Biology of Spirochetes  


  HEADDr PICARDEAU Mathieu / mpicard@pasteur.fr
  MEMBERSPICARDEAU Mathieu / BOURHY Pascale / LOURDAULT Kristel / OLIVEIRA Priscilla / SLAMTI Leyla / POGGI Dante / LEITE Mara / ZININI Farida / COUEILLE Solange / MOREL Viviane / CLEMENT Sabine / CHAUMET Delphine / ETIEMBLE Sylviane


  Annual Report

Spirochetes are the causative agents of several important animal and human diseases such as Lyme disease and leptospirosis. The unit is composed of a research group on Leptospira and the National Reference Center for Leptospira (also WHO collaborating Center for Leptospira).

In 2009, the main research activities included:

i) Mutagenesis in the causative agent of leptospirosis

The L. interrogans genome contain approximately 3400 predicted coding regions (excluding transposases and pseudogenes), of which half were assigned no biologic role, whereas the remaining half were assigned roles that await experimental validation. We recently provided the first evidence of gene transfer in the pathogen L. interrogans, involving the transposition of a transposon of eukaryotic origin (Bourhy et al. 2005). We used this methodology in L. interrogans serovar Lai strain Lai 56601, a pathogenic strain which is entirely sequenced (Ren et al. 2003) and relatively transformable, to construct a library of random mutants. The insertion site of the transposon has been determined in approximately 300 random mutants of L. interrogans strain Lai. We also have generated libraries of random mutants in other pathogenic strains, then generating approximately 1000 mutants with characterized transposon insertion points (Murray et al. 2009). Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. This library provides a valuable resource for the study of gene function in the pathogen L. interrogans.

ii) Animal model for leptospirosis

The use of experimental models of leptospirosis remains a critical component for elucidating disease pathogenesis. Most studies exploring the pathogenicity of Leptospira have used hamsters or guinea pigs as animal models. However the dynamics of leptospirosis infection has been poorly studied. We determined the LD50, the rate of bacterial dissemination, histopathology, and antibodies responses against leptospira following inoculation of L. interrogans strain Fiocruz L1-130 strain in the guinea pig model of leptospirosis (Lourdault et al. 2009). Three routes of infection (i.e. intraperitoneal, conjunctival, and subcutaneous inoculations) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in blood and tissues of infected animals was determined using quantitative real-time PCR (qPCR). Further studies will focus on determining the kinetics of dissemination of various mutant and parental strains into blood and tissues.

Mice are not usually susceptible to leptospirosis. Indeed, like rats, the common domestic mouse is a major reservoir for leptospirosis and bacteria produce an asymptomatic infection. However, progress in mouse genetics, functional genomics and imaging technologies may increase our understanding of the pathogenesis of leptospirosis. Leptospira spp. possess an unusual LPS that has been shown to use TLR2 and TLR4 for signaling in murine cells (Nahori et al. 2005). We showed that TLR2/TLR4 double-deficient mice rapidly died from severe hepatic and renal failure following Leptospira inoculation (Chassin et al. 2009). These experiments are useful for studies of the role of immune mechanisms in the pathogenesis of leptospirosis. This mouse model may be valuable for in vivo studies of the virulence of L. interrogans mutant strains.

iii) Molecular typing of pathogenic leptospires

The main function of the National Reference Center (NRC) for Leptospira is the surveillance of this zoonosis, based on the collection of diagnosis data from laboratories around the country. The NRC is one of the few laboratories that can confirm leptospirosis diagnosis by means of the serological test called MAT in France. The NRC has also a duty to improve diagnostic techniques. We have developed a rapid and simple typing method based on analysis of the polymorphism of variable-number tandem repeats (VNTR) loci in the genome of pathogenic Leptospira species. This method is now into routine used at the NRC. Further improvements to this method are required, so that MLVA (Multiple Locus VNTR analysis) can be applied directly to biological and environmental samples without the need to culture the pathogen which is a slowly-growing bacteria. This typing method would help to improve the surveillance of this underestimated disease. This method should also facilitate the sharing of results and the generation of databases.

Keywords: Spirochetes, Leptospira, epidemiology, genetics, virulence factors

picardeau.jpg

Electronic microscopy of Leptospira biflexa



  Publications

Ristow, P., P. Bourhy , F.W. McBride, C.P. Figueira, M. Huerre, P. Ave, I.S. Girons, A.I. Ko, and M. Picardeau (2007). The OmpA-like protein Loa22 is essential for leptospiral virulence.PLoS Pathog. 3(7):e97

M. Picardeau, D.M. Bulach, C. Bouchier, R. L. Zuerner, N. Zidane, P. J. Wilson, S. Creno, E. S. Kuczek, S. Bommezzadri, J. C. Davis, A. McGrath, D. Roche, M. J. Johnson, C. Boursaux-Eude, T. Seemann, Z. Rouy, J. I. Rood, A. Lajus, J. K. Davies, C. Médigue, and B. Adler (2008). Genome sequence of the saprophyte Leptospira biflexaprovides insights into the evolution of Leptospiraand the pathogenesis of leptospirosis .PLoS ONE 3(2):e1607.

J. Croda, C.P. Figueira, E.A. Jr Wunder, C.S. Santos, M.G. Reis, A.I. Ko, and M. Picardeau (2008). Targeted mutagenesis in pathogenic Leptospira: Disruption of the ligBgene does not affect virulence in animal models of leptospirosis. Infect. Immun.76:5826-33.

G. L. Murray, V. Morel, G.M. Cerqueira, J. Croda, A. Srikram, R. Henry, A.I. Ko, O.A. Dellagostin, D.M. Bulach, R. Sermswan, B. Adler, and M. Picardeau (2009). Genome-wide transposon mutagenesis in pathogenic Leptospiraspp.Infect. Immun. 77:810-816.

A. I. Ko, C. Goarant, and M. Picardeau (2009). Leptospira: The dawn of the molecular genetics era for an emerging zoonotic pathogen. Nature Rev. Microbiol 7:736-47.



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Activity Reports 2009 - Institut Pasteur
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