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  Director : BARANTON GUY (gbaran@pasteur.fr)


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In 2000 studies were devoted to experimental genetics, innate immunity and typing of Leptospira. In Borrelia, antigenic variation have been studied. In Yersinia, the main approaches were pathogenicity island analysis, Y. pseudotuberculosis genome sequencing and antibiotic resistance.



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Introduction

The "Bactériologie Moléculaire et Médicale" unit studies essentially three pathogenic bacterial genera Leptospira, Borrelia (two spirochaetes) and Yersinia (enterobacteria).
Leptospires are responsibles for a zoonosis which causes an acute illness with often severe outcomes in man. Symptoms vary from haemoraghes to meningitis, jaundice and renal failure .. A saprophytic species is used as a model bacterium for developping genetic tools. Other informations are available at the following adress: :http://www.pasteur.fr/recherche/Leptospira/LepE.html

Borrelia are in Europe responsibles for Lyme disease but in tropical regions, other species of Borrelia cause relapsing fevers caracterized by reiterated bouts of fever at regular intervals.

The genus Yersinia is composed of three species pathogenic for humans. Y. pseudotuberculosis and Y. enterocolitica, widely distributed in cold and temperate countries, are transmitted by the oral rout and cause intestinal symptoms. Y. pestis is the agent of plague, a fulminant disease transmitted by fleas and responsible for endemic and epidemic human cases in Africa, Asia and the Americas.

LEPTOSPIRA The first genetic tools for Leptospira I. Saint Girons, P. Bourhy, C. Ottone, M. Picardeau, D. Yelton, R. Hendrix, P. Glaser, N. Charon

Genetic tools for spirochetes were scarce and non-existent for the genus Leptospira.We succeeded in constructing a L. biflexa-E. coli shuttle vector with the following characteristics: (i) a 2.2 kb fragment from LE1 leptophage DNA which contains its origin of replication (ii) a kanamycin cassette from Enterococcus faecalis (iii) an E. coli origin of replication. Furthermore, an allelic exchange was obtained for the flagellin gene (flaB) of L. biflexa, a saprophytic species. The flaB: kanR obtained mutant had a reduced motility. Experiments are under way to inactivate other genes
and to extend these results to pathogenic Leptospira.

Dispersion of the methionine genes on the two chromosomes of Leptospira P. Bourhy, I. Saint Girons

The metF gene belonged to the CII secondary chromosome, in contrast to the previously isolated metY and metX genes, which had been localized to the CI chromosome of Leptospira sp. These data of dispersion of the methionine
genes on the two chromosomes of Leptospira indicate a functional link between the two chromosomes of the genome.

Penicillin binding proteins in Leptospira A. Brenot, D. Trott, I. Saint Girons, R. Zuerner

Six penicillin binding proteins in L. interrogans have been shown to exist by a functional assay. The ponA and pbpB genes had been isolated and encode PBP1 and PBP3 respectively. The L. interrogans PBP1 and PBP3 proteins were
synthesized in E. coli and were modified with ampicillin using a digoxygenin-ampicillin conjugate. These data show that both genes encode functional penicillin-binding proteins.

Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism C. Werts, R.I. Tapping, J.C. Mathison, T.H. Chuang, V. Kravchenko, I. Saint Girons, D.A. Haake, P.J. Godowski, F. Hayashi, A. Ozinsky, D.M. Underhill, C.J. Kirschning, H. Wagner, A. Aderem, P.S. Tobias, R.J. Ulevitch

Bacterial pathogens activate the host innate immune system via "Toll-like" receptors (TLR), which recognize conserved bacterial components as for example endotoxins (LPS) or lipoproteins. Leptospiral LPS is much less
endotoxic than LPS from Gram-negative bacteria. We showed that both leptospiral LPS and lipoproteins activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central
component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also showed that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant
signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.

A clonal subpopulation of Leptospira interrogans sensu stricto is the major cause of leptospirosis outbreaks in Brazil M.M. Pereira, M.G.S. Matsuo, A.R. Bauab, S.A. Vasconcelos, Z.M. Moraes, G. Baranton, I. Saint Girons

Surprisingly, although there is no interhuman transmission, leptospirosis outbreaks (erroneously called "epidemics") occurring during floods in large Brazilian cities, are linked to one largely prevalent serovar. 44 strains from outbreaks in Sao Paulo in 1994 and 1995 were isolated and identified : 38 were icterohaemorrhagiae and 6 canicola. Infective foci can be related to different animals, but infective leptospires are clonal anyway.

Analysis of Leptospira isolates from mainland Portugal and the Azores islands Cao Thi Bao Van, G. Baranton
Leptospirosis cases have been recently recorded in the Azores islands. In collaboration with Portuguese and Dutch scientists, we identified the serovars prevailing in these volcanic islands.

Identification of a 36-kDA fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae F. Merien, J. Truccolo, G. Baranton, P. Perolat
Following the previous work about Leptospira interrogans-host cell interactions showing an adhesion, we looked for a potential adhesin to a protein of the extra-cellular matrix : fibronectin. We succeeded in characterizing membrane interactions between human fibronectin and a 36 kd protein present in L. interrogans. This protein (reacting with proteinase K), lacking in L. biflexa (saprophytic), binds with the "gelatin binding" domain of fibronectin.

Interest of partial 16SrDNA gene sequences to resolve heterogeneities between Leptospira collections : application to L. meyeri D. Postic, N. Sertour, F. Merien, P. Perolat, G. Baranton
Paradoxically, although many methods are used for identification of serovars, Leptospira species identification is not easy, and is not always successful. For instance, in L. meyeri species identification often leads to controversial results. This species involves serovars isolated from frogs, surface waters, or mammals. Generally considered as pathogenic in Europe, in the USA this species was described as saprophytic. A complete study showed that serovar type strains were not the same in Europe and in the USA. In Europe, strains belonging to these serovars could be related to other species, which were actually pathogenic, whereas in the USA these strains, with one exception, were linked to L. meyeri species which phylogenetically belongs to saprophytic group. These results were obtained by sequencing the amplicon used in PCR diagnosis which, on 330 pb of the rrs gene coding for ARNr16S, represents 50 % of the polymorphism of the whole gene. This enables species identification in Leptospira.


BORRELIA

Antigenic variation of Borrelia turicatae Vsp surface lipoproteins occurs in vitro and generates novel serotypes N. Marti Ras, D. Postic, P. Ave, M. Huerre, G. Baranton
Borrelia turicatae is one of the models used to study antigenic variation in relapsing fever Borrelia. Experimentally, as well as in patients, new serotypes, associated to a variable protein Vsp regularly emerge, replacing the previous ones. Each new serotype occurrence corresponds to a febrile access. The selection of a new serotype variant is due to the antibody selective pressure.

We repeatedly cultivated a cloned (by serial dilution) isolate. At passage 50, the bacterial population was cloned again and submitted to Pulsed Field Gel Electrophoresis showing 4 distinct plasmidic rearrangements. Each of these clones Vsp, the protein of which was sequenced, showed it belonged to a given serotype. Inoculated to mice, each of these serotypes exhibited a specific organotropism.

National Reference Center and WHO Collaborating Center for Leptospires E. Fournié, N. Sertour, E. Bellenger, P. Bourhy, http://www.pasteur.fr/sante/clre/cadrecnr/lepto-index.html

In 2000 a low number of cases has been recorded according to the endemic aspect of the disease : 268 in continental France, corresponding actually to a reduced circulation of leptospires, and 266 in overseas departments where leptospirosis diagnosis was not always easily performed. cf. : http://www.pasteur.fr/recherche/Leptospira/Leptospira.html
for data available on the Web.
Several signifying outbreaks were recorded. On their return from the Eco Challenge raid in Malaysia, the 4 members of the French team suffered from leptospirosis (158 out of the 304 participants contracted the disease). Another occurrence concerned 5 militaries who, in Martinique, took part in operations in the country.
In 2000 was published a new bilingual version (English-French) of the Manual : Biological diagnosis : Leptospirosis - Lyme Borreliosis by D. Postic, F. Merien, P. Perolat, G. Baranton, in the collection "Laboratory Methods" http://www.pasteur.fr/infosci/manuels/edlept12ed.html CLRE, Institut Pasteur, Paris, 2e Edition, English : 113 p., French : 135 p. This publication involves in particular protocols for molecular identification and detection of Leptospira and Borrelia.

Legionella and Francisella Laboratory C. Tram

In 2000, a hundred strains, (mainly L. pneumophila) were isolated and identified, particularly regarding their pathogenicity (PCR on MIF gene).

About fifty strains of Francisella tularensis were identifed from cultures of animal samples (Maisons-Alfort) and 5 strains of patients suffering from tularemia (ganglia).

YERSINIA (Head: Elisabeth Carniel)

The genus Yersinia is composed of 3 species pathogenic for humans: the enteropathogens Y. pseudotuberculosis and Y. enterocolitica, and the plague agent Y. pestis.

The main fields of activity of the Yersinia laboratory are:



- The characterization of a pathogenicity island whose presence confers the host bacterium with the ability to cause systemic infections in humans and to be lethal in mice.
- The molecular bases for the exceptional pathogenicity of Y. pestis. Sequencing of the genome of Y. pseudotuberculosis, a bacterium genetically almost identical to Y. pestis but of much lower pathogenicity is underway and will allow a "gene by gene" comparison of the 2 genomes.
- The relations between Y. pestis and its insect vector, the flea.
- The physiopathology of Yersinia infection.
- The evolution of Y. pestis since its recent emergence from Y. pseudotuberculosis.
- The resistance of pathogenic Yersinia to antibiotics and the evaluation of new treatments.
- Public health at the national (French Reference Center) and international (WHO Collaborating Center) levels.


The works published in 2000 dealt with:


1. Characterization of the Yersinia high pathogenicity island

The notion of foreign chromosomal regions termed "pathogenicity islands" which carry virulence genes emerged recently. Their capacity to excise spontaneously from the bacterial chromosome, their GC% different from the remaining of the genome and the presence of a phage-like integrase suggest their acquisition from a bacteriophages, by horizontal transfer. Some pathogenic Yersinia harbor such a structure, termed "high pathogenicity island" (HPI) because they confer their bacterial host with a high pathogenicity potential (systemic infections in humans, lethality in mice). Our laboratory analyzes the HPI of the 3 pathogenic species of Yersini.

1.1. Characterization of repeated sequences present on the Y. enterocolitica HPI I. Guilvout and E. Carniel Collaborators: A. Rakin, S. Schubert, and J. Heesemann

A cluster of 3 repeated sequences are present on the HPI of Y. enterocolitica. Two of these sequences have previously been identified as insertion sequences belonging to the IS3 family. Analysis of the third repeated sequence revealed that, as for the two others (IS1400 and IS1328) it corresponds to an IS (termed IS1329) belonging to the IS3 family. This sequence is itself inserted in the remnants of a fourth IS (IS1222), also belonging to the IS3 family. Therefore, this A+T rich region of the HPI represents a hot spot for integration of IS of the IS3 family.

1.2. Distribution of the Yersinia HPI among enterobacteria.S. Bach, A. de Almeida, E. Carniel

The Yersinia HPI has previously been identified in different pathotypes of E. coli. A search for this island among members of the enterobacteriaceae family demonstrated its presence also in Citrobacter diversus and different species of Klebsiella where it seems to be functional. With very few exceptions, the HPI is well conserved in these bacteria and is inserted, as in Yersinia, in an asnT tRNA locus. The HPI is so far the only example of a pathogenicity island present and well conserved in different bacterial species and genera.

2. Antibiotic resistance and treatment

2.1. Analysis of the ß-lactamases produced by Y. enterocolitica L. Martin and E. Carniel Collaborators: J. Pham and S. M. Bell

Y. enterocolitica harbors chromosomal genes encoding a penicillinase and a cephalosporinase constitutively expressed. An extensive analysis of Y. enterocolitica strains of various biotypes and serotypes indicated that susceptibility to ß-lactamins is directly linked to the presence and types of beta-lactamases produced: enzyme A or A-like for penicillinase, and B or B-like for cephalosporinase. There is a close correlation the types of enzymes produced and the phenotypic characteristics of the strains.

2.2. Evaluation of a new treatment for plague: A. Guiyoule and E. CarnielCollaborators: L. Rahalison, S. P. Bonacorsi, I. Slacanin, S. Chanteau

The antibiotics of choice for plague therapy are streptomycin and tetracyclines. Although efficient, these treatments pose practical problems. The efficacy of single or two-dose injections of oily chloramphenicol was compared with standard multiple injections of reference drugs in a murine plague model. One or two injection of oily chloramphenicol were less effective in reducing mouse mortality than standard therapy. However, because of the marked pharmacokinetics differences between mice and humans, the failure of depot injection of oily chloramphenicol in murine plague treatment is not indicative of its ineffectiveness in human plague.


3. Analysis of the polymorphism in fatty acid composition of Y. pestis cell wall A. Guiyoule and E. Carniel Collaborators: A. Leclerq, M. El Lioui, et J. Decallonne

The cellular fatty acid compositions of various strains of Y. pestis representing the global diversity of this species was analyzed by gas-liquid chromatography to investigate the extent of fatty acid polymorphism in this microorganism. All Y. pestis strains studied displayed some major fatty acids that were extremely homogeneous. The genus Yersinia could be separated into three clusters corresponding to: (i) non-pathogenic Yersinia, (ii) pathogenic Y. enterocolitica isolates, and (iii) Y. pseudotuberculosis and Y. pestis strains. Therefore, our results indicate that the fatty acid composition of Y. pestis is highly homogeneous and very close to that of Y. pseudotuberculosis.


4. National Reference Laboratory and WHO Collaborating Center for Yersinia L. Martin, F. Guinet, E. Carniel

The National Reference Laboratory for Yersinia is involved in research dealing with epidemiology, therapy and diagnosis of yersiniosis. This laboratory participates to the description of unusual clinical forms, the development of new diagnostic tools, and the improvement of the Yersinia taxonomy. The activity of the WHO collaborating center concerns molecular typing of Y. pestis of worldwide origins, maintenance of these strains, provision of biological materials, recommendations, and training of foreign scientists. The center also participates to field works when necessary.


5. Sequencing of the Y. pseudotuberculosis genome V. Chenal, D. Dacheux and E. Carniel Collaborators: E. Garcia, P. Chain

The genome of Y. pestis was recently sequenced at the Sanger Centre (UK). Comparison of the genomes of Y. pestis and Y. pseudotuberculosis (a bacterium genetically closely related to Y. pestis but of much lower virulence) might help identifying genes responsible for the extraordinary pathogenicity of Y. pestis. Therefore, sequencing of the genome of Y. pseudotuberculosis has started at the Lawrence Livermore National Laboratory (US), in collaboration with our laboratory for the pre- and post-sequencing phases. The post-sequencing phase of the project will include identification of species specific alleles and evaluation of the role of these alleles on bacterial pathogenicity.



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  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

DELARUE Nadine, IP

DUVAQUIER Annie, IP

CARNIEL Elisabeth, IP, carniel2@pasteur.fr

GUINET Françoise, IP, fguinet@pasteur.fr

POSTIC Danièle, IP, dpostic@pasteur.fr

SAINT GIRONS Isabelle, IP, isgirons@pasteur.fr

WERTS Catherine, IP, cwerts@pasteur.fr

PICARDEAU Mathieu IP, mpicard@pasteur.fr

BACH Sandrine

BRENOT Audrey

CALDERARO Adriana

DACHEUX Denis

GHNEIM George

HACKER Jörg

LESIC Bibiana

NADOLNY Agathe

ROSSO Marie Laure

SEBBANE Florent

THEODORE George YOUNSI Hend

BELLENGER Elisabeth, IP

BOURHY Pascale, IP

CHENAL Viviane, IP

DENIS Patrick, IP

FOULON Jeannine, IP

GUIYOULE Annie, IP

FOURNIE-AMAZOUZ Edith, IP

GALUPA Isabelle, IP

GUINANDIE Françoise IP

MARTIN Liliane, IP

OTTONE Catherine, IP

PARMIER Martine, IP

SERTOUR Natacha, IP

TOUTTAIN Chantal, IP

TRAM Cuong, IP


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