (greater than or equal to 1:128) in the 18- to 24-month age group suggested that primary infections were acquired at this age. The findings suggested that infection was maintained in possum populations by direct transmission, probably associated with breeding. Focal interstitial nephritis was observed in kidneys of possums greater than 18 months of age and was associated with MA titres to hardjo (P less than 0.001). Serovar balcanica was isolated from possum kidneys from 2 of these populations, suggesting that balcanica infections were responsible for most of the hardjo titres. However, agglutinin-absorption tests indicated that some possums may be infected with a leptospire more closely related to hardjo than to balcanica.

Age Factors↗
PubMed1979

Experimental infections of brush-tailed possums, common wombats and water rats with Leptospira interrogans serovars balcanica and hardjo.

Of 12 brush-tailed possums (Trichosurus vulpecula) inoculated with Leptospira interrogans serovar balcanica 11 developed migroagglutination (MA) antibody to jardjo antigen by 14 days postincubation (PI). Leptospiruria was observed in 2 possums 117 to 145 days PI. Of 6 possums inoculated with serovar hardjo 4 developed low short-lived titres by day 18 PI. Two of 3 wombats (Vombatus ursinus) inoculated with balcanica had high MA titres (greater than or equal to 1:128) by day 16 PI and leptospiruria occurred by day 16. One wombat inoculated with hardjo developed a low MA titre. Low transitory MA titres to hardjo were found in 1 of 3 water rats (Hydromys chrysogaster) after inoculation with balcanica and 1 of 2 given hardjo. Histopathological examination of kidneys revealed mild to moderately severe focal interstitial nephritis in 4 of 8 possums, in 2 wombats and in 2 water rats following experimental infection with balcanica. Similar lesions were observed in 2 of 4 possums, 1 wombat and 2 water rats following experimental infection with hardjo.

Agglutination Tests↗
PubMed1979

Experimental infection of calves and sheep with Leptospira interrogans serovar balcanica.

Two of four calves inoculated with Leptospira interrogans serovar balcanica developed low microscopic agglutinating (MA) titres to serovar hardjo. A third calf had an MA titre of 1:1024 by day 19 post-inoculation (PI). Transient leptospiruria was recorded in one calf on days 12 and 13 PI. An in-contact calf did not seroconvert. None of the calves had fever or other clinical signs of disease. Four ewes inoculated with balcanica developed MA titres to hardjo by day 13 PI, and a transient leptospiruria between days 14 and 25 PI. None of the ewes showed any evidence of clinical disease and three of them delivered healthy lambs 22 to 64 days PI. One ewe had mild lesions of focal interstitial nephritis.

Agglutination Tests↗
PubMed2003

Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

Bacterial Adhesion↗
PubMed2004

Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome.

Comparative genomic analysis of the coding sequences (CDSs) of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequences and operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth, whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other characterized toxin-antitoxin systems of bacteria, i.e., mazEF, relBE and chpIK. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that although toxins were evolved from divergent sources and may or may not follow their speciation paths (as indicated by their 16s RNA sequences), co-evolution with their antitoxins was obvious.

Amino Acid Sequence↗
PubMed1981

Movement of antibody-coated latex beads attached to the spirochete Leptospira interrogans.

Antibody-coated latex beads (Ab-beads) were attached to Leptospira interrogans serovars illini 3055 and icterohaemorrhagiae SC1157. The movement of the Ab-beads relative to the motion of the cells was observed by direct darkfield microscopy or was recorded on videotape. When the Ab-beads were attached to the front end of motile cells, the Ab-beads were displaced towards the back end of the cells. When the cells reversed direction, the Ab-beads also reversed direction. A number of hypotheses were proposed and tested to account for this Ab-bead displacement. The one best supported by the evidence states that the Ab-beads are attached to antigens of the outer membrane sheath. These antigens are dragged laterally through the sheath due to the forward motion of the cells and the retarding forces of the medium acting on the beads. The results obtained provide information on the nature of the outer membrane sheath of L. interrogans, the basis for certain movements of spirochetes, and insight on how spirochetes attach to eukaryotic cells and tissues. In addition, the results indicate that antigens can move laterally through membranes as rapidly as 11 micrometers/sec.

Antibodies↗
PubMed2004

A methylated phosphate group and four amide-linked acyl chains in leptospira interrogans lipid A. The membrane anchor of an unusual lipopolysaccharide that activates TLR2.

Leptospira interrogans differs from other spirochetes in that it contains homologs of all the Escherichia coli lpx genes required for the biosynthesis of the lipid A anchor of lipopolysaccharide (LPS). LPS from L. interrogans cells is unusual in that it activates TLR2 rather than TLR4. The structure of L. interrogans lipid A has now been determined by a combination of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR spectroscopy, and biochemical studies. Lipid A was released from LPS of L. interrogans serovar Pomona by 100 degrees C hydrolysis at pH 4.5 in the presence of SDS. Following purification by anion exchange and thin layer chromatography, the major component was shown to have a molecular weight of 1727. Mild hydrolysis with dilute NaOH reduced this to 1338, consistent with the presence of four N-linked and two O-linked acyl chains. The lipid A molecules of both the virulent and nonvirulent forms of L. interrogans serovar Icterohaemorrhagiae (strain Verdun) were identical to those of L. interrogans Pomona by the above criteria. Given the selectivity of L. interrogans LpxA for 3-hydroxylaurate, we propose that L. interrogans lipid A is acylated with R-3-hydroxylaurate at positions 3 and 3' and with R-3-hydroxypalmitate at positions 2 and 2'. The hydroxyacyl chain composition was validated by gas chromatography and mass spectrometry of fatty acid methyl esters. Intact hexa-acylated lipid A of L. interrogans Pomona was also analyzed by NMR, confirming the presence a beta-1',6-linked disaccharide of 2,3-diamino-2,3-dideoxy-d-glucopyranose units. Two secondary unsaturated acyl chains are attached to the distal residue. The 1-position of the disaccharide is derivatized with an axial phosphate moiety, but the 4'-OH is unsubstituted. (1)H and (31)P NMR analyses revealed that the 1-phosphate group is methylated. Purified L. interrogans lipid A is inactive against human THP-1 cells but does stimulate tumor necrosis factor production by mouse RAW264.7 cells.

Animals↗
PubMed2005

A Leptospira interrogans enzyme with similarity to yeast Ste14p that methylates the 1-phosphate group of lipid A.

Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 (TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group (Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25420-25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the 1-phosphate moiety of E. coli Kdo2-[4'-(32)P]lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene (renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2.

Amino Acid Sequence↗
PubMed2005

Novel conformational states of peptide deformylase from pathogenic bacterium Leptospira interrogans: implications for population shift.

Peptide deformylase is an attractive target for developing novel antibiotics. Previous studies at pH 3.0 showed peptide deformylase from Leptospira interrogans (LiPDF) exists as a dimer in which one monomer is in a closed form and the other is in an open form, with different conformations of the CD-loop controlling the entrance to the active pocket. Here we present structures of LiPDF at its active pH range. LiPDF forms a similar dimer at pH values 6.5-8.0 as it does at pH 3.0. Interestingly, both of the monomers are almost in the same closed form as that observed at pH 3.0. However, when the enzyme is complexed with the natural inhibitor actinotin, the conformation of the CD-loop is half-open. Two pairs of Arg109-mediated cation-pi interactions, as well as hydrogen bonds, have been identified to stabilize the different CD-loop conformations. These results indicate that LiPDF may be found in different structural states, a feature that has never before been observed in the peptide deformylase family. Based on our results, a novel substrate binding model, featured by an equilibrium between the closed and the open forms, is proposed. Our results present crystallographic evidence supporting population shift theory, which is distinguished from the conventional lock-and-key or induced-fit models. These results not only facilitate the development of peptide deformylase-targeted drugs but also provide structural insights into the mechanism of an unusual type of protein binding event.

Amidohydrolases↗
PubMed1998

Identification of Leptospira species in the pathogenesis of uveitis and determination of clinical ocular characteristics in south India.

Uveitis is considered a rare complication of leptospirosis. This report describes an epidemic of uveitis among patients with leptospirosis and provides data, using polymerase chain reaction (PCR) amplification of Leptospira DNA, that the pathogenesis is associated with anterior chamber spirochetes. Forty-six uveitis patients, 49 uveitis controls, and 54 cataract controls were enrolled at Aravind Eye Hospital (Madurai, India). Leptospiral DNA was detected by PCR of aqueous humor; serum antibody titers were determined by ELISA and microagglutination (MAT). Thirty-seven uveitis patients (80%) demonstrated leptospiral DNA compared with 5 controls (8%; P < .001). Thirty-three uveitis patients (72%) had positive serology compared with 10 uveitis controls (20%) and 13 cataract controls (24%; P < .001). This report describes the largest cluster of patients with leptospiral uveitis and identifies six clinical characteristics that provide a diagnostic profile for leptospiral uveitis. This profile will be important for determining treatment regimens in countries where PCR and MAT are not available.

Adolescent↗
PubMed1999

Expression and immunologic characterization of recombinant heat shock protein 58 of Leptospira species: a major target antigen of the humoral immune response.

A clone of Leptospira interrogans serovar lai that was isolated by immunoscreening of a genomic lambda library with sera from convalescent patients with leptospirosis directed expression of a unique 62-kDa protein in Escherichia coli. When examined by SDS-PAGE, the protein comigrated with an immunodominant protein present in leptospiral cell lysate. Determination of the nucleotide sequence of the 2.7-kb insert DNA identified two genes homologous to the hsp58 and hsp10 of L. interrogans serovar copenhageni reported previously. The overexpressed recombinant Hsp58 protein was purified and used to immunize a rabbit to produce a polyclonal antibody. Immunoblot analysis using the rabbit anti-Hsp58 G antibodies showed that the 62-kDa protein was commonly present in lysates of other serovars of leptospires, consistent with the strong sequence conservation between the hsp58 genes of the two serovars. Immunoglobulin G antibodies to the Hsp58 were specifically detected by ELISA in 82% of sera (18/22) from patients with leptospirosis. Deletion analysis of the recombinant Hsp58 protein indicated that a strong antigenic determinant for humoral immune response is located between amino acids 360 and 380 (DREKLQERLAKLAGGVAVIHV) of Hsp58, which are highly conserved among the GroEL family. The strong sequence conservation of the Hsp58 among leptospires and its importance as a major target for the humoral immune response warrant further studies of its potential pathogenetic role.

Animals↗
PubMed1990

The use of immunodeficient male (CBA/N x BALB/c) F1 mice to produce monoclonal antibodies directed to proteins of Leptospira interrogans rather than to immunodominant lipopolysaccharides.

A method, using an immunodeficient mouse strain, for the production of monoclonal antibodies directed exclusively against the proteins in an antigen mixture also containing immunodominant LPS, is described. Male (CBA/N x BALB/c) F1 mice were immunized with an outer envelope antigen mixture from Leptospira interrogans strain Wijnberg containing both lipopolysaccharides and proteins. The immune response in these mice was shown to be predominantly directed against protein antigens. Hybridoma cell lines were generated by fusing spleen cells from a (CBA/N x BALB/c) F1 mouse with BALB/c Sp2/0 plasmacytoma cells. Hybridoma cell lines producing monoclonal antibodies reacting with the outer envelope preparation were identified by ELISA. All epitopes recognized by the monoclonal antibodies are sensitive to proteinase K degradation and resistant to oxidation by periodate indicating that they are located on proteins. All epitopes are located on a 35 kDa protein and specific for the pathogenic L. interrogans species.

Animals↗
PubMed2002

Comparative in vitro activity of five cathelicidin-derived synthetic peptides against Leptospira, Borrelia and Treponema pallidum.

OBJECTIVE: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of five different cathelicidin-derived synthetic peptides (SMAP-29, LL-37, PG-1, CRAMP and BMAP-28) for Leptospira interrogans, Borrelia spp. and Treponema pallidum subsp. pallidum were investigated in vitro. METHODS: The MIC of individual peptides was defined as the lowest concentration able to inhibit the motility of spirochaetes after 2 h of incubation, as detected by dark-field microscopy. The MBC of individual peptides was defined as the lowest concentration at which no spirochaetes were subcultured either in cathelicidin-free medium (leptospires and borreliae) or in hamsters (T. pallidum). RESULTS: The MIC values of peptides for leptospires were highly variable, depending on the compound and the strain used. Of the five cathelicidin-derived peptides, SMAP-29 from sheep and BMAP-28 from cattle were the most active against L. interrogans serovars, with MIC values varying between 3 and 51 mg/L, depending on the strains. The MICs of the remaining synthetic peptides ranged between 4.3 and 224 mg/L. The MIC values of synthetic peptides for T. pallidum ranged between 32.3 mg/L for PG-1 and 449.4 mg/L for LL-37. The MICs of all cathelicidin-derived peptides tested for Borrelia strains ranged between 307 and 449.4 mg/L. The activity of the peptides on the motility of spirochaetes was both dose- and time-dependent. The MBC values of the peptides were the same as the MIC values. CONCLUSION: The results of this study demonstrate that the activity of cathelicidin-derived peptides against spirochaetes is fast and highly variable, depending on the species and the strain.

Animals↗
PubMed1991

Physical map of chromosomal and plasmid DNA comprising the genome of Leptospira interrogans.

The size and physical structure of the Leptospira interrogans genome was characterized using contour-clamped homogenous electric field (CHEF) gel electrophoresis. The L. interrogans genome is approximately 4750 kb in size and is composed of two molecular species of DNA: a 4400 kb chromosome; and a 350 kb plasmid, pLIN1. A physical map of the chromosome was constructed with the restriction enzymes NotI and SfiI. A physical map of pLIN1 was constructed with ApaI, NotI, Sse83871, SgrAI, and SmaI. Both the L. interrogans chromosome and pLIN1 are circular.

Chromosome Mapping↗
PubMed1977

Immunochemistry of extracts from Leptospira interrogans serotype hardjo.

Antigens from Leptospira interrogans serotype hardjo grown in modified Korthof's medium were obtained by ethanol and alkaline extraction procedures and their chemical and serological properties were compared. The protein to polysaccharide ratio in the ethanol extract was 1:1-8 and in the alkali extract was 1-7:1. The lipid content of the latter was twice that of the former (8-5%, w/w). There was an inverse relationship of the protein and carbohydrate contents of the two preparations, the total reducing sugar being higher in the ethanol extract whereas protein was higher in the alkali extract. Both preparations contained arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose and galacturonic acid but in different amounts. No muramic acid or 2-keto-3-deoxyoctonate was detected. Both extracts contained erythrocyte sensitizing substances which, in the passive haemagglutination absorption test, appeared to be closely related antigenically but not identical. In the electron microscope, thin sections of the ethanol extract showed trilaminar outer envelope-like material.

Antigens, Bacterial↗
PubMed1978

'Viscotaxis', a new behavioural response of Leptospira interrogans (biflexa) strain B16.

When in a non-viscous environment and confronted with a viscous one, Leptospira interrogans (biflexa) strain B16 preferentially selected the latter. We have designated this positive response to a viscosity gradient as 'viscotaxis'. Using an originally designed experimental chamber, a pool of leptospires were faced with capillary tubes containing either polyvinylpyrrolidone (PVP) or N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES). Leptospires suspended in HEPES responded positively to 2% (w/v) PVP in capillaries, and migrated into them in large numbers in 1 h. No response was observed when the chamber and capillary tubes contained solutions of the same viscosity. As the viscosity of PVP was increased, a proportionally larger number of leptospires migrated into it. This newly observed aspect of leptospiral behaviour may have ecological significance.

Culture Media↗