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Extracellular proteome analysis of Leptospira interrogans serovar Lai.

ABSTRACT: 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.

SUBMITTER: Zeng L

PROVIDER: S-EPMC3783971 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

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Genome-wide subcellular localization of putative outer membrane and extracellular proteins in Leptospira interrogans serovar Lai genome using bioinformatics approaches.

Project description:BACKGROUND: In bacterial pathogens, both cell surface-exposed outer membrane proteins and proteins secreted into the extracellular environment play crucial roles in host-pathogen interaction and pathogenesis. Considerable efforts have been made to identify outer membrane (OM) and extracellular (EX) proteins produced by Leptospira interrogans, which may be used as novel targets for the development of infection markers and leptospirosis vaccines. RESULT: In this study we used a novel computational framework based on combined prediction methods with deduction concept to identify putative OM and EX proteins encoded by the Leptospira interrogans genome. The framework consists of the following steps: (1) identifying proteins homologous to known proteins in subcellular localization databases derived from the "consensus vote" of computational predictions, (2) incorporating homology based search and structural information to enhance gene annotation and functional identification to infer the specific structural characters and localizations, and (3) developing a specific classifier for cytoplasmic proteins (CP) and cytoplasmic membrane proteins (CM) using Linear discriminant analysis (LDA). We have identified 114 putative EX and 63 putative OM proteins, of which 41% are conserved or hypothetical proteins containing sequence and/or protein folding structures similar to those of known EX and OM proteins. CONCLUSION: Overall results derived from the combined computational analysis correlate with the available experimental evidence. This is the most extensive in silico protein subcellular localization identification to date for Leptospira interrogans serovar Lai genome that may be useful in protein annotation, discovery of novel genes and understanding the biology of Leptospira.

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

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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.

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Global proteome analysis of Leptospira interrogans.

Project description:Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometry complemented with two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. A total of 563 proteins were identified in this study. Altered expression of 65 proteins, including upregulation of the L. interrogans virulence factor Loa22 and 5 novel proteins with homology to virulence factors found in other pathogens, was observed between the comparative conditions. Immunoblot analyses confirmed upregulation of 5 of the known or putative virulence factors in L. interrogans exposed to the in vivo-like environmental conditions. Further, ELISA analyses using serum from patients with leptospirosis and immunofluorescence studies performed on liver sections derived from L. interrogans-infected hamsters verified expression of all but one of the identified proteins during infection. These studies, which represent the first documented comparative global proteome analysis of Leptospira, demonstrated proteome alterations under conditions that mimic in vivo infection and allowed for the identification of novel putative L. interrogans virulence factors.

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