Unit: Spirochetes (Laboratory of)

Director: Isabelle SAINT GIRONS

A random insertional mutagenesis has been developed in Leptospira interrogans, the leptospirosis agent. The determination of the sequence of the LE1 prophage, an extra-chromosomal circular replicon of Leptospira biflexa, was accompanied by the characterization of its replication and partition function. The typing of leptospires, by use of mini satellites (VNTR), which proved to be quite efficient to identify most of the Leptospira interrogans serovars, was developed in the National Reference Center for Leptospira. A new methodology (MLSA) was developed to assess the taxonomy of Borrelia instead of the cumbersome reference method. For Lyme borreliosis, the focus was on two epidemiological aspects: the clinical incidence of the human disease and the study of the vector density (the Ixodes ricinus tick) and its infection rate by Borrelia burgdorferi.

LEPTOSPIRA

Complete nucleotide sequence of the LE1 prophage from the spirochete Leptospira biflexa and characterization of its replication and partition functions (P. Bourhy1, L. Frangeul2, E. Couvé3, P. Glaser3, I. Saint Girons1 and M. Picardeau1)

1 Laboratoire des Spirochètes, 2 Plateau Technique 4 - Intégration et Analyse Génomique, 3 Unité de Recherche de Génomique des Microorganismes Pathogènes, Institut Pasteur, Paris, France.

The first, and only to date, extra-chromosomal circular replicon identified in the spirochete Leptospira is the LE1 prophage from Leptospira biflexa. The 74-kb long LE1 genome has a GC content of 36%, which is similar to the GC content of Leptospira spp. Most of the 79 predicted ORFs showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or in L. biflexa late after infection. Base compositional asymmetry allows the identification of the LE1 replication region and suggests that LE1 replicates via a bidirectional  -like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1 kb region. This minimal replication region consists of a rep encoding protein of 180 amino acids. Upstream from rep, putative partitioning genes, called parA and parB, were found to be similar to the par loci in Borrelia plasmids. A significant increase of plasmid stability in L. biflexa can be seen only when both genes parA and parB were present. These results enable the construction of new shuttle vectors for the genetics of Leptospira. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.

Random insertional mutagenesis of Leptospira interrogans, agent of leptospirosis, using a mariner transposon (P. Bourhy, H. Louvel, I. Saint Girons and M. Picardeau)

Laboratoire des Spirochètes, Institut Pasteur, Paris, France.

The recent availability of the complete genome sequences of Leptospira interrogans, the agent of leptospirosis, has allowed the identification of several putative virulence factors. However, to our knowledge, attempts to carry out gene transfer in pathogenic Leptospira spp. have failed to date. In this study, we show that the Himar1 mariner transposon permits random mutagenesis in the pathogen L. interrogans. We have identified genes that have been interrupted by Himar1 insertion in 35 L. interrogans mutants. This approach of transposon mutagenesis will be useful for understanding the spirochetal physiology and the pathogenic mechanisms of Leptospira which remain largely unknown.

National Reference Center (NRC) for Leptospires, WHO and FAO Collaborating Center for leptospirosis epidemiology (G. Baranton, D. Postic, D. Chaumet, D. Deberne, E. Fournié-Amazouz, MA. Golléty, V. Hossard and N. Sertour, with the collaboration of D. Portnoï)

See: http://www.pasteur.fr/sante/clre/cadrecnr/lepto-index.html

2004 has been considered as a low endemicity year for leptospirosis. However 2005 data show a further decrease in the number of cases. It only reaches 135 cases for 5785 sera (data restricted to the NRC) as compared to 186 in 2004, 242 in 2003, 244 in 2002 and 241 in 2001 in the same conditions. In fact it is the lowest recorded number of cases since 1985. During these last twenty-one years, the Micro Agglutination Test (MAT) has been systematically applied to each suspect serum.

The 2005 decrease of cases is observed all over the year but is specially important during the second semester which does not exhibit the usual "peak" in September. The general context of droughness noticed in 2004 probably contributes to maintain a low level of Leptospira transmission.

It is unfortunately noticeable that 2005 will be the last year with such a diagnosis procedure (systematic MAT) which warrants a representative surveillance of the leptospires circulation level. Indeed, a recent nomenclature change concerning medical biology requires a primary screening of suspect sera by Macro Agglutination, and a MAT only in case of positivity of the screening. An artificial deficit in leptospirosis cases is therefore expected for the next years. The decrease will be heterogeneous since serogroups Grippotyphosa, Australis, Panama … are less detected by screening tests.

The Leptospires NCR published a follow up of epidemiological surveillance of leptospirosis in continental France from 1920 up to 2003. It shows that modifications in diagnosis conditions lead to large fluctuations in the number of recorded cases. Moreover we observe that important "peaks" of one to five years occur at irregular intervals.

The use of VNTR (Variable Number of Tandem Repeats) for L. interrogans isolates typing has been published in 2005 in collaboration with M. Picardeau Group. This method in progress in the NCR allows a serovar identification with 3 PCRs.

Consult also: http://www.pasteur.fr/recherche/Leptospira/LeptospiraF.html

BORRELIA

CNR of Borrelia (D. Postic, E. Ferquel, M. Garnier, V. Hossard and N. Sertour, with the collaboration of C. Pérez-Eid and D. Portnoï)

See: http://www.pasteur.fr/sante/clre/cadrecnr/borrelia-index.html

Lyme borreliosis, transmitted to man by the bite of Ixodes ricinus tick, is due to bacteria within the complex Borrelia burgdorferi sensu lato. Clinical manifestations of the disease are mostly cutaneous, since the more frequent lesion is the erythema migrans. Later manifestations include neurologic, articular or cardiac involvement.

No national surveillance system is available in France. Therefore a network of volunteer physicians was set up by the National Reference Centre for Borrelia in restricted regions of the country. A prospective study was conducted in the department of Meuse from 2002 to 2005, yielding an estimated Lyme borreliosis incidence of 79 and 84 cases/100 000 inhabitants in 2002 and 2003 respectively. A significant increase of the disease incidence was observed in 2004 when 156 cases/100 000 inhabitants were recorded.

A similar study was initiated in Auvergne in 2004. The incidence was estimated to be 37 and 58 cases/100 000 inhabitants in departments of Allier and Cantal respectively. The low participation level of physicians from Haute-Loire did not allow any estimation of incidence in this department.

Another approach of the epidemiology of the disease consists in the study of the density and infection level of the tick vector.

We conducted a survey in three cantons of Alsace where incidence data were available in order to establish correlation between the incidence of the human disease and the density of ticks infected by pathogenic B. burgdorferi sl species. We showed that, in the cantons of Guebwiller and Munster, where the incidence was 219 and 279 cases/100 000 inhabitants, density of infected ticks was 114/100 m2, while it was only 5/100 m2 in the canton of Dannemarie, where the incidence of the disease was 36 cases/100 000 inhabitants. Moreover the level of infected ticks was among the highest reported in Europe.

B. afzelii and B. garinii are the most frequent species identified in I. ricinus ticks in Alsace, followed by B. valaisiana. B. burgdorferi ss is rare in this region. B. spielmanii was identified from only one tick while B. lusitaniae was never found.

We showed a positive correlation between the incidence of Lyme borreliosis and the density of infected ticks, a parameter directly correlated to the risk for man. Therefore the methodology settled in this work should be used for evaluating the risk of human infection in a given region.

Besides, the infection level of ticks by Anaplasma phagocytophilum was very low in Alsace, since 0.37% of nymphs and 1.2% of adults were infected.

The use of MLSA allowed us to propose a new methodology to delineate new B. burgdorferi sl species, instead of the cumbersome DNA-DNA hybridization reference method. This methodology allowed us to validate the new species B. spielmanii.

Consult also: http://www.pasteur.fr/recherche/borrelia/Welcome.html

Keywords: Spirochetes, Leptospira, Borrelia, epidemiology, genetics, mutagenesis


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