Cloning of genes required for amino acid biosynthesis from Leptospira interrogans serovar icterohaemorrhagiae

Leptospira interrogans belongs to a large family of important pathogens, which is part of the order Spirochaetales, a distinct group of eubacteria. In order to obtain a better understanding of the genetic organization of this species, we have constructed a DNA library of the serovar icterohaemorrhagiae, using the Escherichia coli vector pUC13. We have isolated Leptospira DNA fragments containing the genetic information required to complement strains of E. coli with defects in proline and leucine biosynthesis. While a 3.9 kb fragment which complemented proA also complemented proB, a 15 kb fragment complementing leuB could not complement other leu mutations. The L. interrogans origin of the cloned DNA fragments was confirmed by DNA-DNA hybridization. The hydridization was specific to the pathogenic species and was not seen with the saprophytic species L. biflexa.     

Unique organization of Leptospira interrogans rRNA genes.

We cloned Sau3AI fragments containing the rRNA genes for Leptospira interrogans serovar canicola strain Moulton in the BamHI site of lambda EMBL3 bacteriophage DNA. Physical maps of the fragments were constructed, and the locations of the rRNA genes were determined by Southern blot hybridization and S1 protection. Each fragment of the 23S or the 16S rRNA gene contained at least one copy of the 23S or the 16S sequence. Genomic hybridization showed that there were two genes for the 23S rRNA and the 16S rRNA but only one gene for the 5S rRNA on the chromosome of L. interrogans. The results revealed the important fact that each rRNA gene is located far from the other rRNA genes. Our findings, accordingly, also suggest that these rRNA genes are expressed independently in this organism.     

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.     

The immunofluorescent detection of Leptospira interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae infections in human tissues

The identification of Leptospira interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae infections in post-mortem tissues by direct fluorescence microscopy is demonstrated by the use of a serogroup-specific fluorescein isothiocyanate label.     

In vivo apoptosis of hepatocytes in guinea pigs infected with Leptospira interrogans serovar icterohaemorrhagiae

To investigate the contribution of the previously demonstrated in vitro apoptosis to the pathogenesis of leptospirosis, guinea pigs were infected with Leptospira interrogans serovar icterohaemorrhagiae strain Verdun and sequentially killed to collect target organs involved in the natural history of the disease (liver, kidneys, lungs, spleen and heart). The combination of histopathological procedures and a specific TUNEL assay showed a significant Leptospira-induced programmed cell death of hepatocytes with a peak at 48 h post inoculation. Hepatocyte nuclei showed morphological changes including fragmented and condensed nuclei. This phenomenon occurred early in the course of the disease at a time where infecting leptospires were present at a low density between the liver parenchyma cells.     

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.     

Cloning of dapD, aroD and asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd gene.

Metabolites such as diaminopimelate and some aromatic derivatives, not synthesized in mammalian cells, are essential for growth of bacteria. As a first step towards the design of a new human live vaccine that uses attenuated strains of Leptospira interrogans, the asd, aroD and dapD genes, encoding aspartate beta-semialdehyde dehydrogenase, 3-dehydroquinase and tetrahydrodipicolinate N-succinyltransferase, respectively, were cloned by complementation of Escherichia coli mutants. The complete nucleotide sequence of the asd gene was determined and found to contain an open reading frame capable of encoding a protein of 349 amino acids with a calculated Mr of 38,007. Comparison of this deduced L. interrogans aspartate beta-semialdehyde dehydrogenase amino acid sequence with those of the same enzyme from Saccharomyces cerevisiae and Corynebacterium glutamicum revealed 46% and 36% identity, respectively. By contrast, the identity between the L. interrogans enzyme and the Streptococcus mutans or E. coli enzymes was less than 31%. Highly conserved sequences within aspartate semialdehyde dehydrogenase from the five organisms were observed at the amino and carboxyl termini, and around the cysteine of the active site.     

Expression of antigen genes of Leptospira interrogans serovar canicola in Escherichia coli

Leptospira interrogans serovar canicola DNA was cloned into the plasmid pBR322 and introduced into E. coli. Eight out of approximately 10,000 transformants were found to express antigens of canicola by ELISA including colony ELISA blot test using anti-canicola antiserum. The canicola antigens expressed in the transformants reacted with the antisera against the serovars belonging to Canicola serogroup and other serogroups of L. interrogans. They did not react, however, with the antiserum against L. biflexa (with only one exception) nor with the antiserum against Leptonema illini. Thus, the recombinant DNA technique may provide alternative possibilities for preparing antigens of leptospires.     

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.     

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogens serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD 18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.     

Isolation and characterization of the 5S rRNA gene of Leptospira interrogans.

The gene encoding the 5S rRNA for Leptospira interrogans serovar canicola strain Moulton was isolated and sequenced. The 5S rRNA gene occurs as a single copy within the genome and encodes a 117-nucleotide-long RNA molecule. The 5S rRNA gene is flanked at both the 5' and 3' ends by regions of A + T-rich sequences, and the 5'-flanking region contains a promoter sequence. L. interrogans has a unique and remarkable organization of the 5S rRNA gene. The 5S rRNA molecule exhibits a strong similarity to typical eubacterial 5S rRNA in terms of overall secondary structure, while the primary sequence is conserved to a lesser degree. Restriction analysis of the 5S rRNA gene indicated that the DNA sequence including the 5S rRNA gene is highly conserved in the genomes of parasitic leptospires.     

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.     

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.     

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura.

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura were analyzed by thin-layer chromatography, gas-liquid chromatography, gas-mass spectrometry and infrared absorption spectrometry. The virulent cells possessed a characteristic lipid pattern consisting of free fatty acid (FFA) (41.8%), one major unidentified phospholipid (14.8%), phosphatidylethanolamine (PE) (12.9%), cholesteryl ester (CE) (9.3%), lysophosphatidylethanolamine (LPE) (4.9%) and diphosphatidyl-glycerol (DPG) (1.1%). Various fatty acids such as hexadecanoic (26.9%), hexadecenoic (15.4%), octadecenoic (26.5%) and octadecadienoic (27.4%) acids were detected in the FFA. The fatty acid composition of the major unidentified phospholipid distinctly differed from those of other lipids including PE, LPE, DPG and CE, and comprised mainly tetradecadienoic (53.6%), tetradecatrienoic (14.0%) and octadecanoic (13.8%) acids. This phospholipid with a large amount of polyunsaturated fatty acids with chain lengths of 14 carbon atoms was detected only in the lipids of the virulent cells.     

钩端螺旋体内毒素的研究：Ⅴ,乙二胺四乙酸二钠对钩端螺旋体...

The yield of leptospiral LPS was able to increase 1-2 times when the cells were treated with EDTA-Na before extracting with hot phenal-water method. The colours and solubility of L-LPS were improved. When the leptospiral cells were stored for a long period of time, and treated by EDTA-Na, the phase distribution of the LPS changed, i.e. they returned from water phase into phenol phase. Such change was related to the quantity of both the saccharide and lipid.     

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.     

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.     

First human isolate of Leptospira interrogans as serovar bratislava in Italy

A strain of Leptospira interrogans was isolated from a patient suffering from leptospirosis and was typed by the Cross Agglutination Absorption test using monoclonal antibodies prepared against different serovars of the Australis serogroup. This newly isolated strain belonged to serovar bratislava. It is the first reported isolation from man, in Italy, of Leptospira bratislava, thus supporting the emerging role of this serovar in human leptospirosis.