Skin reaction to lipids from avirulent strain Shibaura of Leptospira interrogans serovar copenhageni

Sonically disrupted cells from avirulent strain Shibaura of Leptospira interrogans serovar copenhageni induced a skin reaction characterized by infiltration of polymorphonuclear leukocytes (PMN) associated with some edema in guinea pigs. To determine the substance inducing infiltration of PMN, lipids of avirulent strain Shibaura were extracted with chloroform--methanol--water after washing with acetone. The lipids comprised 28% of the dry weight of the cell. When the lipids were further separated into water--methanol and chloroform fractions, the most severe PMN infiltration of all samples was seen in the skin inoculated with extract recovered from the chloroform fraction. Neutral and polar lipids were detected after thin-layer chromatography of the chloroform extract. Neutral lipids were detected as free fatty acids (FFA). Fatty acids contained in polar lipids were mainly palmitic acid and palmitoleic acid, whereas FFA comprised 66.5% oleic acid. Skin reactions consisting of marked edema with mild infiltration of PMN were elicited by FFA. There was no obvious difference between a commercially available FFA mixture and the FFA from avirulent strain Shibaura. These observations suggest that FFA may play some role in the pathogenesis of leptospirosis.     

Glycolipoprotein cytotoxin from Leptospira interrogans serovar copenhageni

Lipopolysaccharide (LPS), glycolipoprotein (GLP) and lipid extract were prepared from Leptospira interrogans serovar copenhageni. GLP, lipid extract or purified fatty acids from lipid extract produced cytotoxic effects seen as cell enzyme leakage followed by cytotoxic death when tested in mouse fibroblast L929 cells in tissue culture. All extracts also agglutinated mouse erythrocytes but purified LPS was not cytotoxic. Neither GLP nor LPS were pyrogenic but both gelled Limulus amoebocyte lysate. Specific anti-GLP IgG neutralized the cytotoxic and haemagglutinating effect of GLP; however, at higher concentrations it enhanced the cytotoxicity of GLP and mediated lysis of the erythrocytes. A high dose of leptospires (i.e. 10(10) organisms) killed weanling mice causing pathological changes similar to those seen in acute leptospirosis. Similar results were obtained with live, dead, pathogenic and saprophytic leptospires. The results suggest that toxicity is involved in leptospiral infection and that lipid components either of whole leptospires or of a leptospiral GLP may contribute to the pathogenesis of acute leptospirosis.     

An immunoprotective monoclonal antibody directed against Leptospira interrogans serovar copenhageni

A monoclonal antibody (mAb) was prepared by hybridoma technology in BALB/c mice immunized to Leptospira interrogans serovar copenhageni. This mAb agglutinated serovars copenhageni and icterohaemorrhagiae to high titres and protected hamsters, dogs and monkeys against challenge with a virulent strain of serovar copenhageni. The mAb gave protection to hamsters at dilutions up to 1 in 1000; at a 1 in 10 dilution the protective effect lasted for at least two weeks. Biochemical analysis by SDS-PAGE and Western blotting indicated that this mAb reacted with an epitope of a carbohydrate nature.     

Ultrastructure and chemical composition of lipopolysaccharide extracted from Leptospira interrogans serovar copenhageni

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.     

Repetitive sequence of Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1: a sensitive probe for demonstration of Leptospira interrogans strains.

A 4.8-kilobase (kb) repetitive sequence element generated with KpnI digestion was cloned from the Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1. The sequence, repeated in tandem, was located on the 280-kb fragment between the FseI and AscI sites on the chromosome by hybridization using the 4.8-kb fragment as a probe. We cloned the fragment containing the element for the Ictero No. 1 strain in a lambda EMBL3 bacteriophage DNA, and one out of 5 clones was sequenced. Within the sequenced 9-kb segment that partially repeated, 9 putative open-reading frames and 2 transfer RNA genes, for alanine and isoleucine, were identified. A similarity search for the products deduced from the sequenced data revealed that the repeated sequence includes both beta-oxidation enzymes, acyl-CoA dehydrogenase and enoyl-CoA hydratase, and hydroxythiazole kinase protein homologues. Hybridization experiments against different leptospiral strains using the element as a probe showed a similar sequence in the strains of L. interrogans and L. kirschneri, but not in any strains of L. borgpetersenii, L. weillii, L. meyeri or L. biflexa. Results indicated that the highly repeated element in the Ictero No. 1 strain exists as a well conserved sequence, though at a moderate level of repetition, in certain strains of L. interrogans and L. kirschneri. PCR amplification targeting the repetitive element was successful and indicated that the procedure provides a sensitive and specific probe to detect leptospires.     

Modeling of phosphomethyl pyrimidine kinase from Leptospira interrogans serovar lai strain 56601

Many microorganisms, as well as plants and fungi, synthesize thiamin, but vertebrates do not produce it. Phosphomethyl pyrimidine kinase is an enzyme involved in an intermediary step of thiamin biosynthesis from purine molecules. This enzyme is absent in humans. Thus, it is a potential chemotherapeutic target for antileptospiral treatment. Structure of this enzyme from Leptospira interrogans serovar lai strain 56601 has not yet been elucidated. We used the structural template of phosphomethyl pyrimidine kinase from Thermus thermophilus HB8 for modeling the phosphomethyl pyrimidine kinase structure from Leptospira interrogans serovar lai strain 56601 . The model is deposited in Protein Data Bank (PDB ID: 2G53) at RCSB. Thus, we analyse and propose the usefulness of the modeled phosphomethyl pyrimidine kinase for the design of suitable inhibitors towards the treatment of leptospirosis.     

Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae

We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin. A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results. The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin-binding domain. The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction. Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells.     

Surface proteomics and label-free quantification of Leptospira interrogans serovar Pomona

Leptospirosis is a re-emerging zoonosis with a global distribution. Surface-exposed outer membrane proteins (SE-OMPs) are crucial for bacterial–host interactions. SE-OMPs locate and expose their epitope on cell surface where is easily accessed by host molecules. This study aimed to screen for surface-exposed proteins and their abundance profile of pathogenic Leptospira interrogans serovar Pomona. Two complementary approaches, surface biotinylation and surface proteolytic shaving, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) were employed to identify SE-OMPs of intact leptospires. For quantitative comparison, in-depth label-free analysis of SE-OMPs obtained from each method was performed using MaxQuant. The total number of proteins identified was 1,001 and 238 for surface biotinylation and proteinase K shaving, respectively. Among these, 39 were previously known SE-OMPs and 68 were predicted to be localized on the leptospiral surface. Based on MaxQuant analysis for relative quantification, six known SE-OMPs including EF- Tu, LipL21, LipL41, LipL46, Loa22, and OmpL36, and one predicted SE-OMPs, LipL71 were found in the 20 most abundant proteins, in which LipL41 was the highest abundant SE-OMP. Moreover, uncharacterized LIC14011 protein (LIP3228 ortholog in serovar Pomona) was identified as a novel predicted surface βb-OMP. High-abundance leptospiral SE-OMPs identified in this study may play roles in virulence and infection and are potential targets for development of vaccine or diagnostic tests for leptospirosis.     

Re-characterization of an extrachromosomal circular plasmid in the pathogenic Leptospira interrogans serovar Lai strain 56601

In China, Leptospira interrogans serovar Lai strain 56601 （str.56601） is one of main pathogenic strains that cause severe leptospirosis in both human and animals. The genome of this organism was completely sequenced in 2003. However, in 2011, we identified and corrected some assembly errors in the str.56601 genome due to the repeat sequences widely distributed in the Leptospira genome. In this study, we re-analyzed the previously reported mobile, phage-related genomic island in the chromosome and rectified detailed sequence information in both the plasmid and chromosome using various experimental methods. The presence of a separate circular extrachromosomal plasmid was also confirmed, and its location in the genomi~ region was determined relative to the genomic island reported in L. interrogans serovar Lai by a combination of pulsed-field gel electrophoresis -based and plasmid extraction-based Southern blot analysis. This report confirmed that the sep arate extrachromosomal circular plasmid is not integrated into the chromosome of. interrogans str.56601 and markedly improved our understanding of the genomic organization, evolution, and pathogenesis of L. interrogans. In particular, characterization of this extrachromosomal cir cular plasmid will contribute to the development of genetic manipulation systems in pathogenic Leptospira species.     

Biological activities of lipid components ofLeptospira interrogans serovarcopenhageni avirulent strain Shibaura

The gas chromatogram showed that the polar lipids (PL) extracted fromLeptospira interrogans serovarcopenhageni avirulent strain Shibaura consisted mainly of palmitic acid, palmitoleic acid, and oleic acid, and that the free fatty acids (FFA) from the strain contained oleic acid at a concentration as high as 67.5%. The intracutaneous injection of FFA induced much stronger edematous and vascular permeability reactions in guinea pigs than did that of PL. In addition, O ₂ ^– generation of polymorphonuclear leukocytes in vitro was enhanced with FFA to much greater extent than with PL.Among the FFA, oleic acid caused the strongest biological activities such as edematous reaction, vascular permeability reaction, O ₂ ^– -generating activity, and hemolytic activity. It is, therefore, very likely that oleic acid may play an important role in toxicities of leptospiral lipids.     

Serological and chemical interrelationship of antigens from Leptospira interrogans serovar canicola

Antigens of the outer envelope from Leptospira interrogans serovar canicola (Hond Utrecht IV) were extracted by 50% (v/v) ethanol or by sodium dodecyl sulphate and serological analysis suggested that they were identical. The "fraction 4" extracted by alkali was found to contain glycoproteins of high (retentate) and low (filtrate) molecular weight; the latter behaved like a hapten in serology and in animal immunization experiments. Antibodies were raised in rabbits against this hapten by conjugating it to bovine albumin fraction V. The antiserum was found to react with both the low molecular weight and high molecular weight glycoproteins. This anti-hapten serum contained little or no whole-cell-agglutinating antibodies. The fraction 4 retentate behaved like a complete antigen in serological and immunization studies. Fraction 4 retentate and the outer envelope preparations were serologically related but they were not identical. Chemical studies revealed similarities between the carbohydrate component of the outer envelope obtained by ethanol extraction and fraction 4. The outer envelope extracted by ethanol, fraction 4 and its low and high molecular weight glycoproteins contained arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose, glucosamine and glucuronic acid. Three unidentified peaks were observed in gas-liquid chromatographic analysis of the O-trimethylsilyl derivatives of methyl glycosides of all these samples and one of these peaks co-eluted with the O-trimethylsilyl derivative of 3-O-methylmannose.     

Biological activities of lipopolysaccharide-like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton

The biological activities of lipopolysaccharide-like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton by the hot phenol-water method were studied in mice. The addition of 12.5 micrograms/ml or more of LLS fraction increased the incorporation of [3H]thymidine into in vitro cultured spleen cells of C57BL/6 mice, while the activity of the LLS fraction was about 20 times weaker than that of Salmonella typhimurium lipopolysaccharide (LPS). Pretreatment of murine spleen cells with rabbit anti-mouse thymocyte antiserum did not diminish the mitogenic activity of leptospiral LLS, and the LLS could not increase the incorporation of [3H]thymidine into thymocytes, suggesting that LLS acts on a B-lymphocyte population of lymphocytes. When sheep erythrocytes and LLS fraction were injected intraperitoneally into BALB/c mice, LLS exhibited an enhancing effect on antibody response in vivo. However, lethal toxicity of the LLS fraction was about 500 times lower than that of LPS in C57BL/6 mice loaded with galactosamine. No antitumor activity of leptospiral LLS (250-1,000 micrograms/mouse) against the ascites form of Ehrlich carcinoma in ddY mice was observed. The biological activities of the LLS fraction from the organism were weaker than those of gram-negative bacterial LPS, suggesting that Leptospira possesses no typical LPS.     

Chemical properties of lipopolysaccharide-like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton

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.     

Characterization and taxonomic significance of lipopolysaccharides of Leptospira interrogans serovar hardjo

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.     

Functional characterization of GDP-mannose pyrophosphorylase from Leptospira interrogans serovar Copenhageni

Leptospira interrogans synthesizes a range of mannose-containing glycoconjugates relevant for its virulence. A prerequisite in the synthesis is the availability of the GDP-mannose, produced from mannose-1-phosphate and GTP in a reaction catalyzed by GDP-mannose pyrophosphorylase. The gene coding for a putative enzyme in L. interrogans was expressed in Escherichia coli BL21(DE3). The identity of this enzyme was confirmed by electrospray-mass spectroscopy, Edman sequencing and immunological assays. Gel filtration chromatography showed that the dimeric form of the enzyme is catalytically active and stable. The recombinant protein was characterized as a mannose-1-phosphate guanylyltransferase. S _0.5 for the substrates were determined both in GDP-mannose pyrophosphorolysis: 0.20 mM (GDP-mannose), 0.089 mM (PPi), and 0.47 mM; and in GDP-mannose synthesis: 0.24 mM (GTP), 0.063 mM (mannose-1-phosphate), and 0.45 mM (Mg²⁺). The enzyme was able to produce GDP-mannose, IDP-mannose, UDP-mannose and ADP-glucose. We obtained a structural model of the enzyme using as a template the crystal structure of mannose-1-phosphate guanylyltransferase from Thermus thermophilus HB8. Binding of substrates and cofactor in the model agree with the pyrophosphorylases reaction mechanism. Our studies provide insights into the structure of a novel molecular target, which could be useful for detection of leptospirosis and for the development of anti-leptospiral drugs.     

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.     

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.