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

ABSTRACT: 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.

SUBMITTER: Umashankar V

PROVIDER: S-EPMC1891682 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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

Umashankar Vetrivel V Subramanian Gurunathan G Dorairaj Sudarsanam S

Bioinformation 20060617 5

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 py ...[more]

PMID: 17597880

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Extracellular proteome analysis of Leptospira interrogans serovar Lai.

Project description:Abstract Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.

| S-EPMC3783971 | biostudies-literature

A genomic island of the pathogen Leptospira interrogans serovar Lai can excise from its chromosome.

Project description:An examination of the two Leptospira interrogans genomes sequenced so far reveals few genetic differences, including an extra DNA region, 54 kb in length, in L. interrogans serovar Lai. This locus contains 103 predicted coding sequences that are absent from the genome of L. interrogans serovar Copenhageni, of which only 20% had significant BLASTP hits in GenBank. By analyzing the L. interrogans serovar Lai genome by pulsed-field gel electrophoresis, we also found that this 54-kb DNA fragment exists as a circular plasmid. This was confirmed by amplification of a DNA fragment corresponding to that of the predicted fragment if this region excised from the chromosome and its left and right ends joined together. In addition, cloning of the putative rep gene of this DNA region was responsible for autonomous replication in Leptospira spp., therefore generating a new Escherichia coli-Leptospira sp. shuttle vector. Taken together, our results show that this genomic island can excise from the chromosome and form a replicative plasmid. Analysis of the distribution of this genomic island revealed that highly related sequences exist in other L. interrogans virulent strains. This genomic island, containing a high proportion of novel genes, may have an important role in spreading genes, including virulence factors, among bacterial populations.

| S-EPMC1828511 | biostudies-literature

Cloning, expression, and homology modeling of GroEL protein from Leptospira interrogans serovar autumnalis strain N2.

Project description:Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and sequenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indicated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and validated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a protein. The superposition of the Ca traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5 Å. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.

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Expression and characterization of an iron-regulated hemin-binding protein, HbpA, from Leptospira interrogans serovar Lai.

Project description:In an earlier study, based on the ferric enterobactin receptor FepA of Escherichia coli, we identified and modeled a TonB-dependent outer membrane receptor protein (LB191) from the genome of Leptospira interrogans serovar Lai. Based on in silico analysis, we hypothesized that this protein was an iron-dependent hemin-binding protein. In this study, we provide experimental evidence to prove that this protein, termed HbpA (hemin-binding protein A), is indeed an iron-regulated hemin-binding protein. We cloned and expressed the full-length 81-kDa recombinant rHbpA protein and a truncated 55-kDa protein from L. interrogans serovar Lai, both of which bind hemin-agarose. Assay of hemin-associated peroxidase activity and spectrofluorimetric analysis provided confirmatory evidence of hemin binding by HbpA. Immunofluorescence studies by confocal microscopy and the microscopic agglutination test demonstrated the surface localization and the iron-regulated expression of HbpA in L. interrogans. Southern blot analysis confirmed our earlier observation that the hbpA gene was present only in some of the pathogenic serovars and was absent in Leptospira biflexa. Hemin-agarose affinity studies showed another hemin-binding protein with a molecular mass of approximately 44 kDa, whose expression was independent of iron levels. This protein was seen in several serovars, including nonpathogenic L. biflexa. Sequence analysis and immunoreactivity with specific antibodies showed this protein to be LipL41.

| S-EPMC1951163 | biostudies-literature

Complete Genome Sequence of Leptospira interrogans Serovar Bratislava, Strain PigK151.

Project description:Leptospira interrogans serovar Bratislava infection occurs in multiple domestic and wildlife species and is associated with poor reproductive performance in swine and horses. We present the complete genome assembly of strain PigK151 comprising two chromosomes, CI (4.457 Mbp) and CII (358 kbp).

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Leptospira interrogans serovar Lai str. Lai

Project description:Leptospira interrogans serovar Lai str. Lai Genome sequencing and assembly

| PRJNA74197 | ENA

Genome-wide transcriptional analysis of temperature shift in L. interrogans serovar lai strain 56601.

Project description:BackgroundLeptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptation to a range of new environmental conditions in the organs and tissues of the infected host. Several studies have shown that a shift in culture temperature from 28 degrees C to 37 degrees C, similar to that encountered during infection of a host from an environmental source, is associated with differential synthesis of several proteins of the outer membrane, periplasm and cytoplasm. The whole genome of the Leptospira interrogans serogroup Icterohaemorrhagiae serovar lai type strain #56601 was sequenced in 2003 and microarrays were constructed to compare differential transcription of the whole genome at 37 degrees C and 28 degrees C.ResultsDNA microarray analyses were used to investigate the influence of temperature on global gene expression in L. interrogans grown to mid-exponential phase at 28 degrees C and 37 degrees C. Expression of 106 genes differed significantly at the two temperatures. The differentially expressed genes belonged to nine functional categories: Cell wall/membrane biogenesis genes, hemolysin genes, heat shock proteins genes, intracellular trafficking and secretion genes, two-component system and transcriptional regulator genes, information storage and processing genes, chemotaxis and flagellar genes, metabolism genes and genes with no known homologue. Real-time reverse transcription-PCR assays confirmed the microarray data.ConclusionMicroarray analyses demonstrated that L. interrogans responds globally to temperature alteration. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many new insights into its pathogenesis.

| S-EPMC1534042 | biostudies-literature

Isoleucine biosynthesis in Leptospira interrogans serotype lai strain 56601 proceeds via a threonine-independent pathway.

Project description:Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded alpha-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower K(m) (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed beta-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.

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Effects of temperature on gene expression patterns in Leptospira interrogans serovar Lai as assessed by whole-genome microarrays.

Project description:Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20 degrees C, 30 degrees C, 37 degrees C, and 39 degrees C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37 degrees C and 39 degrees C) versus environmental temperatures (20 degrees C and 30 degrees C). Additionally, cultures grown at 30 degrees C then shifted overnight to 37 degrees C were compared with those grown long-term at 30 degrees C and 37 degrees C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.

| S-EPMC1594916 | biostudies-literature

Transcriptional analysis of CRISPR I-B arrays of <i>Leptospira interrogans</i> serovar Lai and its processing by Cas6.

Project description:In the genome of various Leptospira interrogans serovars, the subtype I-B locus of CRISPR-Cas possesses either one or multiple CRISPR arrays. In silico database (CRISPRCasdb) for predicting CRISPR-Cas reveals seven CRISPR arrays in L. interrogans serovar Lai positioned between the two independent cas-operons. Here, we present the redefined repeat-spacer boundaries of the CRISPR subtype I-B locus of serovar Lai. Such refinement of boundaries of arrays in serovar Lai was done after comparison with the characterized array of another serovar Copenhageni and the manual analysis of CRISPR flanking sequences. Using the reverse transcription-PCR (RT-PCR), we account that the seven CRISPR are transcriptionally active in serovar Lai. Our RT-PCR and quantitative real-time PCR analysis of transcripts in serovar Lai indicated that seven CRISPR of subtype I-B transcribe together as a single precursor unit. Moreover, the cleavage of the two miniature pre-crRNA of the subtype I-B by Cas6 demonstrates the biogenesis of the expected size of mature crRNA essential for the guided interference of foreign DNA. This study features insight into transcription direction and the crRNA biogenesis in serovar Lai essential for RNA-mediated interference of invading nucleic acids.

| S-EPMC9372919 | biostudies-literature

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